Transcript Jill Youds Presentation

Post-mortem SNP analysis of CYP2D6
gene reveals correlation between
genotype and opioid drug metabolite
ratios in blood
Levo et al.
Forensic Science International
2003
Pharmacogenetics of Cytochrome
P450
 Factors impacting drug response:
 Dietary intake, age, concurrent drug therapies
 Genetic factors impact drug bioavailability
 Absorption, distribution, clearance
 CYP450 family involved in drug metabolism
 60 genes
 8 are most important for pharmacogenetics
Pharmacogenetics of Cytochrome
P450
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Rogers et al. (2002). Am J of Med 113; 746-750..
 CYP2D6 known as debrisoquine/sparteine hydroxylase
A Note on Nomenclature
Sub-family
(55% sequence similarity)
Cytochrome
P450
CYP2C9*1
Family
(40% sequence similarity)
Allele
Individual
gene
CYP2D6
 Maps to 22q13.1 in gene cluster
 9 exons code for 461 amino acids
 73 known alleles
Cascorbi (2003). Eur J Clin Invest 33 (Suppl. 2); 17-22.
CYP2D6 function
 Catalyzes hydroxylation or demethylation
in the liver
3
Cascorbi (2003). Eur J Clin Invest 33 (Suppl. 2); 17-22.
Tramadol
 Synthetic analogue of codeine
 Analgesic used to treat moderate to severe pain
 Prescribed following surgery or for chronic
conditions
 Generic “Ultram” is tramadol and
acetominophen
 Binds the mu-opioid receptor
 Inhibits reuptake of 5-HT and NE in the CNS
Metabolism of Tramadol
 Major pathway is metabolism to
O-demethyltramadol by CYP2D6
 Secondary pathway is
inactivation to Ndemethyltramadol
 Clearance of metabolites via
kidney
Aims
 How do gene defects lead to adverse drug
effects
 How does variation in CYP2D6 affect
tramadol metabolite ratios in post-mortem
samples
 Relevent to interpretation of forensic
toxicology results
Why Post-mortem?
 Genotyping before prescribing could
prevent toxicity
 Poisoning as a cause of death

Contribution of genetic factors
 Intentional vs. accidental overdose
Subjects
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Autopsies done at U Helinski
Positive test for tramadol in blood
11 males, 22 females
“Unexpected” deaths
Ages 23 to 91 years
Genotyping
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Large deletions or amplifications:
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Blood samples from autopsy records
Extract DNA
PCR for whole CYP2D6, deletion or duplication
Confirm using allele-specific PCR
Genotyping
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SNP typing by RFLP analysis
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Nested PCR entire gene
Amplify specific fragments of gene
RE digest to identify SNPs
Verify SNPs with allele-specific PCR
Tramadol and its Metabolites
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Frozen venous blood samples from autopsy
Tramadol  routine screening via alkaline
extraction and gas-chromatography
O- and N-demethyltramadol  ethanol
extraction, liquid chromatography and mass
spectrometry
Results
 Classed individuals into groups based on
number of functional CYP2D6 alleles
Group 0 = no functional alleles (n=4)
Group 1 = 1 functional allele (n=9)
Group 2 = 2 functional alleles (n=16)
Group 3 = 3 or more functional alleles (n=4)
Some Ratios
 Tramadol/O-demethyltramadol = MR1
 Tramadol/N-demethyltramadol = MR2
MR1 vs. # Functional Alleles
Decreased number of
functional alleles
correlated with more
tramadol and less
O-demethyltramadol
MR2 vs. # functional alleles
Decreased number of
functional alleles
correlated with high
levels of tramadol and
even higher levels of
N-demethyltramadol
Seems like a
straightforward
picture…
However…
Some Qualifying Statements
 Other factors –age, liver or kidney malfunction,
metabolic drug interactions
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Average patient age 70
Some died of disease
Many cases 2 to 10 other drugs were found
One case of CYP2D6 metabolic inhibition
 Post-mortem pharmacokinetic determinations are
one-time samples
 10 cases of high tramadol explained by advanced
age and multiple disease
Conclusion
 Number of functional alleles of CYP2D6
correlated with ratio of parent drug to metabolite
 Dominant role of genetic factors in metabolism
of tramadol visible after death
 In poor metabolizers, N-demethylation pathway
may prevent parent drug accummulation
 In future, genotyping to determine genetic or
intentional causes of overdose
Criticisms
 So many variables, so few controls…
 Age, additional drugs, cause of death, healthy or
diseased, time since taking the drug, time of
death to time of autopsy
 They show a correlation and imply a causation
 Quality of post-mortem samples?
Questions for Discussion
 Is this a practical method for genotyping?
What methods would be better?
 What higher standards for genetic analysis
might be necessary when post-mortem
material is used?
 What are the ethical issues related to
genotyping deceased individuals?
Thanks for your attention.
Questions?
On Pain, Opioids, 5-HT and NE
 5-HT and NE have effects that elevate mood and
moderate pain
 Tramadol inhibits reuptake of 5-HT, NE from
synapse, causing increased activity
 Opioids activate pathways that increase spinal
levels of NE and 5-HT
 Opioid drugs mimic endogenous opioids like
endorphins, enkaphalins and dynorphins
Drugs that inhibit CYP2D6
activity
 Compete with CYP2D6 in binding drug
 Quinidine – essentially causes poor
metabolizer status
 Others include: tricyclic antidepressants,
SSRI’s, methadone, fluoxetine…
Factors Influencing CYP Activity
 Age – decreased CYP expression
 Diet – some nutrients compete for
absorption, grapefruit juice inhibits the
activity of transporters and intestinal
CYP3A subfamily
 Alcohol – transiently increases CYP, long
term decrease due to liver disease