post-mortem snp analysis of cyp2d6 gene reveals correlation between genotype and opioid drug...
TRANSCRIPT
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 bioavailabilityAbsorption, distribution, clearance
CYP450 family involved in drug metabolism60 genes8 are most important for pharmacogenetics
Pharmacogenetics of Cytochrome P450
CYP2D6 known as debrisoquine/sparteine hydroxylase
Rogers et al. (2002). Am J of Med 113; 746-750..
A Note on Nomenclature
CYP2C9*1Cytochrome
P450
Family (40% sequence similarity)
Sub-family (55% sequence similarity)
Individual gene
Allele
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 liver3
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 N-demethyltramadol
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
Autopsies done at U Helinski Positive test for tramadol in blood 11 males, 22 females “Unexpected” deaths Ages 23 to 91 years
Genotyping
Large deletions or amplifications: Blood samples from autopsy records Extract DNA PCR for whole CYP2D6, deletion or duplication Confirm using allele-specific PCR
Genotyping
SNP typing by RFLP analysis Nested PCR entire gene Amplify specific fragments of gene RE digest to identify SNPs Verify SNPs with allele-specific PCR
Tramadol and its Metabolites
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 allelesGroup 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)
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
Some Qualifying Statements Other factors –age, liver or kidney malfunction,
metabolic drug interactions 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?
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…