Rational Pharmacometabolomics & Pharmacogenomics for Pain ...

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


Acknowledgement

• Dr. Bing Pang

• Dr. Lindsay Macnarama

• Dr. Ami Jackson

• Dr. Elizabeth Palavencio

• Dr. Steven Cotten (umbilical cord tissue drug screen)

• William Nell

• Sciex


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)

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


• Pharmacometabolomics


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?


• Turmp opioid program

Aug. 10, 2017


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!)

https://www.statnews.com/2016/12/09/opoid-overdose-deaths-us/

The National Academies of Sciences, Engineering and Medicine

Source: Dr. David Clark

Source: Dr. David Clark


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)

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.

https://www.ncbi.nlm.nih.gov/pubmed/28941590

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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?????

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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

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• 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

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• 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


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


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


• 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


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


Pain Management Panel, Calibration Standard 6


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


• 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


• 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


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


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?


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

?


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


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


Brodifacoum https://emergency.cdc.gov/han/han00410.asp


• 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,”


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


• Mix 200 mL blood, calibrators, QC with phosphate buffer and IS

• Extract with Ethyl acetate• Spin and transfer upper organic layer• Evaporate and reconstitute


• 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


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


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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