Rational Pharmacometabolomics & Pharmacogenomics for Pain Management &
Opioid Addiction Steven H. Wong, Ph.D. DABCC(TC), FAACC. FACs
Prof. Path., Dir. Clin Chem & Tox., Co-Dir, Clin & Translational MS.Ctr
MSSS for Lab Med 8th Annual Conference, Philadelphia, 10.4.2018
Wake Forest Baptist Medical Center
Disclosure
• NIH/NCI – funded Paclitaxel study (1R21CA208968) – Hammond and Thomas PIs, and Wong, Co-investigator
• Sciex LCMSMS grant
• NCI Antibody committee member and APOLLO working group member
Wake Forest Baptist Medical Center
Acknowledgement
• Dr. Bing Pang
• Dr. Lindsay Macnarama
• Dr. Ami Jackson
• Dr. Elizabeth Palavencio
• Dr. Steven Cotten (umbilical cord tissue drug screen)
• William Nell
• Sciex
Wake Forest Baptist Medical Center
Objectives
• Next Generation Clinical Mass Spec for Personalized Medicine and Personalized Justice, Pain Management & Opioid Epidemics
• Umbilical Cord Tissue Drug Screening (Cotten with permission)
• Proactive opportunities applying pharmacometabolomics and PGX for Pain Management and Opioid Addiction – Suboxone, “Tainted” marijuana (BrodifocaumFentanyl)
Wake Forest Baptist Medical Center
Proposed Interrelationship of Molecular biology/biomarkers, Precision/Personalized Medicine and Personalized Justice, Wong 2016
DNA RNA Proteins Metabolites
Genomics Transcriptomics Proteomics Metabolomics
Precision/Personalized Medicine
Environment
Epigenetics/Imprintomics
Microbiome
Toxicity, Sensitivity
Performance, Behavior
Pharmacogenomics
Personalized Justice
Efficacy
Molecular imaging, Tissue imaging/molecular microscopy
PharmacometabolomicsExhalome,
Pharmacoproteomics
Wake Forest Baptist Medical Center
• Pharmacometabolomics
Wake Forest Baptist Medical Center
Next Generation Clinical Mass Spec• Inevitable global, evolution(?) - Product cycles and “ Natural,
ubiquitous, logical and encompassing”, similar to Next Gen Sequencing?
• Frame work by AACC MSSS – 5 years to enable Precision/Personalized Medicine, Cancer Moonshot/APOLLO
• Technological advances:
1. Higher throughput – 100s per day?
2. Total automation – online, sample preparation, IT and report
3. Complementary to immunoassays
4. Cloud based
• Beyond Pathology – with surgery and other clinical departments
• Data – Big, Standardization/harmonized and commutability
• Education – CLS, fellows and other colleagues
• Governmental approval – Lab Developed Tests?
Wake Forest Baptist Medical Center
Wake Forest Baptist Medical Center
• Turmp opioid program
Aug. 10, 2017
Wake Forest Baptist Medical Center
The Opioid Epidemic• 2015 - 22,598 teaths
• States with the highest death rate/1000,000 residents1. West Virginia 28.92. New Hamsphire 28.03. Rhode Island 19.84. Massachusetts 17.05. Ohio 16.4
• 2016 > 50,000 (https://www.statnews.com/2016/12/09/opoid-overdose-deaths-us/)
• Fentyanl intoxication greatly increased 73% from 2014 to 2015 (Prince!)
Wake Forest Baptist Medical Center
The National Academies of Sciences, Engineering and Medicine
Source: Dr. David Clark
Source: Dr. David Clark
Wake Forest Baptist Medical Center
Objectives• Next Generation Clinical Mass Spec for Personalized Medicine and Personalized
Justice, Pain Management & Opioid Epidemics• Umbilical Cord Tissue Drug Screening (Cotten with
permission)
• Proactive opportunities applying pharmacometabolomics and PGX for Pain Management and Opioid Addiction – Suboxone, “Tainted” marijuana (BrodifocaumFentanyl)
Wake Forest Baptist Medical Center
Alternative Matrices and Workflow Outcomes Associated with Neonatal Drug Testing
Steven Cotten, Ph.D. DABCC NACBAssistant Professor PathologyDirector of Chemistry, Immunology, Toxicology,
and Point of CareOhio State University Wexner Medical Center
To Drug Test a Baby
Source: Clin Biochem. 2017 Dec;50(18):1093-1097.
20
PlacentaMaternal Blood
Cord BloodMaternal Oral Fluid
Breast MilkMaternal UrineNeonate Urine
Neonate HairAmniotic Fluid
MeconiumUmbilical Cord
Maternal Hair
Hours Days Weeks Months Years
Choosing a specimen
Window of Detection
Source: Cotten
Umbilical cord tissue: an emerging specimen
• Several laboratories offer drug testing of umbilical cord tissue
• Advantages include:
• Ease of collection
• Less nursing time (compared to meconium)
• Potential cost savings depending upon your institutions screening algorithm.
• Same window of detection?????
21
Pathology departments may need to be aware that cord is removed.Some institutions send placentas and cords for gross analysis on all newborns.Short cords <40 cm can be cited as contributing to factors to other abnormalities.
Source: Cotten
Multistep “extraction” and analysis
22
• Tissue needs to be disrupted
• Various methods, mechanical, bead based….
• Throughput….
• Most 96 well extractions are for genomics
• Drugs and target compounds extracted
• Mixed mode extraction or targeted purification of drug classes…
• Sample cleaned up
• Removal of interfering substances, matrix effects
• Analysis
• Limit of detection, sensitivity based on amount of starting material, EIA vs. Mass Spec, analytes?
Source: Cotten
Summary
23
• We have commercialized a test for toxicology analysis of umbilical cord tissue.
• Birth-to-Result time was significant improved after switching to umbilical cord tissue in-house.
• Sensitivity and NPV for NAS increased based on ICD10 codes.
• The lab should understand who is ordering the test, how the results are used, and what are important expectations.
• Engage the different stakeholders within your organization to find the optimum choice(s).
Meconium Umbilical Cord Tissue
Standardized Collection
Less Sensitive?
Rapid Results
Difficult Collection
No epidural medications
Delayed Results
Source: Cotten
Wake Forest Baptist Medical Center
Objectives• Next Generation Clinical Mass Spec for Personalized Medicine and Personalized
Justice, Pain Management & Opioid Epidemics
• Umbilical Cord Tissue Drug Screening (Cotten with permission)
• Proactive opportunities applying pharmacometabolomics and PGX for Pain Management and Opioid Addiction
1. Suboxone2. “Tainted” marijuana - Brodifocaum3. “Tainted” marijuana - Fentanyl
Wake Forest Baptist Medical Center
Suboxonehttps://www.drugs.com/suboxone.html
• Narcotic (opiate) addiction• Not for pain management• Combination of buprenorphine (opioid, a mu-
opioid ) and naloxone • Sublingual or buccal administration
Wake Forest Baptist Medical Center
• 60 µL hydrolysis/internal standard solution + 100 µL sample
• Incubate for 30 min
• Centrifuge, add: 450 µL 10% acetonitrile + 0.1% formic acid in water, 50 µL sample
• Analyze using LC/MS/MS
Procedure for Sample Prep and LC-MS/MS
Wake Forest Baptist Medical Center
WF Pain Management Drug Panel
11-nor-9-Carboxy-Δ9-THC Codeine
6-Acetylmorphine Desalkylflurazepam
7-Aminoclonazepam Diazepam
7-Aminoflunitrazepam EDDPalpha-Hydroxyalprazolam Estazolam
alpha-Hydroxymidazolam Fentanylalpha-Hydroxytriazolam Hydrocodone
Alprazolam HydromorphoneAmphetamine Lorazepam
Benzoylecgonine MDABuprenorphine MDEAButalbital MDMACarisoprodol MeperidineClonazepam Meprobamate
Methadone OxycodoneMethamphetamine OxymorphoneMorphine PCPN-Desmethyl-cis-tapentadol Pentobarbital
N-Desmethyl-cis-tramadol Phenobarbital
Norbuprenorphine PregabalinNordiazepam Propoxyphene
Norfentanyl Secobarbital
Norhydrocodone Tapentadol
Normeperidine Temazepam
Noroxycodone Tramadol
Noroxymorphone Zolpidem
NorpropoxypheneZolpidem phenyl-4-carboxylic acid
Oxazepam Naloxone
Wake Forest Baptist Medical Center
Pain Management Panel, Calibration Standard 6
Wake Forest Baptist Medical Center
Naloxone buprenorphine (n = 23)Naloxone Bup N-bup Nal/Bup N-Bup/Bup
1430 490 >1000 2.92 >2.04756 565 869 1.34 1.54693 312 585 2.22 1.88658 155 622 4.25 4.01539 184 913 2.93 4.96523 212 893 2.47 4.21451 289 553 1.56 1.91444 330 706 1.35 2.14378 175 515 2.16 2.94289 354 563 0.82 1.59284 427 >1000 0.67 >2.34265 300 477 0.88 1.59247 129 267 1.91 2.07232 82 263 2.83 3.21169 38 78 4.45 2.05126 74 773 1.70 10.45115 87 209 1.32 2.4079 100 380 0.79 3.8050 114 246 0.44 2.1620 31 89 0.65 2.8716 6 37 2.67 6.178 31 27 0.26 0.87
<2 2 51 0.75 25.50
2017-8 Quest Send-out results
Wake Forest Baptist Medical Center
• BNX (Buprenorphine-naloxone co-formulation) subject to parenteral abuse and Lab cannot differentiate therapeutic and illicit use?
• Patient urine samples before (naloxone negative), during stable and unstable treatment phases (positive bup. Norbup, naloxone)
• Naloxone upper limit at 200 ug/L, median Nal/bup ratios higher for high vs low (0.9 vs 0.3)
• Suggest - Naloxone for compliance, high ~ parenteral abuse
Drug Testing Analysis. 2013;27:220-5
Wake Forest Baptist Medical Center
• Patient urine bup. Conc >1000 ng/mL, compliance 4.4%, endorsed and suspected adulteration, 42.9% and 40.6%
• Endorsed and suspected adulteration samples vs compliance – higher bupand lower norbup
• Bup >700 ng/mL
Drug Alc. Dependence 2017 180:46-51
Wake Forest Baptist Medical Center
Movantik and the frequency of positive naloxone in urine
Wotring, Countryman, Wallace, Strickland, Cummings, McIntire. JAT, 2018;42:38-40.
• Movantik (naloxegol) – OIC
• Naloxegol patient samples – 1355 with 14 naloxone, 209 Bup/Norbup(Suboxone or Movantik), 14/187 (7.5% request ) <100 ng/mL or 14/1335 (0.8%)
• 39 yr, male, 210 lbs, normal CYP3A4, 12.5 mg naloxegol, 1.5 hrnaloxone at 12.1 ng/mL (LOD 10 ng/mL)
• Naloxone from Suboxone – average 240 ng/mL* to 299 ng/mL** to for substance abuse patients, 2 patients positive several months apart
• Movantik patient may test positive for low naloxone (<100 ng/mL?)*Cummmings et al. 2016 SOFT Annual meeting **Heikman et al.. Drug testing Anal 2014
Wake Forest Baptist Medical Center
Suboxone/Naloxone – compliance or abuse?
• Naloxone abuse? High >600 ng/mL abuse? Or 240 to 299 ng/mL?
• Bup < 1000 ng/mL?• Treat the patient, not the “ number “• Clinical and forensic cases review approach –
patient history, medication history/drug interactions? toxicological report findings, and previous test result if any
• Future studies and guidelines?
Wake Forest Baptist Medical Center
Naloxone buprenorphine (n = 23)
Naloxone Bup N-bup Nal/Bup N-Bup/Bup1430 490 >1000 2.92 >2.04756 565 869 1.34 1.54693 312 585 2.22 1.88658 155 622 4.25 4.01539 184 913 2.93 4.96523 212 893 2.47 4.21451 289 553 1.56 1.91444 330 706 1.35 2.14378 175 515 2.16 2.94289 354 563 0.82 1.59284 427 >1000 0.67 >2.34265 300 477 0.88 1.59247 129 267 1.91 2.07232 82 263 2.83 3.21169 38 78 4.45 2.05126 74 773 1.70 10.45115 87 209 1.32 2.4079 100 380 0.79 3.8050 114 246 0.44 2.1620 31 89 0.65 2.8716 6 37 2.67 6.178 31 27 0.26 0.87
<2 2 51 0.75 25.502017-8 Quest Send-out results
?
Wake Forest Baptist Medical Center
Outbreak of Life-Threatening Coagulopathy due to Brodifacoum
• March 2018, IL Dep. Health – unexplained bleeding of patient used synthetic cannabinoids
• 202 cases and 5 deaths• Il, 164, MD 20, FL, IN, KY, MS, PA, VA , WI• 95% patient samples positive for brodifacoum• Symptoms – Bruising, nosebleeds, excessive heavy
menstrual bleeding, hematemesis, hemoptysis, hematuria, flank pain, abdominal pain and bleeding gum and mouth
Wake Forest Baptist Medical Center
Recommendations for patients with signs and symptoms of Vit.
K dependent coagulopathy
• Contact Carolina Poison Center• Ask patient synthetic
cannabinoids use – K2, synthetic marijuana, fake and legal weed
• Check INR>2• CDC Health Advisory
Wake Forest Baptist Medical Center
Brodifacoum https://emergency.cdc.gov/han/han00410.asp
Wake Forest Baptist Medical Center
• High level of brodifacoum, rat poison• Added to Spice, K2 (synthetic cannabinoids - Cheap and not
“screened”) to prolong high?• “ Toxin ties up liver enzymes that metabolize drugs, extending their
effects.”• User - “ playing Russian roulette,”
Wake Forest Baptist Medical Center
Wake Forest Baptist Medical Center
Brodifacoum
file:///Users/shwong49/Desktop/10.3.5.18%20AACC%20MS/Brodifacoum%20-%20Wikipedia.webarchive
• E4-hydroxycoumarin vitamin K antagonist anticoagulant pesticides/rodenticide
• Pharmacologically similar to warfarin , “second generation” “superwarfarin”
• Increases permeability of blood capillary – leaking from capillary, caution – penetrate skin
• Vitamin K anti-dose, charcoal, blood transfusion • Bioavailability – 100%, slowly and incompletely
metabolized by liver (vitamin K epoxide reductase) , LD50 270ug/kg (rat)
• T 1/2 = 20-130 days
Wake Forest Baptist Medical Center
• Mix 200 mL blood, calibrators, QC with phosphate buffer and IS
• Extract with Ethyl acetate• Spin and transfer upper organic layer• Evaporate and reconstitute
Wake Forest Baptist Medical Center
• Waters Acquity triple quad and UPLC system
• ESI + mode• Quantitation: 523.2>335.1• Confirmation: 523.2>178.1• Linear range : 2-200 ng/mL, LOD 0.5
ng/mL• Recovery: 70-105 %
Brodifacoum LCMSMS analysis
Wake Forest Baptist Medical Center
Wake Forest Baptist Medical Center
Wake Forest Baptist Medical Center
JAT, 2005;29:590-8.
• Fentanyl/Duragesic patches for pain management such as cancer• Started in late 80’s - abuse of transdermal patches, new or used patches, smoke,
ingest, transdermal, oral, and IV• 25 cases, 2002-2004, fentanyl present , fentanyl toxicity, and mixed drug toxicity• LCMSMS by NMS, and genotyped for CYP3A4*1B and CYP3A5*3 using
Pyrosequencing• Cases :
1. 1 CYP3A4*1b and 3A5*3 heterozygous2. 1 compound CYP3A4*1B and CYP3A5*3 heterzygous3. 22 CYP3A4*1B WT and CYP3A5*3 homozygous4. 1 CYP3A5*3 and CYP3A4*1B WT
• Mean fentanyl and fent/norfent ratio for cases 1and 2, and 3(22 cases) : 12.8 and 1.4 ug/L vs 16.7 and 7.3 ug/L
• CYP3A5*3 – impaired fentanyl metabolism
Wake Forest Baptist Medical Center
Wake Forest Baptist Medical Center
Wake Forest Baptist Medical Center
Conclusions and observations
• Next Gen Clin Mass Spec - Roles of Clinical Chemists for Personalized Medicine?
• Rational complementary omics –pharmacometabolomics and pharmacogenomics
• Enabling rational interpretation and emerging and increasing lab role for Addiction and Pain Management - TDM and PGx?
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