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)

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 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