1. 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

2. 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

3. Pharmacogenetics of Cytochrome P450   CYP2D6 known as debrisoquine/sparteine hydroxylase Rogers et al. (2002). Am J of Med 113; 746-750..

4. A Note on Nomenclature CYP2C9*1 Cytochrome P450 Family (40% sequence similarity) Sub-family (55% sequence similarity) Individual gene Allele

5. 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.

6. CYP2D6 function  Catalyzes hydroxylation or demethylation in the liver 3 Cascorbi (2003). Eur J Clin Invest 33 (Suppl. 2); 17-22.

7. 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

8. Metabolism of Tramadol  Major pathway is metabolism to O-demethyltramadol by CYP2D6  Secondary pathway is inactivation to N- demethyltramadol  Clearance of metabolites via kidney

9. 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

10. Why Post-mortem?  Genotyping before prescribing could prevent toxicity  Poisoning as a cause of death  Contribution of genetic factors  Intentional vs. accidental overdose

11. Subjects  Autopsies done at U Helinski  Positive test for tramadol in blood  11 males, 22 females  “Unexpected” deaths  Ages 23 to 91 years

12. Genotyping  Large deletions or amplifications:  Blood samples from autopsy records  Extract DNA  PCR for whole CYP2D6, deletion or duplication  Confirm using allele-specific PCR

13. 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

14. 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

15. 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)

16. Some Ratios  Tramadol/O-demethyltramadol = MR1  Tramadol/N-demethyltramadol = MR2

17. MR1 vs. # Functional Alleles Decreased number of functional alleles correlated with more tramadol and less O-demethyltramadol

18. MR2 vs. # functional alleles Decreased number of functional alleles correlated with high levels of tramadol and even higher levels of N-demethyltramadol

19. Seems like a straightforward picture… However…

20. 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

21. 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

22. 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?

23. 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?

24. Thanks for your attention. Questions?

25. 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

26. 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…

27. 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