new psychotropic drugs: a review

Introduction to NPS

Volker AuwärterInstitute of Forensic Medicine, Forensic Toxicology, Medical Center – University of Freiburg

BLT weekend – 21.-22. October 2016 – Hotel Bel Air, Echternach

New psychoactive substances (NPS)

Synthetic cannabinoidsHistory

1990s

Increasing popularity of ‘Spice’

as legal cannabis alternative

12/2008

CP-47,497 and JWH-018 identified in ‘Spice’ products

Valinamides(Pfizer patent

2009)AB-PINACA

Indazole core structureAKB-48

~2004

First ‘Spice’ products on the market

20132012

2008

N

O

OH

OH

2015

Carbazole core structureEG-018

N

N

ONH

N

N

ONH

NH2

O

N

O

AKB-48 AB-PINACA EG-018

~ 1965

Synthesis of structural THC

analogues

Discovery of CB1 and CB2

receptors

…

Synthetic cannabinoids: structural modifications

JWH-018

Ki (CB1): 9,0 nM

N

O

AM-2201

Ki (CB1): 1,0 nM

N

O

NAM-1220

Ki (CB1): 0,27 nM

N

O

N

N

N

O

Ki (CB1): ?

N

O

UR-144

Ki (CB1): 150 nM

+

2 x 3 x 7 x 14 = 588 possibilities

Methods of detection indrug material

Methods of detection

Drug material:

- Fast detection, on site(IMS or IR, no color tests available!)

- TLC(-DESI-MS), HPLC-DAD, LC-MS/MS, GC-FID, HRMS

- Gold standard: GC-MS

Problem: Quick identification of new compounds (NMR)

Thermal degradation of carboxylates

N

O

O N

Tsujikawa et al. 2014, Forensic Toxicology

PB-22

N

OO

NH

OO

OH

N

e db

Methods of detection inbiological matrices

n

n (

trac

es)

Med

ian

Mea

n(w

ith

ou

ttr

ace

s)

JWH-081 270 23 0.9 3.5

JWH-210 463 16 1.1 6.1

JWH-122 450 80 0.6 5.7

JWH-018 192 101 0.4 1.8

AM-2201 140 39 0.4 1.8

AB-CHMINACA 116 7 2.7 8.1

MDMB-CHMICA 225 53 0.5 2.9

ADB-CHMINACA 72 7 1.1 3.3

5F-ADB 20 1 0.4 1.3

Concentrations2008 - 2016

Synthetic cannabinoids – serum analysisPrevalence and serum concentrations

Synthetic cannabinoids –post-mortem blood concentrations

e.g. MDMB-CHMICA

Synthetic cannabinoids – urine analysis

N

O

Parent compounds usually notdetectable in urine samples

Detection of parent compounds

Synthetic cannabinoids – urine analysisMetabolism

N

O

JWH-018

N

O

OH

JWH-018 5-hydroxy-pentyl metabolite

O

OHO

OH

OH

OH

OH

O

N

O

O

O

OH

O

OHOH

OH

JWH-018 pentanoic acid glucuronide metabolite

Glucuronic acid

Phase I Phase II

Solubility in water

CYP 450 enzymesFurther oxidation,

glucuronyltransferase

in vitro

IsolatedCYP 450

isoenzymes

Phase Imetabolites

Human liver microsomes

(HLM)

Phase I (+II)metabolites

Human hepatocytes

Phase I+IImetabolites

in vivo

Animal model

Phase I+IImetabolites

Human samples

Phase I+II metabolites

Synthetic cannabinoids – urine analysisIdentification of metabolites

Method updating

HLM assay

(in vitro)

Preliminary main metabolites

(in vitro)

Urine-ScreeningPositive authentic

urine samples

Main metabolites

(in vivo)

New synthetic cannabinoid

Metabolite-identification(LC-MS/MS)

UpgradeLC-MS/MS

screening method

New knowledge of metabolic pathways

Optimized LS-MS/MS screening method

Metabolite-identification(LC-MS/MS)

Synthetic cannabinoids – urine analysis

HLM = Humanliver microsomes

Metabolism of synthetic cannabinoidsSome important facts

N

O

OH

N

O

OH

N

O

OH

N

O

OH

O

N

O

JWH-018

JWH-018 6-hydroxyindole

metabolite

JWH-018 4-hydroxy-

pentyl metabolite

JWH-018 5-hydroxy-

pentyl metabolite

JWH-018 pentanoic

acid metabolite

Most common metabolites

are hydroxylated and

carboxylated compounds!

Hydroxylation

Oxidation

Hydroxylation

Carboxylation

Hydroxylation

HydroxylationOxidation


Similar compounds

can lead to identical

metabolites!

N

O

N

O

F

N

O

OH

N

O

OH

O

N

O

OH

O

N

O

AM-2201JWH-018 JWH-073

JWH-018

5-hydroxypentyl

metabolite

JWH-018

pentanoic

acid metabolite

JWH-073

butanoic acid

metabolite

Hydroxylation

OxidationDecarboxylation +

hydroxylation/oxidation

CarboxylationHydrolytic

defluorination

N

O

N

O O

N

N

O


Unstable bonds in a compound can be cleaved

• by pyrolytic reactions (smoking)

• by enzymes in the organism

• during sample preparation or analysis

OH

N

N

O OH

+

UR-144

PB-22

Isomerisation

Ester

hydrolysis

Pitfalls

Synthetic cannabinoidsAnalytical pitfalls

MDMB-CHMICAC23H32N2O3

384.241293 Da

240

144

N

NHO O

O

100 200 300

5.0e7

1.0e8

1.5e8

240

144

3850.0

m/z [Da]

Intensity [cps]

BB-22C25H24N2O2

384.183778 Da

240

144

100 200 300

1.0e7

2.0e7

3.0e7

4.0e7

5.0e7

240

144

3850.0

m/z [Da]

Intensity [cps]

N

OO N

1. Same nominal mass and identical fragment ions

2. Isobaric compoundse.g. 5F-PB-22 vs. 5F-SDB-005

N

OO

F

N

5F-PB-22C23H21FN2O2

376.158706 Da

NN

OO

F

5F-SDB-005C23H21FN2O2

376.158706 Da

Synthetic cannabinoidsMetabolite vs. artefact

N

N

NHO OH

O

AB-CHMINACA Valine

N

N

NHO NH2

O

AB-CHMINACA

N

OHO

F

5F-PB-223-Carboxyindol

N

OO

F

N

5F-PB-22

∆T

∆T

External contamination via side-stream smoke!

Are immunoassays helpful?

Synthetic cannabinoids – immunoassayIA vs. LC-MS/MS

LC-MS/MS

confirmation

Positive Negative

IAPositive 1,0 % 0,5 %

Negative 49,0 % 49,5 %

THJ-018

N

N

O

N

N

ONH

NH2

O

AB-CHMINACA

N

N

ONH

NH2

O

F

AB-FUBINACA

N

OO

N

F

5F-PB-22ADB-CHMINACA

N

N

NHO NH2

O

MDMB-CHMICA

N

NHO O

O

AM-2201

N

O

F

Pharmacology and toxicology

Acute toxicity (clinical cases)

Most symptoms similar to cannabis intoxication- Tachycardia- Reddened eyes- Anxiousness- Mild sedation- Hallucinations, acute psychosis- Memory deficits

Symptoms not typically seen after cannabis intoxication

- Seizures- Hypokalemia - Hypertension- Nausea/vomiting- Coma- Agitation, violent behavior

Schneir 2012 J Med Tox

Rosenbaum et al. 2012 J Med Chem

Forrester et al. 2011 J Add Dis

Hermanns-Clausen et al. 2012 Addiction

Quantification of SC’s in drug material

HPLC-DAD:

Analysis of 313 packets (31 brands) from one single online retailer

‘Complete‘ analysis of 34 packets (21 brands)

Inter-package variability

313 packets analyzed (27 brands):

• Highest standard deviation in product “Summerlicious” (AM-2232): 26 – 100 mg/g (n=23)

• Five brands with the most packets investigated:

• MNK (n=55)

• Monkees go Bananas (M.G.B. n=33)

• Summerlicious (n=23)

• OMG (n=22)

• ACME (n=19)

Risks - Inhomogeneity

Product weight: 2 gAliquots: 200 mgSC: JWH-210Ki CB1: 0.46 ± 0.03 nM

Relative harm of drugs – Synthetic cannabinoids

Nutt et al., Drug harms in the UK: a multicriteria decision analysis, Lancet 2010

SC


ONH2

Cathinone

Designer stimulantsHistory

Phenethylamines

Amphetamine marketed as

‘Benzendrine’

MDPV(patent 1969)

PIHKAL (1995)

Alexander Shulgin

1893

First synthesis of methamphetamine

(Nagai)

Since 20081930s

Mephedrone(first synthesis 1929)

1920s

Use of methamphetamine as stimulant (medical?)

drug (‘Philopon’, later ‘Pervitin’)

1912

Merck patent:MDMA

1970s

MDMA self-experiments

1887

First synthesis of amphetamine

(Edeleanu)

NH2

NH

NHO

O

1980s

Ban of MDMA, -> wave of designer

stimulants

…

Relative harm of drugs – Stimulants


SC

Stimulants

Designer benzodiazepinesHistory

1957

First synthesis by Sternbach

(Chlordiazepoxid, Hoffmann-La Roche)

1960s

Market introduction of the first

benzodiazepines2011

First designer benzodiazepine

(pyrazolam)

Designer-Benzodiazepine

NH

N

O

NO2 Cl Br

e.g.

NO2: Nitrazepam 5-10 mgCl: Nordazepam 10 mg

H F Cl

e.g. (Cl at pos. 7)H: Diazepam 5 - 10 mgF: Fludiazepam 0,25 mgCl: Diclazepam 2 mg

Methyl- Cyclopropylmethyl- …

e.g.CH3: Diazepam 5 - 10 mgCPM: Prazepam 10-20 mg

(Prodrug)

e.g. H: Nordazepam 10 mgOH: Oxazepam 20 mg

H OH CH3

CH3

z.B.Nordazepam 10 mg

H: Estazolam 1 - 2 mgCH3: Alprazolam 0,5 mg

Structure-activity-relationships (SAR)

N

N

N

N

Cl

Drug design and potency

NN+

NH

O-

O

O

NN+

NH

O-

O

S

NN+

N

O-

O

NH

NH2

NN+

N

O-

O

N

N

P4S10 (H2N)2 MeC(OMe)3

Nitrazepam Nitrazolam

5-10 mg ~ 1 mg

Flubromazepam Flubromazolam ca. 5,0 vs. 0,2 mg

Clonazepam Clonazolam ca. 0,5 vs. 0,2 mg

Bromazepam Pyrazolam ca. 5,0 vs. 2,0 mg

Dosing: drugs.tripsit.me

Interpretation of the analytical results

1.) Metabolites of prescription benzodiazepines sold as research chemicals

• Nifoxipam (Metabolite of Flunitrazepam)• Fonazepam (= Norflunitrazepam) (Metabolite of Flunitrazepam)• Desalkylflurazepam (Metabolite of Flurazepam)• 3-OH-phenazepam (Metabolite of Phenazepam)

2.) Metabolites of designer benzodiazepines identical to prescription benzodiazepines (metabolites)

• Diclazepam (Lorazepam, Lormetazepam, Delorazepam)• Adinazolam (α-OH-alprazolam, estazolam)• Cloniprazepam (Clonazepam, 7-Aminoclonazepam)

Immunoassay – Benzodiazepines

• Cross-reactivities usually sufficient

• LC-MS/MS method up-to-date?

• Serum: CAVE: highly potent compounds(e.g. Flubromazolam)

• Urine: Forensic cut-offs vs. clinical cut-offs

• Advantage: Repeated uptake accumulation

Immunoassay – Benzodiazepines

Relative harm of drugs – Benzodiazepines


SC

Ben

zod

iaze

pin

es

Stimulants

Drug design - Phenidates

Methylphenidate

3,4-CTMP

Methylnaphtidate (HDMP-28)

4-Fluoro-methylphenidate4-Methyl-methylphenidate

Ethylphenidate

NH

O O

NH

O O

NH

O O

F

NH

O O

Cl

Cl NH

O O

NH

O O

Ethanol consumption

Pitfalls phenidatesArtefact methylphenidat-transesterification

2 4 6 8 10 12 14 16 18 20 22 24 26

2.0e5

6.0e5

1.0e6

1.4e6

1.8e6

2.2e6

2.6e6

3.0e6

3.4e6

3.8e6

4.2e6

4.6e6

Intensität [cps]

Zeit [min]

Methylphenidate

Isopropylphenidate

2 4 6 8 10 12 14 16 18 20 22 24 26

2.0e4

6.0e4

1.0e5

1.4e5

1.8e5

2.2e5

2.6e5

3.0e5

3.4e5

3.8e5

Zeit [min]

Intensität [cps]

O

O

NH

O

O

NH

Sample prep:

Automatic SPE

Restriction (N2, 40°C)

AdditionIsopropanol:HCl (3:1)

Evaporation to dryness

Reconstitution

Dissociatives

Ketamine Methoxetamin PCP

NH

O

Cl

O

NH

O

N

O

N

Methoxphenidine

Dissociative anestheticsHistory

19621950s

First synthesis of ketamine (US)

2013

Phencyclidine (PCP),developed as an

i.v. anesthetic (US) Methoxphenidine

2010

Methoxetamine(patented 1966)

1960s

PCP misuse

Relative harm of drugs – Dissociatives


SC

Dis

soci

ativ

e

LSD

-an

alo

gue

s

Ben

zod

iaze

pin

es

Stimulants

OpioidsHistory

1930s

First synthesis of VA 10820, later marketed as methadone, and pethidine

(I.G. Farben, Hoechst)

1970s … … …

1960

Synthesis of Fentanyl

1960s/70s

Discovery and characterization of

the opioid receptors

1968

Buprenorphine patented

AH-7921, U-47700 (US)

Opioids

Fentanyl derivatives

e.g. furanyl-fentanyl nasal spray (0.4 mg per stroke)

Opioids

e.g. U-47700

Relative harm of drugs – Opioids


SC

Opioids

Dis

soci

ativ

e

LSD

-an

alo

gue

s

Ben

zod

iaze

pin

es

Stimulants

Interpretation of (qualitative/quantitative) results

-> semi-quantitative estimation does make sense;

fully validated quantitation not really necessary

Prevention work / legislation

New concepts needed

Prevention: web-based approaches, analytical methods (!), drug checking

Legislation:

- Substance-by-substance approach: slow, ineffective

- Generic definition of structural drug classes: much better, but may be

circumvented by more exotic structures (e.g. indole -> indazole ->

carbazole -> …) with unknown additional risks

- ‘Analogue’ laws (focusing on effects rather than structure): vague, may

be challenged in court, constitutional restraints (principle of legal

certainty)

- Decriminalization of drug users / focus of repression on supply chains

- Strictly regulated, legal market for less harmful drugs (New Zealand)?

Résumé

- Most NPS originate from pharmaceutical research

- Many NPS pose a serious threat to public health (potency, toxicity,

missing declaration)

- NPS give a lot of trouble to forensic toxicologists (analysis,

interpretation)

- Prevention measures and legal regulations have to be adapted

- Some of the new drugs seem to be less harmful than classic illicit

drugs with similar effects

- There might be some useful therapeutic drugs among the new

compounds

new psychotropic drugs: a review

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