Pharmacogenetics: What is it How do I measure it What does it mean to me

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Pharmacogenetics What is it? How do I measure
it? What does it mean to me?

• Joseph S. Bertino Jr., Pharm.D.
• Associate Professor of Pharmacology
• Columbia PS
• Bertino Consulting
• sbertino_at_ix.netcom.com

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Goals and Objectives

• Review the concept of pharmacogenetics and
  pharmacogenomics
• Discuss how genetics and the environment affects
  the activity of drug metabolizing enzymes and
  transporters
• Discuss examples on how pharmacogenetics will
  lead to individualization of drug therapy
  (personalized or individualized medicine)

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Pharmacogenetics What is it?
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Important Definitions

• Pharmacodynamics is an expression of
  pharmacokinetics (i.e., drug effect)
• Pharmacokinetics is an expression of
  pharmacogenetics (i.e. ADME)
• Pharmacogenomics Study of hereditary basis for
  differences in a populations response to a drug
• Pharmacogenetics (effect of genetics and
  environment) includes CYP450s, Conjugative
  reactions and Transporters

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Important Facts

• Human genome 22 chromosome pairs and 1 pair of
  sex chromosomes
• Functional unit of the genome gene
• 2 of genes code for proteins, remainder is
  structural for DNA
• Entire genome 3 billion DNA pairs, with 30,000
  protein coding genes
• DNA sequence of a pair of genes varies between
  each individual gene in that pair

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Important Facts

• SNPs (single nucletide polymorphisms) account for
  sequence variation between people (1SNP/1000 base
  pairs of DNA)
• 1 of SNPs affect protein coding region of DNA
  sequence
• SNPs can change the way protein is made or be
  stop codons that result in functionally
  inactive or reduced activity protein
• Gene deletion can also be seen with no protein
  made

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Important terms

• Chromosome is the largest unit
• Gene is the next unit
• Allele codes for a gene, different alleles
  determine different characteristics. An alleles
  is one member of a pair that makes up a gene
• 2 same alleles are homozygous (one allele on each
  part a pair of chromosomes)
• 2 different alleles are heterozygous

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Pharmacogenetics and Evolution

• Plant-Animal Warfare Explains the variety of
  enzymes capable of destroying naturally occurring
  toxins
• Person-to-person variability is prominent
  (genetic distances)
• Allelic variants have high frequency due to
  mutations

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Phase I reactions The CYP system
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Cytochrome P450 Enzymes
Brain

• Phase I oxidative enzyme system
• Present in various organs in the body
• Liver is the major source

Lungs
Kidneys
Intestines
Monocytes Macrophages Lymphocytes
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Cytochrome P450 enzyme system terminology
CYP2C9 12

• CYP - P450 for all mammalian species
• 2 - family (17 - 14 human)
• C - subfamily (42 in humans)
• 9 - enzyme/gene (55 genes, 29 pseudogenes in
  humans
• 12 -Allele pattern

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What do the CYPs do?

• Drug metabolism throughout the body
• Adapt the organism to the environment (plant
  animal warfare)
• Activate drugs
• Detoxify substances and activate non-toxic
  substances into toxic substances

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ADM Kashuba
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Intersubject Variability in Drug Metabolizing
Enzymes

• Degree of enzyme activities are different between
  individuals
• Genetic Polymorphism
• Variable gene expressions that are found in gt1
  of population
• Numerous metabolizing enzymes are polymorphic
• CYP2C9, CYP2C19, and CYP2D6 polymorphismcan be
  clinically significant

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Phase II (conjugative) enzymes (can be
polymorphic)

• UDP-Glucuronosyltransferase UGTs
  (glucuronidation)
• UGT1 and UGT2 families
• Glutathione S-Transferase GST
• Sulfontransferase (Sulfation) SULT
• Methyltransferase (Methylation) MT
• Epoxide Hydrolases (Oxidation) EH
• NAT

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Drug Transporters

• PGP P-glycoprotein, ATP dependent efflux
  transporter
• MDR Gene family that codes for PGPs, multidrug
  resistance genes (MDR1 only)
• ABC transporter (ATP binding cassette)
• OATP Organic Anion Transport Protein
• OCTP Organic Cation Transport Protein
• OATP and OCTP are influx transporters

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What do Drug Transporters Do?

• PGP Wide range of functions and substrates
  (unlike most ABC transporters)..it is an efflux
  transporter
• Functions as a pump in the gut to act as
  transport facilitator (located in the lining of
  epithelial cells, increases throughout gut)
  Activation keeps drugs in the gut
• Pump functions in tumor cells to mediate
  resistance
• Assists in excretion of drugs into bile
• Functions as a pump in other areas of body

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What do Drug Transporters Do?

• OATP/OCTP Functions just being uncovered,
  appears to function in the gut as an influx pump.
  May be more important than PGP in some instances
  (inhibited by apple and orange juice)
• Influx transporters

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How do I measure it?
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Investigation of DME and Transporter Activity
Genotyping

• investigation of genetic codes variant alleles
• PCR-based test using DNA from peripheral
  leukocytes or any DNA source (100 specific, 80
  sensitive)
• investigation of the manifestation of genetic
  code differences (due to drugs, environment,
  diet)
• pharmacologic test using a safe, enzyme specific
  drug marker that can be easily quantitated
  (urine, blood, etc)..A Probe

Phenotyping
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Genetics and Genotype
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Molecular mechanisms of altered drug metabolism
Deleted Gene Single Gene
Multiple Gene
Higher enzymes amts Increased Metabolism CYP2D62x
N
Normal Metabolism CYP2D61 CYP2C91 CYP2C191
Altered substrate specificity Other
metabolites formed CYP2D617 CYP2C93
No enzyme No metabolism CYP2D645 CYP2C1923
Unstable enzyme Reduced Metabolism CYP2D610
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Ethnic Evidence of Genetic Variation in CYP
DME..Different patients need different doses
Allele Ethnic group and occurrence

• CYP2C192 or3
• CYP2D62xN
• CYP2D610
• CYP2D617
• CYP2A6del

• Asians 13-20
• Ethiopians/Saudis 30
• Asians up to 70
• African-Americans 5
• Asians 15

reduced activity..needs less drug to get
effect, increased activityneeds more drug
to get effect
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Implications of Pharmacogenomics

• In the population, genetics determines drug
  metabolizing and transporter activity
• Most individuals in the range of normal
  activity (lots of interindividual variability)
  EMs (can use standard dosing)
• Some individuals will have much higher activity
  URMs (need bigger doses)
• Some individuals will have significantly reduced
  activity IMs (need reduced doses)
• Some individuals will have little or no activity
  PMs (need greatly reduced doses)
• However, in the population, phenotype is more of
  a continuum

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Gene-Dose-Sex Effect CYP2C19 Activity

• Females
• 0.92 0.38
• Males
• 1.80 1.34
• Males with CYP2C191/1
• 1.07 0.52

Kim et al, CPT 2002
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Phenotyping
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Examples of CYP phenotyping probes

• Caffeine
• Dextromethorphan
• Midazolam
• Omeprazole
• Warfarin Vitamin K

• CYP1A2
• CYP2D6
• CYP3A4/5/7/43
• CYP2C19
• CYP2C9

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Phenotypic manifestations of Genetic Polymorphism
Antimode
³ 2 Distinct Populations
Metabolic Ratio of Probe Drug
Prevalence of least ³ 1
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Genotype-Phenotype Relationship of CYP2D6
polymorphism
Genotype
or
or
or
PM (5-10)
URM (5-10)
EM (65-80)
IM (10-15)
Phenotype Frequency (Caucasians)
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Effect of Pharmacogenetics

• Other exogenous factors will affect drug
  metabolizing and transporter activity
• Diet -Infx diseases/cytokines
• Drugs -Sex (maybe)
• Pollution -Menstrual cycle (rare)
• Concurrent other diseases
• Age (1st year of life)

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Implications of Pharmacogenetic Variability

• Drug dosing needs individualization
• Prediction of toxic side effects
• Prediction of therapeutic effects
• Prediction of drug interactions

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What does it mean to me?
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Clinical Implications of Pharmacogenetics
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Drug Interaction Mechanisms

• Protein Binding Displacement
• Inhibition of the activity of DMEs
• Induction or allosteric activation of DME activity

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Persons with either genetically or
environmentally reduced enzyme activity will be
at less risk of drug-drug interactions due to DME
inhibition Persons with genetically or
environmentally induced DME activity are at
greater risk of drug-drug interactions
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Use of EMs for DDI studies Venlafaxine and CYP2D6
Eap et al, Pharmacogenetics 20031339-47
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Use of EMs vs. PMs for DDI studies Venlafaxine
Quinidine and CYP2D6
4-12 fold increase
No increase
Eap et al, Pharmacogenetics 20031339-47
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How is pharmacogenomics being used in treating
patients?

• 5 drugs where genetic testing is suggested
• Warfarin (CYP2C9 and VKOR1C)
• Atomoxetine (CYP2D6)
• 6MP or azathioprine (TPMT)
• Tamoxifen (CYP2D6)
• Abacavir (HLA-B5701 for hypersensitivity)

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Example 1 Omeprazole (the Purple Pill) and
Diazepam (Valium)

• Diazepam is used as a sedative
• Omeprazole is used in the treatment of GERD and
  is metabolized by CYP2C19
• Omeprazole inhibits its own metabolism
• After one week of combination therapy, Caucasian
  pts require a 50 decrease in diazepam dose
• Asians require a 25 decrease in diazepam dose
  after 1 wk of combo therapy
• WHY THE DIFFERENCE?

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Ethnic DifferencesOmeprazole Inhibits Diazepam
Metabolism

• Effect of omeprazole on diazepam metabolism in
  Caucasian vs. Chinese
• Randomized, double blind crossover study
• Inhibitory Effect larger in Caucasian subjects
• Due to inhibition of CYP2C19 activity
• Less CYP2C19 activity in Asians

Caraco et al. Clin Pharmacol Ther 199455169.
Abst.
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Example 2 Metoprolol in MI patients

• Metoprolol is used post-MI to prevent further
  cardiac damage
• Metoprolol is metabolized by CYP2D6
• The observation was made that Asian patients
  require 65 less of a dose (even when weight
  corrected) than Caucasians

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Metoprolol Exposure with Equal Weight
Doses-Reduced Metabolism in Asians

• 10 EMs (Caucs) and 6 PMs (Asians) took metoprolol
  daily for 5 days
• PMs had substantial ? in metoprolol AUC vs. EMs
• PMs had more toxicity (bradycardia and
  hypotension)

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Example 3 HIV patients

• 3 drug regimen is therapy of choice for HIV
  infection (HAART therapy)
• Protease Inhibitors provide the most active
  anti-HIV drugs
• Continued viral suppression is related to
  adequate drug exposure
• Protease inhibitors are metabolized by CYP3A

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Example 3 HIV patients

• 24 yo hispanic male with HIV disease started on
  HAART therapy with Indinavir (IDV) as the PI
• Patients viral load responds well with drop from
  gt 100,000 to lt 50 copies/ml of HIV
• Pt depressed, starts St. Johns Wort on advice
  from internet web site
• At next visit, 4 weeks later, viral load 50,000
  copies, phenotype of HIV shows IDV resistance
• Why??

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St. Johns Wort ? Decreased Indinavir (Crixivan)
Concentrations

• 8 healthy subjects took indinavir alone and with
  St. Johns wort for 2 weeks
• St. Johns wort reduced indinavir AUC in all
  subjects (mean 57)

Piscitelli SC. Lancet. 2000355547-548.
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Example 4 Grapefruit Juice and Statins

• Statins are used to reduce cholesterol and thus
  reduce CHD risk
• Some statins such as simvastatin and atorvastatin
  are metabolized by CYP3A in the gutreducing the
  amount of drug that gets absorbed
• Toxicity of statins includes muscle pain and
  rhabdomyolysis

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Example 4 Grapefruit Juice and Statins

• 49 yo wm with a history of CAD, currently on
  Atorvastatin 20 mg daily
• Patient is trying to maintain a low fat, healthy
  diet and eats only a grapefruit for breakfast
  each day
• One week into his diet, he notices that he is
  sore all over but especially in his legs
• In 4 more days, he notices that his urine is
  red..he goes to the ED
• Pt admitted for rhabo

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Example 7 Grapefruit Juice and Statins
Grapefruit juice inhibits the breakdown of
atorvastatin in the gut
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Summary

• gt 25 hepatic cytochrome P450 enzymes identified
  in man to date
• efficacy may be due to rapid metabolism in EMs or
  to inadequate metabolic conversion in PMs
• toxicity may be due to accumulation in PMs give
  standard medication doses
• morbidity due to drug interactions may be avoided
  with the knowledge of specific enzymes involved
  in drug metabolism
• Every branch of medicine is affected by
  pharmacogenetics

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Questions and Comments jbertino_at_ordwayresearch.or
g Or jjb5_at_columbia.edu