STAKEHOLDER PANEL ON ALKALOIDS OF KRATOM Background & Fitness for Purpose (Mitragynine & 7‐OH Mitragynine) Chair, Corey J. Hilmas, MD/PhD Senior VP Scientific and Regulatory Affairs (NPA) September 5, 2014

STAKEHOLDER PANEL ON ALKALOIDS OF KRATOM

Background & Fitness for Purpose

(Mitragynine & 7‐OH Mitragynine)

Chair, Corey J. Hilmas, MD/PhD
Senior VP Scientific and Regulatory Affairs (NPA) September 5, 2014
Boca Raton Resort & Club
Boca Raton, Florida

Kratom Roadmap

• Background
• Significance
• Regulatory Guidance
• General Analytical Needs
• Existing Methods and Techniques • Challenges
• Fitness for Purpose Statement

Background (Agency History)

• 2006 (street drug language in labeling)
• 2011 – Develop tox regulatory strategy for “low

  hanging fruit” using NDI adulteration charge  no safety data or safety concern based on MOA  “Legal High” Ingredients
   Centrally Active Ingredients
   Analysis of importation

• 2012 – Dietary Supplement (54) Import Bulletin written for Kratom
• Thousands of kilos of kratom detained/refused
• “Operation Log Jam” – not a massive nationwide crackdown

  on kratom

 Biak-biak  Katawn

 Krton  Krypton

 Kakuam  Mambog

 Madat Maeng Da

 Thom  Ithang

 Thang
 Ketum
 Kedemba

Red Vein/White Vein
 Nauclea
Gratom, Cratom, Krathom

Background (Synonyms)

(Mitragyna Speciosa Korth)

Background

 Botanical (fits within 201(ff))

•   Leaves are traditionally chewed and ingested
•   Trees grow 12-30 ft, leaves are 4-7 inches long
•   Kratom contains over 25 different alkaloids

  (mitragynine and 7-hydroxymitragynine)

 Different geographical varieties – vary in their alkaloid composition and potency

Malasia and Borneo – have more red vein Sumatra and Bali – have more white vein Thailand has Maeng Da variety (potent red vein)

Background

 Each leaf – 0.5% alkaloids

 20 leaves – 17 mg mitrag

•   White Vein/Red Vein
•   White – euphoric, energetic & stimulating

 Red – more sedating

•   Whether stimulating or sedating – depends mostly on

  the alkaloid dose ingested

•   Alkaloid amounts can vary markedly within the white or red vein leaf varieties at different harvest times and due to variations in climate

Background

Country or region where leaves were picked (ie. Thai, Indo, Maeng Da, Sumatran, Malaysian, Bali)

“Bumblebee” Origin – Vietnam
 Maeng Da (premium) – Thailand  Rifat Red Vein – Indonesia

Country on product name tells you the variety  Sumatra, Bali – more white vein
 Malasia, Borneo – have more red vein

Grade of Mitragyna speciosa (ie. Premium, Super, Ultra, extract, concentrated, Pimp Grade)

Background

Whole Leaf Crushed Leaf

 Powder

Capsules, Tablets

 Extracts

 Tinctures

Relaxation DS “Drinks”

 Shots  Mists

 Teas
Resins & Brownies

Background (Matrix)

Background (Claims)

Euphoria, Stress Relief at low doses
 Pain relief and opiate withdrawal at higher doses

 Depression
 Muscle aches and pains
 Flu-like symptoms
 Combat nausea and diarrhea  Insomnia
 Restless leg syndrome

Background (Public Health Interest)

 Kratom – banned in Thailand – Kratom Act 2486

 2nd most abused substance in Thailand today

 Not scheduled by DEA but on their list of Drugs and Chemicals of conern

 botanical marketed as a “legal high”, “Not for Human Consumption”, “Incense”, and “Ethnobotanical” that are coming through US ports coded as dietary supplements and dietary ingredients

ingested products with centrally active alkaloids

 products with a clear CNS antinociceptive profile

 sold in head shops (they are the new K2, Spice, and Bath Salts)

Background (Public Health Interest)

products marketed for their alkaloid content
 products with cloaked language in labeling (“has long legs”)  the shipper and consignee do not want to be known during

detention process and after refusal
 multiple forms: liquid DS/capsules, extracts, raw plant material (parachuted)
sold with capsule filling machines

Refusals
 Detained and refused 35,000 (low estimate) kilos kratom in 30

months
 IB (2012, 2013) and IA (2/2014)

12

12

Background (Public Health Interest)

New warning about Kratom

use
Local recovery clinic sees increase in addicts Updated: Sunday, 12 Aug 2012, 5:00 PM CDT Published : Saturday, 11 Aug 2012, 11:15 PM CDT AUSTIN (KXAN) – A local drug treatment facility has seen a sharp surge in people checking in for addiction to a substance called Kratom.

“Its a legal opiate- its a legal heroin,” said Angela Vickrey, Director of Admissions for the Austin Recovery Center, where they have treated seven patients for Kratom addiction in the last 90 days. “It basically has the same effect that opiates do- opiates being heroin and pain medication in large quantities- in low dose- its a stimulant.”

Background (Kratom Alkaloid Breakdown)

Analyte

Alkaloid %

Alkaloid %

Isomitraphylline
Mitraphylline
Mitraciliatine
Isospeciofoline
Ajmalicine (Raubasine) Akuammigine
Epicatechin
Isopteropodine 9‐Hydroxycorynantheidine <1 Mitraversine <1 Speciophylline <1 Stipulatine <1 Tetrahydroalstonine <1 Speciofoline <1

Analyte

Mitragynine
Paynantheine
Speciogynine 7‐Hydroxymitragynine Speciociliatine
Mitragynine oxindole B Mitrafoline
Isomitrafoline
Ciliaphylline
Corynantheidine (δ‐yohimbine) Corynoxeine

Corynoxine Rhynchophylline Isorhynchophylline

66
8.6 ‐ 9 6.6 ‐ 7 2
0.8 ‐ 1

<1 <1 <1 <1 <1 <1 <1 <1

<1 <1 <1 <1 <1 <1 <1 <1 <1

Chelsea College Pharmacognosy Research Laboratories (1961-1970)

Background (Structures)

C23H28N2O4 MW 396.479

C23H30N2O4 MW 398.495

Mitragynine

C23H30N2O5 MW 414.49

7‐Hydroxymitragynine

Paynantheine Ajmalicine (δ‐yohimbine or raubisine)

C21H24N2O3 MW 352.43

(S) (S) (S) (S)

(S) (R)

Speciociliatine

Significance (Public Health Interest)

• LeavesofMitragynaspeciosaareusedto suppress pain and mitigate opioid withdrawal
• Knownsubstituteforopium(bannedinThailand)
• Number2abusedplantinThailandandMalaysia
• Dietaryingredientsshouldnotbeaddictive
• Readilyavailablepsychoactiveplant
• Mostdetained/refusedentrybyFDAoverthe past 3 years – largest hauls are $200k+
• ImportAlert(2/2014),previouslyImportBulletin

• Methoxyl group at C9 – supposedly responsible
for analgesic activity of mitragynine/7‐hydroxymitragynine

• Corynantheidine (9‐demethoxymitragynine)
• Chromophore + wavelength shift

Corynantheidine

• Alkaloid substitution for kratom minus alkaloids marketed for relaxation?
• Yohimbe and Rauwolfia alkaloids (corynantheidine, corynanthine, yohimbine)

Significance (Public Health Interest)

• IA66‐41(importalertasadrug)–inplaceformany years

• WhydidtheproblemgetworsedespiteanFDAIA?

• –  The IA 66‐41 = covered plant material and product imports coded as a drug or drug ingredient entry (66) or for street drug language on labeling
• –  Overseas suppliers ‐ switched to code it as 54
• –  The new IA 54‐14 covered finished supplements and raw botanical material without making drug claims

Significance (Public Health Interest)

• Itisthedietaryingredientof2014andbeyond

•   3‐14‐2014 voluntary recall of kratom capsules, powder, maeng da leaf powder and extracts by Smiley National Inc. (Class II Recall)
•   Who will be caught in the dragnet next?
•   Open ended seizures?

Regulatory Guidance

Slot kratom appropriately (comfrey, ephedra) Kratom could fit under 201(ff) as an herb or other

botanical (alkaloids with opioid activity are the issue) Kratom (mitragynine or 7‐hydroxymitragynine)

= adulterated based on 402(f)(1)(B)
Kratom (mitragynine and 7‐hydroxymitragynine removed)

= allowable? (all other alkaloids left ‐ cause relaxation)  At what level would they be acceptable?
 Zero tolerance? Agency has to make that case

Regulatory Guidance

Kratom – burden is on the Agency

• Labelingaskratomoroneofitssynonymsalonedoesnot

  imply adulteration

• FDAmustconfirmpresenceofthealkaloidswithquantitative level

   Remember:
  • Comfrey minus pyrrolizidine alkaloids • Ephedra minus alkaloids
  • Rauwolfia minus reserpine
  • RYR minus lovastatin

Regulatory Guidance (Import Alert)

• IA54‐15DETENTION/REFUSAL

Label (kratom, Mitragyna speciosa, mitragynine extract, biak‐biak, cratom, gratom, ithang, kakuam, katawn, kedemba, ketum, krathom, krton, mambog, madat, Maeng da leaf, nauclea, Nauclea speciosa, or thang)

+

Quantitative level for mitragynine and 7‐ hydroxymitragynine

General Analytical Needs

• Mitragynine and 7‐hydroxymitragynine are exclusive to speciosa – the only 2 alkaloids likely of interest to the Agency (safety)

  – Physiology/pharmislaggingbehindontheotheralkaloids(bindingdata)

• Currently only 1 FDA lab performs all analyses for kratom
• Need ‐ a quantitative method for these 2 alkaloids
• NPA submitted FOIA for FDA’s quantitative method
• FDA has not used a screening method
• Matrix can vary – finished forms (tablets, capsules), liquid extracts, concentrated extracts, dry whole leaf, powder (detentions have mostly involved raw botanical form and liquids)

Existing Methods ‐ General

Three independent chromatographic methods coupled with 2 detection systems

1) GC with mass spectrometry

2) Supercritical fluid chromatography with diode array detection

3) High‐performance liquid chromatography with mass spectrometry and diode array detection

Techniques Used for Detection of Mitragynine and Other Indole and Oxindole Alkaloids in Kratom

Chromatographic Methods  HPLC (most common)  GC
 SFC

Detection Systems for Indole Alkaloids

•   Diode Arrays
•   Mass‐specific

   Quadrupole
  Linear ion trap Triple quadrupole MS

Existing Methods

LC‐MS with quadrupole mass spec (scan mode)

 Analytes:

• Mitragynine
• 7‐hydroxymitragynine
• other indole alkaloids
   Direct quantitation
   gradient elution with MeOH and water (46 min analysis time)  Matrix

Example:

• raw plant material
• commercial products of kratom

‐ Kikura‐Hanajiri, R., et al., Simultaneous analysis of mitragynine, 7‐ hydroxymitragynine, and other alkaloids in the psychotropic plant “kratom” (Mitragyna speciosa) by LC‐ESI‐MS. Forensic Toxicol. 27 (2009) 67‐74.

Existing Methods

Linear Ion Trap MS

 Analytes:
• Mitragynine, Paynantheine

 HPLC for metabolic studies, Matrix (urine)
 Ion Trap – MS for quantitation and detailed structural

analysis of metabolites in 20‐30 min

Examples:

• ‐  Philipp, A.A., et al., Use of liquid chromatography coupled to low‐ and high‐resolution linear ion trap mass spectrometry for studying the metabolism of paynantheine, an alkaloid of the herbal drug Kratom in rat and human urine. Anal. Bioanal. Chem. 396 (2010) 2379‐91.
• ‐  Philipp, A.A., et al., Metabolism studies of the Kratom alkaloids mitraciliatine and isopaynantheine, diastereomers of the main alkaloids mitragynine and paynantheine, in rat and human urine using liquid chromatography‐linear ion trap‐mass spectrometry. J. Chromatogr B. Analyt. Technol Biomed. Life Sci. 879 (2011) 1049‐55.
• ‐  Philipp, A.A., et al., Studies on the metabolism of mitragynine, the main alkaloid of the herbal drug Kratom, in rat and human urine using liquid chromatography‐linear ion trap mass spectrometry. J Mass Spectrom. 44 (2009) 1249‐61.

Existing Methods

Triple Quadrupole MS

Analyte (mitragynine) in biological samples
 Internal standard (Ajmalicine)
 Extraction with methanol/water and C18 column in 30 min  ESI/MS/MS
 Detection limit of 0.02 ng/mL
 LOQ 100 ng/mL

Example:

‐ Lu, S. et al., Quantitative analysis of mitragynine in human urine by high performance liquid chromatography‐tandem mass spectrometry. J. Chromatogr. B 877 (2009) 2499‐2505.

Existing Methods Triple Quadrupole MS

Analyte (mitragynine) in biological sample
Internal standard (amitriptyline), liquid‐liquid one step extraction (MTBE),

separated on Acquity UPLC column, isocratic elution

LOQ 1 ng/mL (range 1 – 5000 ng/mL) Example:

‐

Vuppala, P.K. et al., Simple, sensitive, high‐throughput method for the quantification of mitragynine in rat plasma using UPLC‐MS and its application to an intravenous pharmacokinetic study. Chromatographia. 74 (2011) 703‐710.

mitragynine and amitriptyline extracted with hexane‐isoamyl alcohol, resolved on Lichrospher RP‐SelectB column (9.8 and 12.9 min respectively)

LOQ 0.2 ng/mL (range 0.2‐1000 ng/mL) Example:

‐ De Moraes, N.V. et al., Determination of mitragynine in rat plasma by LC‐MS/MS: Application to pharmacokineticsJ. Chrom. B. 877 (2009) 2593‐2597.

Existing Methods

Triple Quadrupole MS

Analyte (7‐hydroxymitragynine) in biological sample (rat plasma) Internal standard (tryptoline)

isocratic elution, separation on Acquity UPLC(TM) BEH C18 column (2.5 min run time)

LOQ 10 ng/mL (range 10‐4000 ng/mL) Examples:

‐ Vuppala, P.K. et al., Development and validation of a UPLC‐MS/MS method for the determination of 7‐hydroxymitragynine, a μ‐opioid agonist, in rat plasma and its application to a pharmacokinetic study. Biomed. Chromatogr. 27 (2013) 1726‐1732.

GC – MS Method

Existing Methods

Analyte (mitragynine)

 no quantitation or validation attempted

 HP‐5 capillary column at high temp (200 ̊C)

 mitragynine eluted as symmetrical peak in < 17 min

 identification: comparison of experimental mass spectra with

library

‐ Chan, K.B. et al., Psychoactive plant abuse: the identification of mitragynine in ketum and in ketum preparations. Bull. Narc. 57 (2005) 249‐256.

Examples:

GC – MS Method

Existing Methods

Analytes (metabolites of mitragynine, paynantheine, speciogynine, speciociliatine)

 Matrix (urine)
 enzymatic cleavage of conjugates, solid‐phase extraction, and

trimethylsilylation

 LOQ: 100 ng/mL

 GC‐MS analysis of underivatized mitragynine and other indole alkaloids requires high temp.

 little control available for optimization of resolution of the analytes Example:

‐ Philipp, A.A. et al., Monitoring of kratom or Krypton intake in urine using GC‐MS in clinical and forensic toxicology. Anal. Bioanal. Chem. 400 (2011) 127‐135.

SFC Method

Existing Methods

historically used for analysis of indole alkaloids in Madagascar periwinkle faster,moreeconomical,environmentalconsideration(betterthan HPLC)  Analytes (mitragynine, 7‐hydroxymitragynine, others)
 Matrix (leaves of M. speciosa)

 Head‐to‐head comparison (SFC‐DAD, UHPLC‐MS‐DAD, GC‐MS)  First SFC method for analysis of mitragynine in plant material  elution (7 min) with 15% methanol as eluent
 mitragynine – resolved from speciogynine and speciociliatine

Example:

‐ Wang, M. et al., Comparison of three chromatographic techniques for the detection of mitragynine and other indole and oxindole alkaloids in Mitragyna speciosa (kratom) plants. J. Sep. Sci. 37 (2014) 1411‐1418.

Challenges

• GC Methods

• –  notcommonlyappliedforM.speciosa,particularlyinthewide

  variety of matrices encountered

• –  Hightemperaturesrequiredtoelutethealkaloids–restricts parameter adjustment for resolution of any alkaloid mixture
• –  inadequateresolutionofmitragynineandspeciociliatine
• –  Relianceonmass‐specificdetection–EI,ESI,andESIMS/MS spectra of mitragynine, speciogynine, and speciociliatine are nearly identical
• –  Interferenceofspeciociliatine–wouldprobablynotbedetected by either DAD or MS detectors
• –  Methodsshouldaccountforinterference
• –  derivatization may be necessary to overcome poor chrom. res.

Challenges

• LC Methods

• –  ClassicalLCwithbondedstationaryphasesandaqueous/organic

  eluents coupled to mass detection

• –  ViableforM.speciosaalkaloidsinplantmaterialandvarious fluids (Kikura‐Hanajiri, 2009)
• –  ResolutionofdiastereoisomersisOK(eluentpHdependentorder)
• –  MS/MSincreasessensitivityandselectivity
• –  problemswithmassspecificdetection(asstatedpreviously)
• –  Pureanalyticalstandards(laborintensiveisolationfromnatural products)

Challenges • SFC (plant material)

• –  SFCwithliquidcarbondioxidemodifiedwithorganicliquidaseluent
• –  Unexplored,effective
• –  Advantages of mass‐specific detection and MS/MS are applicable to SFC and HPLC
• –  RequireslessorganicliquidsthanHPLC
• –  Successfullyappliedforseparationofenantiomersand

  diastereoisomers, resolves diastereoisomer resolution in M. speciosa

• –  Faster,betterresolution,separationsorthogonaltoHPLC

Fitness for Purpose Statements

• Screening Method?
• Identification/Confirmation Method

Fitness for Purpose Statement “Screening Method”

The method must be able to screen known and unknown (unexpected) analytes of concern in various dietary supplement matrices.

No unknown analytes
Only mitragynine and 7‐hydroxymitragynine Not seeing economic adulteration
What does it buy you? Added cost, no bang FDA does not use screening here

Fitness for Purpose Statement

“AOAC Kratom Method”

The method must be able to identify known indole alkaloids, which adulterate the product, in a broad range of matrices, including dry and liquid finished products, concentrated extracts, and dry leaf material. The method should be able to quantitate the analytes for which appropriate standards are available and account for interfering compounds.

Questions for SPDS Discussion

“Identification/Confirmation Method”

• Arming decision making – (combination of alkaloids)
• Limit of quantitation?
• Range?
• Acceptable level(s) – Agency concern – below

• List of targeted analytes? Just 2 for now?

• Resolution of diastereoisomers?
• Handle wide variety of matrices?
• Cost of the analysis

QUESTIONS??

Thanks

NPA CEO and Executive Director Dr. Daniel Fabricant Darryl Sullivan
Dawn Frazier