Interpreting Drug Levels Steven B. Karch, MD EAPCCT, ATHENS 2007

1. Interpreting Drug LevelsSteven B. Karch, MDEAPCCT, ATHENS 2007

2. Q: Okay Dr X, did you essentially determine the cause of death based solely on the toxicology screen? A: Cause of death is determined after review of medical records, gross and microscopic findings and the toxicology report. Q: Are the blood levels you measured of X and Y, is that the main reason you draw the conclusion that X overdose was the cause of death? A: That and the absence of any significant disease process that would explain something else occurring. Absence of Disease

3. The postmortem blood levels accurately reflect levels at time of death There can be no other explanations for high postmortem levels besides taking too much drug? Can an autopsy be called negative if histology was limited or not done at all? Or if genetic testing was not performed What this ME assumes

4. The greater the volume of distribution, the greater the concentrations increase after death Postmortem measurements are site dependent, values in LV>>RV Blood drawn from femoral vessels is NOT immune to redistribution Not study has EVER shown that postmortem measurements accurately reflect measurement made in life Redistribution

5. Redistribution • Morphine (and cocaine) have high Vss (2-6 for MS and 2-3 for cocaine) • Metabolites of both drugs have very low Vss. M3G and M6G on order of .5 • As consequence, most of the parent compound is in tissue and most metabolite in blood, which means that only a small amount of M need move back into blood to completely alter ratio of parent and metabolite

6. Moveable Feasts • Glucuronides, particularly of morphine DO NOT REMAIN STABLE after death • Free Morphine rises and concentration may double or triple in first 8 hours • Initially vitreous levels are HIGHER than blood levels. J Anal Toxicol. 2006 Nov-Dec;30(9):651-8.

7. Unable to metabolize drug Metabolize too much drug Invisible viruses Produce abnormal channels and receptors Don’t produce enough target DNA Diagnoses

8. CYP Activity • Activity of P450s is genetically determined and varies from patient to patient, and race to race • Depending on amount of activity, patients are classified as: • Ultra-rapid • Extensive • Intermediate • Poor

9. Normal full term male; mother given codeine 30 mg with acetaminphen 500 mg for episiotomy pain q 12H On Day12 poor color and not feeding Found dead On Day 13 with free MS of 70ng/ml Typically, infants of mothers taking codeine have MS levels of 0-2.2 and American Academy of Pediatrics says codeine users cans safely breast feed Morphine Poisoning

10. Mother had stored Day #10 milk, MS level was 87 ng/mg (typically 1.9 - 20 ng/ML) Infants have impaired ability to excrete M Testing showed mother heterozygous for CYP2D6*2A allele with CYP2D6* 2x2 duplication - an ultrametabolizer. So were other family members Not homicide Lancet 2006,368:704

11. Was This Malpractice? • 62-year-old male Hx chemotherapy, also taking valproate for seizure disorder • Admitted with bilateral yeast pneumonia; patient treated with ceftriaxone, clarithromycin, and voriconazole for a concurrent yeast infection, and codeine for cough. • Suddenly became comatose - blood tests showed toxic levels of codeine, morphine and morphine metabolites NEJM, 351:2827-2831, 2004

12. It Wasn’t… • Testing showed multiple duplications of two P450 enzymes CYP2D6 and CYP3D4 • Normally 2D6 converts 10% of codeine to morphine, in this case much more was converted • Worse, 2D4, which converts morphine to inactive metabolites wasn’t functioning

13. Was this homicide? • A 9-year-old with multiple developmental disorders admitted to hospital with seizures. He was taking methylphenidate, Clonidine, and Prozac • He died of uncontrollable seizures, and other children were removed from the household J Child Adolesc Psychopharmacol. 2000 Spring;10(1):27-34.

14. Absolutely Not • Samples of the decedent’s liver were tested for P450 abnormalities • Child had no functional CYP2D6 and could not form the main metabolite of Prozac, causing Proxac concentrations to reach dangerous levels • Investigation was dropped

15. Who Poisoned Her? • A 6-month pregnant woman diagnosed with severe rheumatoid arthritis; treated with methadone and antidepressants • Found dead New Years Eve by husband • No anatomic findings at complete forensic autopsy • “Heart blood” Methadone, 700 ng/mL, Amitiptyiline, 1500 ng/mL, Nortriptyline, 2200 ng/ml J Forensic Sci. 2003 Nov;48(6):1406-15.

16. She Did! • CYP2D6*4 homozygous = poor metabolizer • Therefore unable to hydroxylate methadone or amytriptyline or nortriptyline • She couldn’t clear the drug from her system and, essentially poisoned herself

17. Patchwork • Young woman, polydrug user, hurt knee and took one of room mates Duragesic patches • Found dead next AM with blood fentanyl of 19 ng/mL plus, norfentanyl 7.6 +DPHN (80 ng/ml, tramadol (.060 ng/mL), citalopram 220 ng/mL • Decreased CYP3A4*1B + CYP3A5*3, could not metabolize fentanyl J Anal Toxicol. 2005 Oct;29(7):590-8.

18. PCR Arrays Use of PCR arrays may provide many surprising answers but they are costly

20. Myocarditis • At autopsy, incidence is 0.1% to 5.5%, but may be much higher in selected series • True incidence not known because infection is not always clinically evident

21. Is this Myocarditis?

22. Yes, Actually • 24 consecutive patients admitted within 24 hours of ACS, including ST elevation and/or T-wave inversion, but negative angiography • Each had RV biopsy - half used for routine histological grading (Dallas Criteria), and half analyzed with nested polymerase for all viruses known to cause myocarditis

23. Histologic Dx of Myocarditis • 14 hearts with histologically proven myocarditis • Biopsied at autopsy with Stanford Bioptome • Only 11 could be diagnosed and that required an average of 17 samples per patient JACC 1989;14;915-920

24. Some Virused Can Only be Detected with PCR • In all 24 cases CPKMB, Troponin-T, and C-reactive protein were elevated at admission and increased while in hospital, even after optimal therapy • One patient with EB was positive by Dallas Criteria, none of the others were even borderline positive by histological criteria

25. Editorial

26. Channelopathies

28. Na-K exchange

29. Implications • What if a viral myocarditis goes undiagnosed (as would happen most of the time) and drug X is present “in therapeutic range.” Would you agree with the doctor in his deposition? • Or what if there were multiple duplications of an important P450 enzyme. Without doing DNA testing, a pathologist would not make the diagnosis, no “significant abnormalities” would be reported, and death would be blamed on a drug, no matter how much of the drug was present (post hoc ergo propter hoc)

30. Free Morphine • Regina v David Moore: Alleged Homicide, cancer patient on morphine • Very high free MS found in heart blood and the accused was seen giving injection 20 minutes before death • Crown argued that high ratio of free to total MS proved injection just before death - otherwise it would all be bound

31. Uridine diphosphate-glucuronosyltranferases • Morphine is converted to glucuronides (bound) by UGT2B7 • This enzyme is polymorphic (79 SNPs at last count) • There are many reasons for increased free MS postmortem, and polymorphism could be one - depending on how many copies of the abnormal gene are present, either too much or too little morphine could be conjugated

32. Autopsy in SCD

33. Hypertrophic Cardiomyopathy Long QT syndromes (LQT1-LQT8) Drug HERG (KCN2) interactions Brugada Syndrome (SCN5A) Catecholaminergic (RyR2) Genetic Causes of SCD

34. Major problem is acquired LQTS syndrome caused by drugs that block human hERG Result is delay cardiac repolarization and increase risk of torsades de pointes arrhythmia (TdP). Not all hERG channel blockers induce TdP because they can also modulate other channels that counteract the hERG channel-mediated effect (like aresnic) However, hERG channel blockade is an important indicator of potential pro-arrhythmic liability. Drug-HERG

35. Symptoms • One-third of symptoms are indistinguishable from those of acute heart attack • One-third simply die suddenly • Of those dying suddenly, as many as one-half have had no previous symptoms of any illness AND THEY VERY WELL MAY HAVE A NEGATIVE AUTOPSY

36. Warfarin • FDA about to change Warfarin labeling to recommend genetic screening • CYP2C9, 3-20% of USA is deficient, which means they cannot metabolize • VKORC1, 50% of USA is deficient (“Vitamin K epoxide reductase complex subunit”) warfarin’s target

38. Warfarin Metabolism

39. Methadone Compliance • Methadone is substrate for P-glycoprotein transporter encoted by ABCB1 • Individuals with two copies of wild-haplotype need higher doses than patients with only one copy • Conversely, carriers of the AGCTT haplotype need lower dose

40. Conclusions • Anatomically normal heart is not necessarily a normal heart • Mere detection of a drug does not imply causation, especially if autopsy is incomplete • High drug levels pre- or postmortem do not prove excessive ingestion.

41. Things to Think About • How can a pathologists testify with any certainty that a heart is normal if the heart has not been examined completely? • If there is even a 10% chance of being mistaken, then when is it possible to testify “beyond all reasonable doubt?”

42. Moral & Forensic Issues • If 10-15 % of SIDS are due to channelopathy, shouldn’t testing be included for family members if no one else