Applications of Pharmacogenetics in Forensic Pathology

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Applications of Pharmacogenetics in Forensic
Pathology

• Jeffrey M. Jentzen M.D.
• Professor Department of Pathology
• Medical College of Wisconsin
• Milwaukee County Medical Examiner

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Quincy Saves the Day
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The CSI Effect
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The Legacy of Forensic Science in Hennepin County
Calvin Brandt, MD
John I. Coe, MD
Garry F. Peterson, MD
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Postmortem Drug Interpretation

• Microbial Metabolism
• Postmortem Diffusion
• Physico-Chemical Factors
• Antemortem Metabolism

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Postmortem Redistribution

• The first study to bring attention to the
  fact that postmortem redistribution of drugs may
  be an expected problem of interpretation of TCAs
  concentration in blood was Bandt in 1980. Bandt
  surmised that the increase in the concentration
  of TCA in the blood was due to release of the
  drug from liver and subsequent diffusion to the
  heart via the venous system.
• Anderson and Prouty, Postmortem
  Redistribution of Drugs, Advances Anal Tox,
  1989270-101.

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Venous System
Subclavian vein
Superior vena cava
Aorta
Hepatic vein
Inferior vena cava
External iliac vein
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Landmarks in Pharmacogenetics
New Gene-Based Drugs
Personalized Medication
1865
1985
?
2015
1925
1955

Discovery of genetic variation in liver enzymes
Early Genetics
Weber SOFT 2002
Inception of Modern Pharmacogenetics
Pharmacogenomics Emerges
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Collins et al., A vision for the future of genome
research. Nature 422835, 24 April 2003
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Systems Biology Approach
GP,2003231(Beyond Genomics, Inc.)
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Pharmacogenomics

• The study of the linkage between an individuals
  genotype and that individuals ability to
  metabolize a foreign compound.
• Linder, M.W., et al. Clinical Chemistry 1997
• 43254-266.

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Pharmacology/Toxicology Paradigm
Pharmcokinetics, PK/Toxicokinetics, TK
Pharmacodynamics, PD
Pharmacogenetics, PG/Toxicogenetics, TG
Modified from Linder
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Cytochromes P450 (CYP)

• Most xenobiotics are metabolized by CYP to some
  extent.
• CYP drug metabolism results in
• Detoxification and elimination
• Activation of the prodrug
• CYP display a wide interindividual variation in
  expression and/or catalytic activity.
• CYP variation can be due to
• Transient causes such as enzyme
  inhibition/induction
• Permanent causes such as genetic
  mutations/deletions.

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CYP 450 Enzymes

• Some 50 distinct CYP 450 Enzymes
• Human CYP450 are classified into 17 families
• Enzymes function as drug metabolism,
  steriodogenesis and endogenous functions
• CYP2C9, CYPC19 and CYP2D6 account for 40 of drug
  metabolism mediated by P450.
• CYP2D6 phenotype varies with race
• Caraco Y., Genes and the Response to Drugs, NEJM,
  2004, 35127 2867-2869.

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CYP450 Nomenclature

• CYP cytochrome pigment at 450 nm
• Family designated by Arabic number
• Subfamily designated with letter
• Italic Gene, non-italic enzyme e.g.. CYP2E1
  codes for the CYP2E1 enzyme.

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Human CYP450s
Note 75 of all drugs metabolized by two enzymes
CYP3A4 CYP2D6
REF Merck Sharp and Dohme, Watt AP. Neuroscience
Research Centre
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Frequency Distribution
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Categorization of Genetic Polymorphisms

• Ultrarapid metabolizers multiple copies of a
  gene
• Extensive metabolizers (wild-type) single copy
  of a gene.
• Intermediate metabolizers (heterozygotes)
  exhibit decreased enzymatic activity.
• Poor metabolizers (homozygotes/double
  heterozygotes) have no detectable activity
• Benet, L.Z., et al., Goodman Gilmans the
  pharmacological basis of therapeutics, 9th
  edition (1996) pp. 3-27.

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PG of CYP 2D6
Weinshilboum R. Inheritance and Drug Response.
NEJM2003348(6)529-537
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Lightcycler (Roche Diagnostics)
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Assay Principles
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Metabolism of Codeine
CYP2D6
CYP3A4
Caraco et. al., 1996. JPET, 2781165-1174
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Cytochromes P450

• January 2002. David A. Flockhart M.D., Ph.D.
• http//www.drug-interactions.com

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Amitriptyline metabolism
FMO
CYP 2D6

2C193A4
CYP 2D6
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Adverse Drug Reactions

• Acetaminophen toxic intermediary
• Codeine bioactivation combined with drug-drug
  interaction
• Morphine genetic variations possibly leading to
  sudden death
• Dextromethorphan genetic mutation may produce a
  protective drug effect

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Jan. 23, 2004
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Acetaminophen Toxicity

• Tylenol is a potent analgesic available OTC
• The most commonly used drug in overdoses
• The most common cause of acute liver failure in
  the United States
• Accounts for gt56,000 ER visits and 200 deaths per
  year.

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CYP1A2 CYP3A4
Acetaminophen Metabolic Pathway
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Alcohol and Acetaminophen Tylenol Combined
Toxicity

• CYP450 2E1 enzyme are strongly induced by ethanol
• Ethanol induces CYP2E1 accelerating bioactivation
  of acetaminophen to N-acetyl-p-benzoquinoneimine
  (NAPQI).
• NAPQI is responsible for the hepatic damage.

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CYP1A2 CYP3A4
Acetaminophen Metabolic Pathway
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Codeine Intoxication Associated with Ultrarapid
CYP2D6 MetabolismGasche Y, et al., NEJM,
2004,35127 2827-2831

• 62 y.o. male with chronic leukemia presents with
  fever and cough
• Diagnosis of bilateral pneumonia (Candida)
• Treated with clarithromycin, voriconiazole and a
  small dose of codeine (25 mg tid)
• Four days later, patient develops coma
• Coma reversed with naloxone

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• Conversion of codeine to codeine-6-glucuronide
  and norcodeine represents 80 codeine clearance
• Conversion of codeine to morphine represents 10
  codieine clearance
• Morphine and morphine-6-glucuronide have potent
  opioid activity
• Glucuronides excreted by kidney

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• Clarithromycin and voriconazole inhibit CYP3A4
• Ultrarapid metabolism of CYP2D6 increases
  demethylation to morphine
• Renal failure decreases morphine excretion

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Methadone-related deaths

• States Study years Increase
• Florida 1998-2001 1850
• Maine 1997-2002 450
• Maryland 1997-2001 1000
• N. Carolina 1997-2001 800
• First half of 2002
• Winecker, Clin For Tox News(AACC), June 2003

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Methadone Metabolism
3A4 2C9 2C19 2D6



2D6
UGT
Winecker, Clin For Tox News(AACC), June 2003
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Pharmacogenomics as Molecular Autopsy - Adjunct
for Forensic Pathology/Toxicology
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Methadone Case Load 2002-3

• British Columbia 134
• Cuyahoga 33
• Miami 33
• Milwaukee 55
• New Hampshire 68
• North Carolina 475
• Washington, D.C. 46
• Washington 400
• Wayne Detroit 71
• Total 1315
• Methadone related death only 2003 only

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CYP 2D6 Prevalence
Paired t-test NS N 57
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Prevalence ( n 60 )
CYP 2C19
CYP 2C9

Paired t-test NS
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Frequency Distribution
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Genotype Phenotype
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Methadone Metabolism
3A4 2C9 2C19 2D6



2D6
UGT
Winecker, Clin For Tox News(AACC), June 2003
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Phenotypes of All Genes in Methadone Users
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Dextromethorphan

• Antitussive in cough syrups, tablets and
  capsules,
• Recreational with alcohol as cough syrup
• Synthetic analogue of codeine
• Binds to opiate sigma receptors abuse
  potential?
• O-demethylated by polymorphic CYP 2D6
• T 1/2 3.2 3.6 h
• Dose 20 to 120 mg

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Coricidin HBP skittles abuse

• Teenagers in high schools. Skittles
• Typical ingestion 6 tablets increased to gt30!
• May be addictive?
• Dextromethorphan, 15 mg per tablet
• Acetaminophen, 0.5 g per tablet
• 6 tablets 3 gms acetaminophen
• 30 tablets, 15 gms acetaminophen

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Dextromethorphan toxicity

• Sedation, respiratory depression
• Acute OD toxic psychosis
• PCP like
• Possibly due to the O-demethylated metabolite
  dextorphan
• CYP 2D6 deficient - protective?

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All patients with same diagnosis
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Remove non-responders and toxic responders
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Treat Responders and Patients Not
Predisposed to Toxicity
Evans and McLeod
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A converging continuum?

• Drug therapy
• Treatment
• Addiction
• Pain management
• 78.3
• ( vs Control 85)

Toxicity Pain OD Addiction - OD
Forensic Pathology/Toxicology
PG?
PG?
Drugs Wt Antidepressants 73
Oxycodone 73 Methadone 71
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• Weinshilboum R. Inheritance and Drug Response.
  NEJM2003348(6)529-537

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All patients with same diagnosis
3 Drug related deaths
1
Remove non-responders and toxic responders
2
Treat Responders and Patients Not
Predisposed to Toxicity
Evans and McLeod, modified by Wong
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• The tangible benefits of forensic science have
  always been to advance antemortem medical care.

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