Forensic Toxicology - MLSCI 466 Combined Notes PDF

Graham R. Jones, Ph.D. Former Chief Toxicologist Office of the Chief Medical Examiner & Clinical Professor, Faculty of Medicine and Dentistry [email protected] Forensic - for legal purpose; pertaining to the law. Deals with the living and the dead:  Postmortem cases - cause & manner of death ...

Graham R. Jones, Ph.D. Former Chief Toxicologist Office of the Chief Medical Examiner & Clinical Professor, Faculty of Medicine and Dentistry [email protected] Forensic - for legal purpose; pertaining to the law. Deals with the living and the dead:  Postmortem cases - cause & manner of death  Impaired driving; sexual assault  Workplace drug testing  Sports (e.g. Olympics; horse-racing)  Criminal cases: ◦ Impaired driving  Not just motor vehicles ◦ Homicides  Victim, perpetrator ◦ Drug facilitated assault  Assailant, victim  Civil issues/cases: ◦ Non-criminal accidents (e.g. falls, drowning) ◦ Workplace drug testing (Usually urine)  Condition of Employment / Site access  Witness in court ◦ Usually only testifies to facts, observations ◦ Medical technologists usually in this category ◦ What they did (or would normally have done)  Expert witness in court ◦ Must be “qualified” by the judge to give “opinion” evidence (based on education, training, experience) ◦ Doesn’t have to have done any lab work on the case  Clinical: ◦ emergency screening (e.g. overdose) ◦ therapeutic drug monitoring (TDM) (limited menu of drugs, defined concentration range) Testing only done if it is likely to influence treatment of the patient.       (Immunoassay) (HPLC/UV) GC (mainly alcohols) GC/MS(/MS) LC/MS(/MS), LC/(Q)TOF (AA; ICP-MS) (rare!) Similar instrumentation to clinical labs, but: 1. Forensic labs do NOT rely only on immunoassay. 2. Must use better extraction and chromatography methods than clinical labs because using whole blood – sometimes decomp!  Testing not usually ‘limited’ ◦ Drugs (prescription, non-prescription and ilicit) and other poisons (e.g. pesticides, other chemicals) ◦ May be “high” concentration – e.g. overdose ◦ May be very low concentration - e.g. sexual assault ◦ May use many methods for a single “drug screen”  e.g. immunoassay, GC-MS, LC-(Q)TOF, LC-MS/MS  Most forensic testing performed on whole blood or urine ◦ Most clinical testing performed on serum, plasma or urine  Whole blood specimens more difficult and give more interference than fresh serum or plasma  Matrix: is the fluid or tissue in which the analyte is contained: ◦ ◦ ◦ ◦    Proteins (blood or may be solid tissue) Lipids, phospholipids Electrolytes Water Analysis can be difficult or impossible without extraction of the analyte out of the matrix But it depends on the instrumentation Most forensic toxicology is performed on whole blood and/or tissue (e.g. criminal, postmortem)   Most samples have to be processed or extracted in some way prior to analysis There are two main purposes: ◦ Minimize or eliminate matrix interference (often involves extracting out the analyte) ◦ Concentrate the analyte to improve sensitivity of the assay  For immunoassay, the degree of sample processing depends on the assay design (e.g. homogenous enzyme immunoassay versus ELISA). Most clinical immunoassays are designed for urine or serum/plasma.  ELISA plates (96 wells per plate) are best suited for forensic work as they are optimised to work with diluted whole blood. Tissues must be homogenized and diluted.  Tissue can rarely be analyzed directly – need to make fluid sample  Use homogenizer ◦ e.g. Polytron homogenizer ◦ 1+3 tissue:water The type of column inlet is critical!!  GC ◦ Column inlet is heated to vaporize the solvent and the analyte; many drugs will not ‘chromatograph’ ◦ Will cause pyrolysis of some analytes and the matrix, causing interference ◦ More sample prep. needed than for LC-based methods  LC ◦ Inlet is cool (on-column), causing little or no destruction of the analyte or matrix ◦ Sample is introduced to MS spray chamber as liquid where it is then vaporized and ionized ◦ Less sample preparation may be needed  1. Extract the analyte from the sample and leave the matrix behind ◦ For example, liquid-liquid or SPE extractions ◦ Most common approach for GC-based assays  2. Remove the interferences from the sample and leave the analytes behind ◦ For example, protein ‘crash’ extracts ◦ Increasingly common approach for LC-MS (and LC-TOF) ◦ MUST CONSIDER THE ANALYTICAL INSTRUMENTATION  Dilution ◦ To “dilute out” proteins in plasma or blood sample to allow direct analysis  e.g. Immunoassay   Headspace analysis  e.g. Alcohols by GC Protein ‘crash’ (e.g. acetonitrile, methanol)  Some highly selective LC-MS(MS) and LC-(Q)TOF assays    Used for “volatiles” (alcohols and solvents) Only volatiles from air above the sample get injected on to the GC Very clean with little interference Headspace GC Diagram Step 1 Sample reaches equilibrium Step 2 Sample is extracted from headspace Step 3 Sample is injected into GC Can use simpler methods such as a “protein crash” if the analytical methods are specific enough!  Add cold acetonitrile and/or methanol to the blood ◦ Can also use for serum or plasma    Proteins and peptides in whole blood are denatured; centrifuge to form a pellet Clear supernatant injected (sometimes after a concentration step) (aka “dilute and shoot”) Increasingly used for LC-MS/MS and LC-(Q)TOF analysis  Caution: analytes can be trapped in the precipitation matrix  Need good, validated methods with deuterated internal standards  Does NOT remove salts, lipids or phospholipids 4) Remove supernatant 5) Analyze supernatant, often after dry down and reconstitution     Principle: analyte more soluble in solvent than aqueous specimen Add solvent to liquid specimen Mix to temporarily “blend” phases Centrifuge to separate     Un-ionized analyte is more lipid-soluble Ionized analyte (e.g. salt form such as hydrochloride or sulfate) is more water soluble Need to get analyte into UN-ionized form in order to extract into organic solvent Use buffers to do this (i.e. adjust pH) Cantharadin Ibuprofen Methamphetamine Specimen + pH 12 Buffer + Solvent Mix & Centrifuge Organic to GC/LC Aqueous (waste) To extract acidic drugs add an acidic buffer instead of pH 12 buffer Specimen (+ pH 12 buffer) (+ solvent) Mix & Centrifuge Organic Aqueous (waste) + Acid pH 2 Mix & Centrifuge Organic (waste) This extraction scheme extracts basic drugs, but leaves neutrals (e.g. fats) and acidic drugs behind Aqueous + Base pH 12 + Solvent Mix & Centrifuge Organic (to GC/LC) Aqueous (waste)  Amphoteric Functions ◦ Phenolic ◦ Amino  e.g. morphine  Zwitterion Functions ◦ Carboxylic ◦ Amino  e.g. GABA OH OH OH H CH3 N CH3 pH 2 N CH3 N O O O OH OH O pH 8 (e.g. morphine) pH 12  Depends on: ◦ ◦ ◦ ◦ ◦ ◦ ◦ Specimen type (e.g. urine vs. whole blood) Instrumentation used/available (e.g. GC/MS vs. LC/MS) Robustness and precision of the assay Purpose of the test (quantitative vs. quick screen) Processing time (urgency and speed of test) Type of analyte/drug being tested Cost of reagents (SPE uses less solvent than liquid/liquid, but SPE columns can be expensive)             GC – gas chromatography LC – liquid chromatography (aka HPLC) MS – mass spectrometry MS/MS – tandem MS LC-MS(MS) refers to single MS or MS/MS TOF – time of flight (accurate mass MS) QTOF – combination “MS” and TOF (highly selective) FID – flame ionization detection NP(D) – nitrogen phosphorus (detection) UV/VIS – ultraviolet/visible detector ELISA – enzyme linked sorbent immunoassay (96-well plates) SPE – solid phase extraction          B.Sc. Pharmacy 1971 Registered as U.K. Pharmacist 1972 Ph.D. Drug Metabolism and Drug Analysis 1976 Post-doctoral Fellowship U. of Alberta Pharmacy 1976-78 Head, Commonwealth Games Drug Testing Lab 1978 Clinical Toxicologist, Univ. Hospital Edmonton 1978-81 Toxicology Lab Director /Chief Toxicologist, Alberta Medical Examiner’s Office 1981 – 2018 Chair, Universiade Games Drug Testing Program1983 Now semi-retired but very active professionally plus private consulting 33 Postmortem Toxicology: An Overview Graham R. Jones, Ph.D. Former Chief Toxicologist Office of the Chief Medical Examiner & Clinical Professor, Faculty of Medicine and Dentistry [email protected] Death Investigation • Medical Examiner system • Alberta • Manitoba • Nova Scotia • Newfoundland • Coroner system • • • • • • British Columbia Saskatchewan Ontario Quebec New Brunswick, PEI NWT, Nunavut, Yukon Medical Examiner system headed by a forensic pathologist Coroner system may or may not be headed by a physician Cause and Manner of Death • Cause of Death • Immediate MEDICAL cause of death • NOT circumstances • e.g. “blunt force trauma” rather than “motor vehicle accident” • Manner of Death • Specific categories • • • • • • Homicide Suicide Accident Natural Undetermined Unclassified (MAID deaths) Time of death? Not usually possible to determine with any accuracy. Examples… • Young man found dead in front of car in closed garage, with hood open and tools laid out. Death caused by carbon monoxide poisoning. • What is the manner of death? • Young woman with long history of depression crosses into the oncoming lane and collides with a semi-trailer headon. • What is the manner of death? Alberta Office of the Chief Medical Examiner • Offices • Edmonton and Calgary • Cases ~6500 / year • Total Alberta deaths ~32000 • Homicides ~80–100 /year • Toxicology • ~50 alcohol or CO only • ~5000 “full” toxicology Alberta – OCME Investigated Deaths ~6500 Medical Examiner Cases annually: Homicidal Suicidal Accidental Undetermined Natural ~2% (80 – 100) 12% 49% ~2% 34% Cases have increased substantially over the past 6 – 10 years!!! Other Forensic Laboratories • RCMP Labs in Vancouver, Edmonton, Ottawa • DNA, trace evidence (e.g. hair and fibre, accelerants), documents, firearms • RCMP Handle ONLY criminal cases • Ontario and Quebec have their own labs (and provincial police services) • Toxicology in other provinces handled by various other arrangements • Local hospital; provincial laboratory, U.S. private laboratory Why is Toxicology Important vs. Autopsy? Myth: • All drug overdoses will have evidence of “pill fragments” in the stomach. Fact: • Most fatalities due to drugs or poisons will not show specific signs at autopsy. An autopsy can rule out natural disease or trauma as the cause of death. Sometimes natural disease may be contributory. Postmortem: Cannot use serum or plasma • Whole blood • Vitreous • Urine • Liver • Stomach contents • Other tissues (sometimes: e.g. decomposition) • Hair (sometimes: e.g. criminal case) • Other “exhibits” (syringes, drinking glass) Blood • Most consistently available, abundant “fluid” • Postmortem blood usually partially to fully hemolyzed • Often not “homogeneous” throughout body • Not usually useful for biochemical tests • Drug concentrations can go up or down after death (depends on drug) • Drug concentrations should not be interpreted “in isolation” (more later) Vitreous Humor • Clear fluid from inside eye • Limited volume (~3 mL per eye); usually available • Very useful for alcohol • Can be useful for some drugs (e.g. cocaine, heroin, digoxin) • Cannot interpret as you would blood • Drug concentrations often lower than in blood – especially highly lipid soluble and protein-bound drugs (e.g. benzodiazepines, tricyclic antidepressants) Urine • Clear fluid, no protein and very little lipid (exception – decomposed bodies) • Urine not always available in postmortem cases • Concentrations of some drugs very high • Some drugs not excreted into urine due to extensive metabolism, but metabolites can be detected • Little or no relationship between urine drug concentration and pharmacological effect (exceptions alcohol and maybe some metals) • Cannot relate urine drug concentration and degree of impairment Liver • Always present, large amount of specimen • Need to make liquid preparation (homogenize) • High protein and lipid content can interfere • Drug concentrations don’t change much after death • Some exceptions: drugs may diffuse form stomach into liver if concentrations high; some drugs are unstable • Liver drugs concentrations are often much higher than in blood • Often useful to have BOTH liver and blood concentrations • Liver widely used in the past – but less now! Stomach Contents • Very variable from clear fluid to a “meal” • Drug concentrations can be very high • Need to relate drug concentration to total volume • Interpretation: total AMOUNT, not concentration • Drug (and/or metabolite) can be in stomach after parenteral use (e.g. i/m, i/v) due to secretion in gastric juice • Presence in stomach does not prove oral use Stomach - Example • Therapeutic range for phenytoin in blood is 10 – 20 mg/l • Gastric concentration is 150 mg/l – toxic? NO! • 150 mg/l may be 15 mg/100 ml, if volume of gastric is 100 ml • Could mean partially absorbed 100 mg dose… Other Specimens… • Bile, CSF or virtually any fluid • Lungs, kidney, muscle or any solid tissue • Bone, nails, hair • Injection sites • Need “control” site away from suspected injection site • Can be difficult to prove injection site if elapsed time – especially if i/v injection • Principle: most drugs are distributed to virtually all fluids and tissue in the body!!! Other “exhibits”… • Syringes • Can indicate what was injected (useful if heroin) • Drinking glasses • Can be useful in drug facilitated sexual assault cases • Can indicate manner of death in suicide • Bottles • Can indicate source of a poison Chain of Custody • A means of tracking exhibits (e.g. specimens) from the point and time of collection to analysis and ultimately disposal. • Involves place/location, date, time and persons (e.g. analysts). • Purpose: to demonstrate that analysis and reporting was performed on the stated exhibit/specimen. • Usually involves a reasonable degree of physical security, such as locked storage (e.g. fridge/freezer, locker), secured facility (e.g. laboratory) and restricted access (authorised personnel; escorted visitors). • Physical or electronic log of those entering and leaving the facility. • Authorised staff may have an electronic key-card. Preventing Deaths - Carbon Monoxide Some are obvious… Carbon monoxide binds to hemoglobin 200x stronger than oxygen! Carbon Monoxide Deaths Some sources are less obvious… Why postmortem toxicology is “different”… Severe motor vehicle accident: Ethanol not detected, but… Urine METHANOL 530 mg% Liver 190, 300 mg% Spleen 20, 70 mg% Explanation? Postmortem Fermentation Blood Vitreous Blood ON ITS OWN is UNRELIABLE as a specimen for assessing the presence of alcohol at the time of death. Can get postmortem BAC up to “legal limit” due to fermentation; in RARE circumstances >300 mg%. Postmortem Redistribution Main mechanism • Release and diffusion of drugs from the major organs • Occurs as cells die, pH changes and protein binding weakens • Time and concentration dependent • Candidates: • Drugs with a high volume of distribution typically >5L/kg • Drugs with ‘Basic’ character (e.g form HCl salts) • Increases of 2 – 10 fold or greater possible • Order of increasing magnitude of PM redistribution: • Cardiac > subclavian > femoral > antemortem Example of the Problem of Postmortem Redistribution  48 y.o. woman with severe heart disease and history of depression found unresponsive at home  Taken to hospital but could not be resuscitated (i.e. dead)  Blood taken at hospital by local medical examiner  However, local ME could not decide whether COD was natural or drug overdose – autopsy ordered  Blood also taken again at autopsy (12 – 18 h later) Example of the Problem of Postmortem Redistribution - Results Antemortem blood (DOA) Femoral blood (at hospital) Cardiac blood (at autopsy) Liver Stomach Amitriptyline 0.10 0.20 2.20 18 trace Nortriptyline 0.34 0.37 5.10 29 trace Concentrations of amitriptyline and nortriptyline ~10x higher in blood taken at autopsy than blood collected at the hospital COD is “natural” – elevated autopsy blood drug concentrations due to postmortem redistribution! Delayed Deaths  Depressants (narcotics, sedatives) with or without alcohol can cause hypoxic brain damage ◦ Resuscitation and hospitalization for hours or days can lead to low or near absent levels ◦ Even without resuscitation, drugs can lead to prolonged coma prior to death, and low drug levels ◦ Near-fatal ethanol can clear in <24h  Delayed deaths also a factor with acetaminophen, ethylene glycol and methanol  Antemortem specimens important!! Impaired Metabolism • Can be due to: • Genetic impairment due to enzyme deficiency • e.g. cytochrome P4502D6 in 7-10% Caucasians • Drug-Drug impairment of enzyme system • e.g. impairment by SSRIs of CYP4502D6 • Impairment due to high single drug concentration • Impairment due to reduced liver function (age, alcohol) Accumulation of ADHD Medication in a Young Boy      7 year old boy with severe attention deficit disorder Medications: imipramine (150 mg hs); valproate (250 mg hs). Collapsed suddenly at school; resuscitation unsuccessful. No prior history of serious side effects Autopsy negative; toxicology indicates “imipramine poisoning”??? Imipramine Desipramine Cardiac blood 1.1 13 Femoral blood 0.47 6 Liver 35 449 Antemortem Se 0.069 0.942 (15 min after collapse) mg/l or mg/kg Juvenile Imipramine Death 10- Hydroxyimipramine 2- Hydroxyimipramine HO (OH) N Hydroxydesipramines CPY3A4 (and conjugates) CYP2D6 N N N Imipramine CYP3A4 N Hydroxydesipramines Desipramine CPY2D6 (and conjugates) NH Fatal Drug Accumulation in a Young Manic Depressive • 21 y.o. university student living with parents • History of manic depression, but well controlled • Watching TV with father, but feeling unwell and went to bed early; 2 h later complained of nausea • 4 h later was heard to collapse and taken to hospital; died en route; resuscitation failed • All medication accounted for (no indication of “overdose”) • Prescribed: 900 mg chlorpromazine, 125 mg imipramine, 10 mg amphetamine • No cause of death at autopsy; toxicology… Fatal Drug Accumulation – Toxicology Results Femoral blood Liver Stomach Chlorpromazine 0.92 111 trace Imipramine 0.60 37 trace Desipramine 3.74 261 trace Results: mg/l or mg/kg 1. Liver levels VERY high (especially metabolite desipramine 2. Very high desipramine (metabolite) to imipramine suggests accumulation 3. Likely genetic (P4502D6) and/or drug-drug induced accumulation 4. All medications accounted for (none missing) Tolerance / Opiate Deaths • Interpretation of postmortem narcotic blood levels VERY VERY tricky! • Need to have some idea of degree of tolerance • If naïve user, interpretation easier • If prescribed, need medical record and/or pharmacy record • Need to know dose and duration of dosage • Preferably need “medication count” Methadone – Two Cases • Case 1: • 26 y.o. with dubious history of opiate addiction; new in methadone program • Dies over w/end after taking 4 x 40 mg methadone in ~48h • PM Blood methadone 0.34 mg/l (liver 4.6 mg/kg) • Witness evidence of sedation, loud snoring etc. • Found dead; no COD Case 1: Little or no tolerance • Case 2: • Young male; well established in methadone program • Daily dose 130 mg • Routine blood methadone 0.83 mg/l • Little or no sedation • Alive!! Case 2: Substantial tolerance Sudden opioid death? No! • 46 year old known drug dealer was rushed to hospital by an acquaintance; he died. WHY? HOW? • Postmortem blood • • • • • • • • • carfentanil = 0.73 ug/l fentanyl = 93 ug/l xylazine = 8.4 ug/l furanylfentanyl = detected acrylfentanyl = detected (urine only) methadone = 0.17 mg/l morphine / 6-MAM = detected (heroin) methamphetamine = 0.20 mg/l Cocaine / BZE = detected • Obviously very high opioid tolerance!! COD was gunshot wound!!! ‘Medical’ Tolerance of Fentanyl • Starting dose 25 ug/hour for chronic pain • Potentially fatal for non-tolerant patients – 100 ug/hour • This patient had 34 x 100 ug/h fentanyl patches • No clinically significant respiratory depression! • Admission blood fentanyl 173 ng/mL • (therapeutic ~0.5 – 5 ng/mL) European Journal of Pain (2001) 5: 325±331 Inefficacy of high-dose transdermal fentanyl in a patient with neuropathic pain Bleeker, Bremer, Dongelmans, van Dongen and Crul Interpreting Postmortem Toxicology is VERY Different from Clinical Toxicology! Difficulties of interpreting postmortem data Don’t know when drug last taken; rate of metabolism; rate of excretion, AND Don’t know if postmortem blood level of drug represents blood level at time of death. Some concentrations increase postmortem; some decrease. Therefore cannot reliably calculate dose. Don’t know tolerance of person to toxic effects of the drug. MUST consider autopsy findings, medical history, circumstances of death. How should you interpret toxicology results? • Interpret with FULL consideration of: • Circumstances of death • Post-death investigation including: • • • • Medical history Medication history Autopsy findings Toxicology on alternate specimens!! • If you don’t have enough information, either DON’T offer an interpretation, or give clear caveats! “Medication Counts” • For each important medication determine: • • • • • Number prescribed Date dispensed Dosage (e.g. tablets per day) Number remaining at death Calculate meds “unaccounted for” • Why? Suicide vs. “Build-up”… 1 Cannabis - Forensic Toxicology Graham R. Jones, Ph.D. Former Chief Toxicologist, OCME & Clinical Professor, U of A Faculty of Medicine and Dentistry Edmonton, Alberta, Canada What do most forensic labs measure? • Delta-9-tetrahydrocannabinol (∆9-THC) • Highly lipid soluble • Usually measured in whole blood • Majority of the research was conducted in serum or plasma • THC blood:plasma ratio ~0.5 - 0.6 • 11-Hydroxytetrahydrocannabinol (11-OH-THC) • Pharmacologically “active” • Concentrations are higher after oral ingestion (first pass effect) • 11-Carboxytetrahydrocannabinol (THC-COOH) • Inactive metabolite • Forms water soluble glucuronide metabolite • Majority present as the glucuronide • Most labs measure the unconjugated THC-COOH (in blood) • Most urine testing labs (workplace) measure the total THC-COOH • Most forensic labs do NOT measure cannabidiol (CBD) ‘Emerging’ in the last couple of years* • Delta-8-tetrahydrocannabinol (∆8-THC) • Low concentrations natural present in Cannabis • Slightly less potent than ∆9-THC abd similar side-effects than ∆9-THC • Challenging to separate from ∆9-THC analytically • Delta-10-tetrahydrocannabinol (10-THC) • Positional isomer of ∆9-THC not usually present in Cannabis • Less potent than ∆9-THC(?) • Tetrahydrocannabiphorol (THC-P) • Much more potent (~30x) than ∆9-THC; not present in Cannabis • O-Acetyl-delta-9-tetrahydrocannbinol (THC-O) • Up to 3 x more potent than ∆9-THC; not present in Cannabis • May have hallucinogenic effects at high doses Other “Cannabis” Cannabinoids • Over 100 cannabinoids naturally present in cannabis • Cannabidiol (CBD) • Can be up to 40% of cannabis plant extract • Has been studied to treat anxiety, cognition, movement disorders, pain and epilepsy disorders • Little if any psychoactivity • May modify the effects of THC if both are present • Legal in the USA (cannabis extracts are not) • Cannabinol (CBN) • Mildly psychoactive, but present in only small amounts in cannabis • Is a metabolite of delta-9-THC Effects of Δ-9-Tetrahydrocannabinol • Psychoactive effects include • a state of relaxation, perhaps euphoria • facility for philosophical thinking, introspection • anxiety and paranoia • increase in heart rate and hunger, reddening of eyes • reduces nausea and vomiting • impaired motor skills; • Impaired judgment of time and distance Note: unless otherwise stated, any reference to “THC” in this lecture refers to ∆9-THC Interpretation...not as ‘easy’ as with alcohol • For alcohol (ethanol) there is a generally ‘direct’ correlation between blood concentration and “effect” (e.g. impairment) • Ethanol: can estimate “dose” from a blood level and can estimate a blood level from a dose • For THC, the correlation Data from: https://www.infrastructure.gov.au/roads/safety/publications/1997/pdf/Speed_Risk_1.pdf between blood level and “effect” is very poor except in the very early stages of smoking Pharmacokinetics of THC & metabolites • The “peak” ∆9-THC represents cessation of smoking • Note, the “research” cigarette only 3.55% THC • In most people, duration of the ‘high’ lasts 2 – 6 hours, i.e. much longer than the blood THC concentration is elevated Huestis MA, Henningfield JE, Cone EJ. 1992 Delta-9-THC • Short distribution half- life • Long elimination halflife resulting in “baseline” blood THC concentrations up to 7 ng/mL after heavy chronic use CH Ashton, Br J Psychiatry, Volume 178: 101-6, 2001 EW Schwilke, EL Larschner, RH Lowe, WD Darwin, MA Huestis, SOFT Abstract 23, NC Oct 2007 8 Cannabis – Forensic / Postmortem Aspects Canadian Criminal Code - Cannabis • Part I of Bill C-46, came into force on June 21, 2018: • an offence for (low-level) THC concentrations of 2 ng/mL to • • • • less than 5 ng/mL, within 2 hours of driving. an offence for higher-level THC concentrations of 5 ng/mL or more, within 2 hours of driving. an offence that recognizes the effects of combined marijuana and alcohol consumption; 50 mg of alcohol per 100 mL blood plus 2.5 ng/mL or more of THC within 2 hours of driving. “per se” limits that have little correlation with “effect or degree of actual impairment”. Recognizes that THC has a short half-life in blood and that it could take police up to 2 hours to get a blood sample drawn. 11 But with THC it can get complicated in civil cases • THC has a relatively short half-life in blood • But a longer half-life in the brain • i.e. where THC has its primary effect related to impairment • And it has a MUCH longer half-life in the body • Lawyers will ask: • What does the blood THC level mean? • Is there a blood alcohol equivalent? • Does a blood concentration of ‘X’ ng/mL mean they were impaired? 12 Delta-9-THC: The Past ‘Wisdom’* • The following used to be held as accepted facts: • THC always drops to near zero a few hours after last smoke • “Blood” THC >2-3 ng/mL consistent with recent use (within 6 h) • You can use THC:THC-COOH ratio to estimate the time of last use • Little postmortem change • …NOT ANY MORE Huestis, M.A and Smith, M.L 2007 13 THC: What we now know … • Baseline blood THC several hours after smoking: • typically < 2 ng/mL in light smokers (<1 after 24 h) • can be > 5 ng/mL in heavy smokers (may be >10) • in one study 1.2 – 5.5 ng/mL 7 days after last use • very high body burden of THC slowly released into blood • Refers to LIVING people! 14 THC Postmortem – It’s More Complex • THC undergoes postmortem redistribution (i.e. can increase after death) • PM femoral blood THC can be MUCH higher in postmortem blood than pre-mortem (‘clinical’) blood • THC concentrates in muscle tissue and may be redistributing postmortem • But much larger effect may be the presence of fat in the postmortem blood drawn 15 Case 1: Aircraft Pilot with THC* • Young commercial pilot flew a small passenger plane into a hillside: “controlled flight into terrain” • Pilot killed on impact or soon after; 2 passengers survived • Pilot toxicology positive for cannabis: • Pleural blood: THC 11.9 ng/mL; carboxy 41.8 ng/mL • Femoral blood: THC 50.1 ng/mL; carboxy 21.6 ng/mL • Pilot could not have been smoking for at least 1 hour before the crash and probably closer to 2 hours • Did the blood THC reflect concentration in flight? • Was the pilot impaired by THC? 16 Case 2: ATV Death with THC* • Middle-aged man involved in an ATV roll-over • Taken to hospital with relatively minor injuries • Includes broken clavicle • Dies from lacerated subclavian vessel and massive bleed • Postmortem toxicology negative except for cannabis: • Antemortem blood THC 14 ng/mL; carboxy 110 ng/mL • Time: 2 hours post-accident • PM femoral blood THC 31 ng/mL; carboxy 19 ng/mL • Time: 68 hours post-accident, after 60 – 66 hours in ICU 17 Case 3: Stabbing victim with THC* • Young man stabbed to death; short survival time • PM Toxicology (blood): • Ethanol, cocaine, levamisole, phenacetin, cannabinoids • Central blood THC 3.5 ng/mL; carboxy 11.8 ng/mL • Femoral blood THC 37.1 ng/mL; carboxy 8.4 ng/mL • Huh? • Elevated femoral blood THC likely due to contamination with “fat” containing sequestered THC from current and past smoking Postmortem blood is not always a reliable sample Remember – THC is very fat soluble!! LEGALIZATION OF CANNABIS Interpretation can be quite complex and may affect both criminal and civil case litigation! SYNTHETIC CANNABINOIDS (AKA CANNABIMIMETICS; AKA “SPICE”, “K2” ETC) 21 “Spice” – Synthetic Cannabinoids • Aka Cannabimimetics • Chemicals impregnated into various dried plant materials • Synthetic chemicals that may or may not be closely related to the structure of delta-9-THC – Interacts with the cannabinoid receptors – Partial agonist activity at the cannabinoid receptor CB1 located mainly in the central nervous system – and the CB2 receptor, mainly in cells of the immune system • Four primary series – JWH (John W. Huffman, funded by NIDA for medical research) – AM (Alexandros Makriyannis, Northeastern University) – CP (research chemists at Pfizer) – HU (Raphael Mechoulam, the Hebrew University) • Many do not have true “THC” effects 22 Cannabimimetic Properties • Similar to Cannabis (hence “…mimetic”), but some may have more intense effect in ability to produce psychosis. • Psychoactive effects include • a state of relaxation, perhaps euphoria • facility for philosophical thinking, introspection • anxiety and paranoia; also • increase in heart rate and hunger, reddening of eyes • reduces nausea and vomiting • impaired motor skills; judging time and distance 23 Adverse effects of synthetic cannabinoids • Psychiatric • Psychosis (new-onset or exacerbation of pre-existing disease), agitation, anxiety, irritability, confusion, aggression, suicidality, memory changes, tolerance, withdrawal, dependence • Cardiovascular • Hypertension, tachycardia, ST-segment changes, chest pain, myocardial infarction, tachyarrhythmia • Neurologic • Generalized seizures, somnolence, brisk reflexes • Gastrointestinal • Nausea, vomiting, anorexia, increased appetite • Other • Hypokalemia, conjunctival injection (‘blood shot’ eyes), hyperglycemia, acute kidney injury, xerostomia (dry mouth), diaphoresis • Ref: Five things to know about synthetic cannabinoids.CMAJ, February 18, 2014, 186(3) 24 * 25 * 26 Cannabimimetic Analysis • Can be difficult! • Very low blood concentrations • Extensively metabolized • Over 200 possible structures • They keep changing as regulations change • Screening methods do not detect all • Need high-end methods for confirmation • e.g. LC/MS/MS • Relatively difficult and time-consuming to develop 27 Metabolism of JWH-18 – Clearance from blood* 28 Prevalence of different cannabimimetics over 3.5 years * 29 Risk Factors for Users • Downside: don’t know what you are getting • May vary from batch to batch even with the same name and label – substance and dose • Toxicity will vary tremendously depending on dose and the individual • For most ‘designer’ drugs little is known about toxicity and longterm effects 30 Canada vs. USA – The Differences • Canada: • Federal drug laws (Controlled Drugs and Substances Act – CDSA) • Specific named substances, but chemically related drugs that may have a similar pharmacological effect are similarly controlled (specific substances do not always have to be named) • USA: • DEA (Drug Enforcement Administration) has “federal” rules, but slow to add new substances • Did have Federal Analogue Act 1986 but sometimes difficult to get a prosecution • e.g. what is an analogue (how similar is it to a scheduled drug) • Stop the Importation and Trafficking of Synthetic Analogues Act of 2017 Individual states can make individual drugs illegal, but that was a slow “ad hoc” process that did not usually address “analogues” Fentanyl and related opioids Graham R. Jones, Ph.D. Former Chief Toxicologist Office of the Chief Medical Examiner and Clinical Professor, Faculty of Medicine and Dentistry [email protected] Opiate vs. Opioid Opioid Refers to any chemical that activates mu-receptors (mainly in the spinal cord and brain) • Endogenous neuropeptides • e.g. endorphins (endogenous ‘morphine’) • Opiates • Opium-derived, found naturally in opium poppy (e.g. morphine, codeine) • Semi-synthetic opioids • Derived from thebaine (from opium poppy), but not naturally occurring • e.g. hydromorphone, hydrocodone, oxycodone, oxymorphone • Synthetic opioids • Not usually structurally related to morphine • Fentanyl, methadone, tramadol, meperidine Opioid Tolerance Tolerance • The ability to withstand increasing doses with static or decreasing side effects • Tolerance can enable a person to take doses of an opioid that would kill most non-tolerant persons • Tolerance is “relative” (i.e. dose related), not absolute • Applies to any opioid • Cross-tolerance from one opioid to another occurs • e.g. if a person develops tolerance to heroin, they will also have a degree of tolerance to fentanyl • The degree of cross-tolerance can be difficult to predict Reversing Agents: Naloxone • Most common is naloxone (trade name Narcan) • Competitive antagonist binds the mu-receptor, displacing opioids that are already bound there, without activating the receptors (i.e. no analgesia) N F N F N N N N N N N Binds to mu-receptor but no activation Reversing Agents: Naloxone Naloxone half-life (t1/2) ranges from 30 to 80 minutes • Fentanyl t1/2 = 3-12 hours, so repeat naloxone injection often required • If minimal or no response within 2–3 minutes, dosing may be repeated every 2 mins to a maximum 10 mg • (e.g. 0.4 mg per injection  25 injections for 10mg) Minimal toxicity if opioids not present • (i.e. very low risk for repeat administration) Medical administration intravenous or intramuscular • Intra-nasal formulation has been available for ‘public’ use • Orally – very poor oral bioavailability, takes too long to act Are some opioids tolerant to naloxone? • Probably not… • Effectiveness depends on the dose of naloxone administered versus potency and dose of the opioid • Highly tolerant abusers using high doses will require higher doses of naloxone to reverse a coma • Some newer opioids may be more dangerous because users are not familiar to the ‘right’ dose and potency of the specific batch being trafficked • Benzodiazepines will increase CNS depression (e.g. etizolam) • Benzodiazepines NOT reversed by naloxone Medical uses of fentanyl • Short term anesthesia • Acute surgical procedures such as intubation (i.e. inserting an airway) • 50 – 200 ug intravenous • Induction of anesthesia • Often with midazolam • Adjunct to anesthesia in longer surgical procedures • Dose depends on type of surgery • Airway must be supported for higher doses and longer surgeries • Transdermal patches for long-term chronic pain • 12.5, 25, 50 and 100 ug/hour • NOT for acute or post-surgery pain • Sublingual lozenge for breakthrough pain • Dose 0.1 – 1.6 mg *Fentanyl is not “new” • Developed in 1960 by Dr. Paul Janssen • Marketed and increasingly used in the 1960s by Janssen Pharmaceutica (trade name Sublimaze) • Properties of fentanyl and other analogues studied repeatedly with animal testing in 1970-80s • Analogues included carfentanil and 3-methylfentanyl • Fentanyl and some of the original analogues are still in legitimate use • sufentanil, alfentanil, remifentanil; carfentanil (not humans) Fentanyl abuse • Highly addictive narcotic; Injectable fentanyl can be abused by medical personnel with easy access (e.g. anesthetists, nurses) • Fentanyl 50 – 100 x more potent than morphine; 25x >heroin • Transdermal patch – for chronic pain (e.g. Duragesic) – Used to be widely abused: gel patch or matrix patch (oral suck/chew); when frozen and cut called ‘chicklets’ • Abuse also as illicit tablets and powder • Local or through the Internet in non-pharmaceutical form (e.g. powder; fake tablets) • Found in counterfeit ‘Oxycontin’ 80 mg tablets 9 *Fentanyl in Alberta 2011- 2023 • Pre-2011 used to see mostly patch-related fentanyl deaths – Abused, stolen, otherwise diverted • Now almost entirely illicit non-patch related – Fake green ‘oxycontin’ tablets were common – Mostly tablets crushed or ‘shaved’ and snorted; some injected; – Now lots of “fake heroin” containing fentanyl and/or carfentanil, with little or no heroin Rapid increase in cases due to fentanyl“abuse” 2011 (6); 2012 (29); 2013 (66); 2014 (127) 2015(279); 2016 (363); 2017 (565); 2018 (667); 2019 (513); 2020 (1055); 2021 (1541); 2022 (~1500+); 2023? Look for the cement mixers… 11 How is fentanyl abused? Snorting: powder or crushed tablet TD patch: inject, chew ‘Smoking’ Injecting Less-conventional modes of abuse Nasal Spray E-Cigarette *Apparent illicit opioid overdose deaths 2011-2021 400 363 Deaths per year 350 900 800 300 Fentanyl deaths 250 Cumulative total deaths 279 700 600 200 500 150 400 127 300 100 66 50 0 * 1000 200 29 100 6 2011 0 2012 2013 2014 2015 2016 Cumulative total deaths *2021: 1547 – a 258-fold increase from 2011 *Number of apparent unintentional non-prescribed opioid poisoning deaths 2018 – Sept 2023 (mostly fentanyl and carfentanil). Alberta substance use surveillance system Updated November 2023 Note: 2022 and 2023 data likely incomplete! Alberta deaths due to an apparent drug overdose related to non-prescribed opioids, by sex and age - 2020* *Alberta substance use surveillance system 2021 Fentanyl alone? – Rarely. • Most fentanyl fatalities include a stimulant • Usually methamphetamine or cocaine, some have both • Many fentanyl related deaths include ethanol and/or prescription or illicit benzodiazepines and/or other prescription drugs • About 35% 2015 cases included xylazine (veterinary sedative) • Now making a huge ‘comeback’ across Canda and the US • Also: • Carfentanil, fluorofentanyl, acetylfentanyl, butyrylfentanyl, 3-methylfentanyl, furanylfentanyl, cyclopropylfentanyl, methoxyacetylfentanyl, etc. • Now also “nitazines” and brorphine *Fentanyl poisoning deaths (unintentional) in Alberta, by most common additional substances causing death and year. Fentanyl Cocaine Alberta substance use surveillance system Updated November 2022 Carfentanil Methamphetamine Some perspective on fentanyl dose… • Estimated fatal dose of fentanyl (non-medical) • 0.5 – 1.0 mg (presuming low tolerance and IV administration) • will be lower with alcohol or depressant drugs • will be higher with tolerance, >2-3 mg • 1 mg is not a lot of powder! • ‘fake Oxys’ used to contain variable amounts of fentanyl (1-4 mg) • But not always fentanyl. May be carfentanil or another drug. Why fentanyl? • Highly addictive and popular • Easy to buy over the Internet • Dose for dose is about 1/1000th weight of cocaine • Dose for dose is about 1/20th weight of uncut heroin • Relatively easy to traffic in pure form • Canadian and U.S. fentanyl originally originated mainly from Asia (mostly China?). Now extensively from Mexico and some other countries. • Precursor chemicals shipped from China and fentanyl synthesized in Mexico 20 Carfentanil • Estimated 100x more potent that fentanyl (based on animal studies) • Very restricted veterinary use for large animals • Fatal doses could be as low as 10 – 20 MICROGRAMS!! Carfentanil Fentanyl 21 *When did carfentanil become an abused drug? • Reports of carfentanil abuse in eastern Europe 2012 & 2013 • May 2016: 4th International Conference Novel Psychoactive Substances discussed carfentanil abuse in Europe. • June 27 2016: 1 kilo of carfentanil seized by Canadian Border Services in Vancouver, destined for Calgary (10 – 20 ug can be fatal!) • July 2016: start(?) of media reports of deaths from carfentanil tainted heroin in Ohio, later Michigan, Florida • August 2016: first Alberta death due to carfentanil • 402 carfentanil related deaths Q3 2016 – Q1 2020 *Fatal dose of carfentanil?? More recent opioids – “nitazenes” • Isotonitazene was first detected in an Alberta death in 2019 • • • • • Estimated to be approx. the same potency as fentanyl Some nitazenes estimated to be up to 20x more potent than fentanyl Classed as benzimidazole opioid analgesics Benzimidazoles investigated as analgesics in the 1950s by CIBA / Novartis Several nitazenes now identified across Canada / U.S. since 2019 • Isotonitazene, butonitazene, etodesnitazene, metonitazene, flunitazene, protonitazene, • desethyl isotonitazene, N-pyrrolidino etonitazene, and brorphine (chemically related) Isotonitazene Benzodiazepines as fentanyl adulterants • Pharmaceutical benzodiazepines: • Alprazolam, bromazepam, clonazepam, diazepam, nitrazepam • Illicit benzodiazepines (illicitly synthesized or not licenced in Canada): • Adinazolam, bromazolam, clonazolam, desmethylgidazepam, desalkylflurazepam, deschloroetizolam, diclazepam, estazolam, etizolam, flualprazepam, flualprazolam, flubromazolam, flubromazepam, fluclotizoam, meclonazepam, pyrazolam, …etc. Case Report: Very sudden death of an opioid addict • Middle-aged male, long-standing opioid/fentanyl addict • Lapses into opioid withdrawal (shakes, sweating, nausea) • Girlfriend contact their drug dealer and arranges a supply • Girlfriend fetches the supply, ‘cooks’ the powder in a spoon and the male then injects himself • Male almost immediately loses consciousness and shortly after stops breathing; girlfriend calls 911 after a few minutes • Paramedics able to partially resuscitate the addict (ROSC), but male declared dead after about 2 hours • Toxicology on postmortem blood: • Fentanyl 25 ng/mL, etizolam 5 ng/mL, flualprazolam 15 ng/mL, xylazine 30 ng/mL, methamphetamine 45 ng/mL, plus metabolites and naloxone • Cause of death: fentanyl toxicity contributed to by benzodiazepines and xylazine How to increase the success of finding the more potent fentanyl and other opioid analogues: • Seizing and analyzing drug paraphernalia • Higher concentrations make for easier detection • Provides authentic drug source for new drugs • Optimizing and/or developing new analytical methods • Applying to purchase more sensitive equipment • Investigating screening urine samples (likely more concentrated) • Information gathering • • • • • Keeping in touch with colleagues (Canada, USA and elsewhere) Contact with police agencies, Health Canada Reading current scientific literature, news articles Following popular drug related ‘blogs’ Looking at ‘research chemicals’ that are being offered • Now many more sources of information that at the start of the opioid “epidemic” Other ‘Novel’ Psychoactive Substances Stimulants, Psychedelics & Hallucinogens • Cocaine • Amphetamines • Phenethylamines • 2C-X series and N-BOMe drugs • Cathinones (aka “Bath Salts”) • Tryptamines • Others (LSD, PCP, mescaline, psilocin) 29 Cocaine • Powerful stimulant, local anesthetic, highly addictive • Used routinely in lower doses in (some cultures in South America) • Still very widely abused • Widely adulterated: – [Diltiazem – cardiac drug] – [Hydroxyzine – antihistamine / sedative] – Levamisole - anticancer / veterinary de-worming agent • Life-threatening blood discrasias (agranulocytosis / neutropenia) – Phenacetin – Obsolete OTC analgesic (“Superbuff”) • Kidney damage (and carcinogenic?) 30 Amphetamines • Methamphetamine / amphetamine – Methamphetamine may be “crystal meth” – Amphetamine a metabolite and used for ADHD, narcolepsy, weight-loss • “Ecstasy” group – MDMA (methylenedioxymethamphetamine) – MDA (methylenedioxyamphetamine) – MDEA (methylenedioxyethamphetamine) – PMMA (paramethoxymethamphetamine) – PMA (paramethoxyamphetamine) – Many others (e.g. DOB, DOM, TMA) • Methamphetamine has become a MAJOR contributing factor in the opioid crisis 31 Psychopharmacology of cocaine and methamphetamine • Cocaine and methamphetamine both stimulate release and block reuptake, to varying degrees of dopamine, serotonin and norepinephrine, causing increased stimulation of the CNS, cardiovascular and other systems. • Abuse leads to tolerance and eventually massively increasing doses, sleep disturbances, psychosis, potentially violent and aggressive behavior (esp. meth!). • The biggest difference between the drugs is half-life: cocaine 0.5 – 1.5 h; methamphetamine 10 – 15 h. • A methamphetamine “binge” can last 2 – 3 days and involve doses of up to 1000 mg or more a day (may be in 100 – 250 mg doses; ‘therapeutic’ 5 – 20 mg). • Users will “crash” after 1 – 3 days depending on “supply” and onset of increasing fatigue due to lack of sleep, even though blood methamphetamine may be high. • Neurotransmitter levels in the brain may be depleted and sensitivity of neurons to methamphetamine decreased. Cocaine, methamphetamine and other stimulants can cause psychosis • Psychosis • A severe mental disorder in which thought and emotions are so impaired that contact is lost with external reality. • Can be a form of medical illness unrelated to drugs, or can be caused by alcohol or drug use. Can include: • Delusional behavior • Where a person has strong beliefs that are not shared by others • A common delusion is someone believing there's a conspiracy to harm them (aka paranoia). • Hallucinations • Where a person hears, sees and, in some cases, feels, smells or tastes things that do not exist outside their mind but can feel very real to the person affected by them; a common hallucination is hearing voices. • Delirium • A temporary mental state characterized by confusion, anxiety, incoherent speech, and hallucinations. *Case #1: Police shoot man who threatened them with a firearm • Police saw a man sleeping in his vehicle by the side of the road. He seemed harmless, so they continued on patrol. • Sometime later the same patrol found the vehicle was still in the same position and the man still appeared to be sleeping. • The police knocked on the car window to make sure than man was OK. • The man woke up, glared at the police shouted “f**k you police” and started to pull a rifle from between his legs in a threatening manner. • Police fired multiple shots at the man, killing him. *Case #1: Autopsy and toxicology • Autopsy: confirmed multiple gunshot wounds including to the heart and other organs. • Toxicology (femoral blood): • • • • • • • Ethanol – 40 mg/100 mL Methamphetamine – 1880 ng/mL* Amphetamine – 180 ng/mL Cocaine – detected (less than 20 ng/mL) Cocaethylene – trace amount Benzoylecgonine – 130 ng/mL Delta-9-THC and carboxy-THC detected in cavity blood (*Peak blood level ~20 ng/mL after 12.5 mg methamphetamine) • But how can a person with such a high blood methamphetamine sleep – possibly for “hours”? *Case #2: MV Accident causing death of passenger • A family was traveling by car very early in the morning when it left the road and crashed into a tree, with children in the back seat (one of the children died). • Collision analysts said the vehicle appeared to leave the road well prior to a bend with no evidence of braking. • The driver was unresponsive while fire department personnel were extracting him from the car, but woke and became incoherent and very agitated when EMS personnel tried to assess him. They subsequently sedated him with midazolam. He suffered non-life threatening injuries. • Police found methamphetamine in the vehicle. • Toxicology testing on the driver’s blood drawn about 3 hours after the accident found methamphetamine (160 ng/mL) and amphetamine (21 ng/mL). Midazolam and ketamine were also detected, resulting from medical treatment. • It appears the driver fell asleep and left the road despite having a significant concentration of methamphetamine in his blood. *Case #3: Cocaine excited delirium death • Police were called to a downtown location because a man was reported to be causing a disturbance and acting in an excitable and very erratic manner. • Notably, he was trying to attack a public transit bus with a baseball bat. • Police were finally able to apprehend him and paramedics were able to sedate him and transfer him to a gurney. • However, he was still violently struggling so they restrained his arms and legs. • He died suddenly while he was being transported to the emergency department and could not be resuscitated. Excited delirium – What is it? • A potentially fatal state, involving extreme agitation and delirium, and potentially violent behavior (aka: “agitated delirium”). • Often related to the acute abuse of cocaine, methamphetamine or other powerful stimulants, but not always • May involve “super-human strength”. • Deaths are not necessarily related to blood concentrations of drugs (if involved). • Deaths may be related to restraint by police or medical personnel. • Not necessarily restraint that causes asphyxia. • Recognized by NAME and the American College of Emergency Physicians. • Not recognised by the American Medical Association “as a medical diagnosis unless clear diagnostic criteria are validated”. • AMA opines that apparent “excited delirium deaths” frequently involve heavy police restraint (sometimes dangerously so, such as “hog tying”), and disproportionately persons of color. Methamphetamine vs. MDMA vs. PMMA • ALL can kill at higher doses • Initial effects: stimulant, wakefulness, hyperactivity, risk-taking, mydriasis • Toxicity: jerky movements, muscle rigidity, hyperthermia, reduced consciousness, rhabdomyolysis, coma, death • MDMA so-called ‘love drug’ (serotonin effects) • PMMA has similar effects, but: • Slower to act; less potent initially • Users may take additional doses • Increased lethality compared to MDMA 39 *Ecstasy / Molly= MDMA? • Not necessarily • Can be • Methamphetamine • Dextromethorphan • Benzylpiperazine &/or TFMPP • Cocaine • Ketamine • Nicotinamide • Caffeine • Anything else, or Nothing • Dose may vary enormously • In 2012…PMMA caused 20 deaths in Alberta and 7 in BC. 40 Other Toxic Phenethylamines • Phenethylamine – Structurally related to amphetamine – Has been used as a ‘weight’ loss ingredient – Putrefactive amine seen in decomposing samples • Substituted phenethylamines have stimulant and psychedelic properties – Most common are 2C-X series: 2C-B, 2C-C, 2C-I etc. • Detection: not as easy as methamphetamine, but a good lab should be able to detect if they know what to look for (need a current mass spectral database) • What distinguishes these drugs? • Degree of psychedelic properties and overt toxicity 41 Phenylethylamine Cathinone 42 *2C- structures vs. phenethylamine • Substitute different elements or groups in the 3 and 4 positions • 2 and 5 positions are 2, 5methoxy • Numerous different combinations 43 ‘N-Bombs (NBOMe) • Very potent derivatives of the 2C-X family of phenethylamines with effects similar to LSD • Strongly hallucinogenic, toxic, lowdose drugs • (sub-milligram doses 0.05 – 0.8 mg) • 25I-NBOMe is the most common, but also • 25B-NBOMe, 25C-NBMe, 25HNBOMe, 25N-NBOMe • Very difficult for laboratories to detect in body fluids!! 44 *NBOMe Cases – from the literature • Case 1: 21 y.o. took ‘acid’ and developed a sudden rage, flailing about violently and eventually unconsciousness. Had multiple external injuries due to the flailing, but none fatal. • Blood 25I-NBOMe 4.7 ng/mL • Case 2: 15 y.o. took ‘acid’ at a rave, developed bizarre behavior, multiple external contusions, hyperthermia, deteriorated and died. • Blood 25I-NBOMe 16 ng/mL • Case 3: 19 y.o. took ‘acid’, developed bizarre, paranoid behavior and fell (flew?) from 7th floor of a building; died from injuries. • Blood 25I-NBOMe 0.4 ng/mL 45 “Bath Salts” – the Cathinones • Cathinone – found in Khat • Used by some African cultures, much as some South Americans chew coca leaf and ‘Europeans’ use caffeine • The ‘Bath Salts’ designer drugs are substituted and modified cathinone • At least 30 are/were relatively common • Stimulants with some psychedelic activity • Examples: • methylenedioxypyrovalerone (MDPV), mephedrone, pyrovalerone • ethcathinone, butylone, fluoromethcathinone, methedrone 46 Canada vs. USA – The Legislative Differences • Canada: • Federal drug laws (Controlled Drugs and Substances Act – CDSA) • Controls specific named substances, but also chemically related drugs that may have a similar pharmacological effect • USA: • DEA (Drug Enforcement Administration) has “federal” rules, but slow to add new substances • Did have Federal Analogue Act 1986 but sometimes difficult to get a prosecution • e.g., what is an analogue (how similar to a scheduled drug) • Also state control of illicit substances, but huge variation from state to state, plus states are also slow to add substances and on a very ad hoc basis 47 Summary of the Non-opioid ‘Designer Drugs’ Cocaine: still a major abused stimulant Amphetamines: (mainly stimulant, some with “feel good” properties) Methamphetamine (MAJOR resurgence) 3,4-Methylenedioxymethamphetamine (MDMA) 3,4-Methylenedioxyamphetamine (MDA) Paramethoxymethamphetamine (PMMA – much more toxic than MDMA) Phenethylamines: (stimulant plus psychedelic) Phenethylamine (illicit diet medication) 2C-X series (at least 50 known, some extremely toxic) N-BOMe series (also substituted phenethylamines, but highly potent and toxic hallucinogens with LSD-like effects) Cathinones: (Khat: mild to moderate stimulant related to ephedrine) Designer cathinones (aka ‘Bath Salts’) are more potent and have more psychedelic activity [Tryptamines: (chemical analogues of the neurotransmitter tryptamine) Stimulants with some hallucinogenic activity; at least 55 described in the literature e.g. 5-Methoxy-diisopropyltryptamine (“Foxy”), psilocybin/psilocin, LSD, ibogaine LSD analogues are illicitly available] – not covered in these lectures PCP, ketamine: (anaesthetics with hallucinogenic and dissociative side-effects) Synthetic analogues illicitly available Cannabinomimetics: (synthetic cannabinoids; 100s known) May or may not have cannabis-like effects Most are untested in humans and potentially very toxic 48 Drug Testing in Sports Graham R. Jones, Ph.D. Former Chief Toxicologist Office of the Chief Medical Examiner and Clinical Professor, Faculty of Medicine and Dentistry 1 [email protected] History • Doping in sports dates back centuries to Greek and Roman events • In 1800s, strychnine, cocaine and caffeine known to be used • Modern ‘dope testing’ started in mid-1960s after British cyclist Tommy Simpson collapsed and died after a grueling race: dehydration and methamphetamine use • Initial ‘dope testing’ focused mainly on stimulants 2 Current Regulation • International Olympic Committee (ICO) • World Anti-Doping Agency (WADA, 2002) • International Sporting Agencies (e.g. IAAF) • Regional Events (e.g. Pan American Games, World University Games, Commonwealth Games) • National (e.g. Canadian Olympic Committee), other regional, local 3 Terms • Prohibited Substances • Some sports specific • Some allowed out-of-competition • Monitoring Program • To monitor potentially abused drugs • Therapeutic Use Exemption (TUE) • Specific exemption for medically justified medications that would otherwise be banned 4 Therapeutic Use Exemption • Certain drugs may be allowed in some formulations if recommended by physician AND approved by WADA • Approved, screened use of specific medications for legitimate medical purposes • Must be approved BEFORE use (during or out-ofevent) • Athlete’s physician must apply to WADA medical team 5 Monitoring Program • To monitor incidence and concentrations of drugs not banned outright, but subject to abuse • 1. Stimulants: In-Competition only: Bupropion, caffeine, nicotine, phenylephrine, phenylpropanolamine, pipradrol and synephrine. • 2. Narcotics: In-Competition only: Hydrocodone, mitragynine, morphine/codeine ratio, tapentadol and tramadol. • 3. Glucocorticoids: In-competition (by routes of administration other than oral, intravenous, intramuscular or rectal) and Out-of- Competition (all routes of administration) • 4. Beta-2-agonists (Bronchodilators) – any combination of two. 6 Stimulants • Sympathomimetic effects: raised blood pressure, increased heart rate, greater endurance, combats fatigue, improved endurance • “Amphetamines” and most other drugs with stimulant effects (cocaine, strychnine, methylphenidate, cathinones…) • Used to be an absolute ban on all stimulants including mild stimulants but now ephedrine*, pseudoephedrine**, phenylpropanolamine and some other mild stimulants now allowed (but in monitoring program) • *Ephedrine allowed if <10 mg/l in urine • **Pseudoephedrine allowed if <150 mg/l in urine 7 Caffeine • Allowed now, but urine concentration originally was to be less than 12 mg/l • In 2009 on “monitored” list (still is) • Can be abused: • By non-coffee drinkers • Abstinence followed by use • Main desired “stimulant” effect is increased aggression 8 Bronchodilators (β2-agonists) • Most banned, except salbutamol, salmeterol, terbutaline and formoterol allowed in inhaled form if athlete is granted TUE for asthma or similar condition • Even with TUE, salbutamol must be <1000 ng/ml in urine, unless athlete can prove they have abnormal metabolism! 9 Narco