Evaluating the abuse potential of psychedelic drugs as ...€¦ · drugs with mainlycentralnervoussystem action, and with effects said to be the expansion or heightening of consciousness,

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Neuropharmacology

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Invited review

Evaluating the abuse potential of psychedelic drugs as part of thesafety pharmacology assessment for medical use in humans

David J. Heal*, Jane Gosden, Sharon L. SmithRenaSci Ltd, BioCity, Nottingham, NG1 1GF, UK

a r t i c l e i n f o

Article history:Received 7 November 2017Received in revised form7 January 2018Accepted 31 January 2018Available online xxx

Keywords:Psychedelic5-HT2A agonistNMDA antagonistk-Opioid agonistDrug-discriminationSelf-administrationDependenceAbuse testing

Abbreviations: 5-APB, 5-(2-aminopropyl)-benzofudimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine;bromophenethylamine; 2C-C, 2,5-dimethoxy-4-chloroation and Research; 2C-E, 2,5-dimethoxy-4-ethylphenN, 2,5-dimethoxy-4-nitrophenethylamine; 25CN-NBcarbaxypiperazine-4-yl) propyl-1-phosphonic acid; Cpylthiophenylethylamine; D-CPP'ene, D-3-(2-carboxypand Human Services; DiPT, N,N-di-isopropyltryptamidimethoxy-4-chloroamphetamine; DOI, 2,5-dinitrophenylisopropylamine; DPT, dipropyltryptamineand Drug Administration; FR, fixed ratio; GLP, Good LInternational Committee on Harmonisation; 25I-dimethoxy-4-iodophenylethylamine; 25I-NBD, N-(2,3intravenous self-administration; LSD, lysergic acid dMDA, 3,4-methylenedioxyamphetamine; MDE, N-momethylenedioxypyrovalerone; 5-MeO-DMT, 5-metho[MMAI], 5-methoxy-6-methyl-2-aminoindan; MMAPMPTIQ, 8-(2-methoxyphenyl)-1,2,3,4-tetrahydroisoqumethyl-D-aspartate; N,N-DMT [DPT], N,N-dimethyltryDevelopment; 8-OH-DPAT, 8-hydroxy-2-dipropylamiratio; 1-PTIQ, 1-phenyl-1,2,3,4-tetrahydroisoquinolineserotonin/noradrenaline reuptake inhibitor; SSRI, seleD9-THC, D9-tetrahydrocannabinol; WDS, wet dog sha* Corresponding author.

E-mail addresses: [email protected] (D.J. H

https://doi.org/10.1016/j.neuropharm.2018.01.0490028-3908/© 2018 Published by Elsevier Ltd.

Please cite this article in press as: Heal, D.Jassessment for medical use in humans, Neu

a b s t r a c t

Psychedelics comprise drugs come from various pharmacological classes including 5-HT2A agonists, indirect5-HT agonists, e.g., MDMA, NMDA antagonists and k-opioid receptor agonists. There is resurgence indeveloping psychedelics to treat psychiatric disorders with high unmet clinical need. Many, but not all,psychedelics are schedule 1 controlled drugs (CDs), i.e., no approvedmedical use. For existing psychedelics indevelopment, regulatory approval will require a move from schedule 1 to a CD schedule for drugs withmedical use, i.e., schedules 2e5. Although abuse of the psychedelics is well documented, a systematicpreclinical and clinical evaluation of the risks they pose in a medical-use setting does not exist. We describethe non-clinical tests required for a regulatory evaluation of abuse/dependence risks, i.e., drug-discrimination, intravenous self-administration and physical dependence liability. A synopsis of the exist-ing data for the various types of psychedelics is provided and we describe our findings with psychedelicdrugs in thesemodels. FDA recently issued its guidance on abuse/dependence evaluation of drug-candidates(CDER/FDA, 2017). We critically review the guidance, discuss the impact this document will have on non-clinical abuse/dependence testing, and offer advice on how non-clinical abuse/dependence experimentscan be designed tomeet not only the expectations of FDA, but also other regulatory agencies. Finally, we offerviews on how these non-clinical tests can be refined to provide more meaningful information to aid theassessment of the risks posed by CNS drug-candidates for abuse and physical dependence.

© 2018 Published by Elsevier Ltd.

ran; 6-APDB, 6-(2-aminopropryl)-2,3-dihydrobenzofuran; 25BN-BOMe [NBOMe-2C-B, BOM-2CB], 2-(4-bromo-2,5-BZP, 1-benzylpiperazine; m-MeO-BZP, 1-(3-methoxybenzyl)-piperazine; 2C-B, 2,5-dimethoxy-4-

phenethylamine; 2C-D, 2,5-dimethoxy-4-methylphenethylamine; CD, controlled drug; CDER, Center for Drug Evalu-ethylamine; CHMP, Committee for Medical Products for Human use; 2C-I, 2,5,dimethoxy-4-iodophenethylamine; 2-C-OH, 2-([2-(4-cyano-2,5-dimethoxyphenyl)ethylamino]methyl)phenol; CNS, central nervous system; CPP, (±)-3-(2-SS, Controlled Substance Staff; 2C-T-2, 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-7, 2,5-dimethoxy-4-(n)-pro-iperazine-4-yl)-1-propenyl-1-phosphonic acid; DEA, Drug Enforcement Administration; DHHS, Department of Healthne; 2,5-DMA, 2,5-dimethoxyphenylisopropylamine; DOB, 2,5,dimethyoxy-4-bromophenylisopropylamine; DOC, 2,5-methoxy-4-iodoamphetamine; DOM, 2,5-dimethoxy-4-methylamphetamine; DON, 2,5-dimethyloxy-4-; EDA, 3,4-ethylidenedioxyamphetamine; EMA, European Medicines Agency; 4-FA, 4-fluoroamphetamine; FDA, Foodaboratory Practice; 5-HTP, 5-hydroxytryptophan; MHRA, Medicines and Healthcare products Regulatory Agency; ICH,NBOH, (2-({[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]amino}methyl)phenol); 25I-NBOMe, N-(2-methoxybenzyl)-2,5--methylenedioxybenzyl)2-5-dimethoxy-4-iodophenylethylamine; IDA, 3,4-isopropylidenedioxyamphetamine; IVSA,iethylamide; MBDB, 3,4-methylenedioxy-N-methyl-a-ethylphenylethylamine; mCPP, 1-(m-chlorophenyl)-piperazine;noethyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane; MDMA, 3,4-ethylenedioxymethamphetamine; MDPV, 3,4-xy-N,N-dimethyltryptamine; 3-MeO-PCP, 3-methoxy-phencyclidine; 4-MeO-PCP, 4-methoxy-phencyclidine; MMA, 1-(3-methoxy-4-methyphenyl)-2-aminopropane; 1-MPTIQ, 1-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline; 8-inoline; NFLIS, National Forensic Laboratory Information System; NIH, National Institute for Health; NMDA, N-ptamine; N-OH-MDA, N-hydroxy-1-(3,4-methylenedioxyphenyl)-2-aminopropane; OECD, Economic Co-operation andnotetralin); PCP, phencyclidine; PCPA, p-chlorophenylalanine; pCPP, 1-(p-chlorophenyl)-piperazine; PR, progressive; PTSD, post-traumatic stress disorder; Ro 60e0175, (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine; SNRI,ctive serotonin reuptake inhibitor; TEDS, Treatment Episode Data Set; TFMPP, 1-(3-trifluoromethylphenyl)-piperazine;kes.

eal), [email protected] (J. Gosden), [email protected] (S.L. Smith).

., et al., Evaluating the abuse potential of psychedelic drugs as part of the safety pharmacologyropharmacology (2018), https://doi.org/10.1016/j.neuropharm.2018.01.049

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D.J. Heal et al. / Neuropharmacology xxx (2018) 1e272

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Which drugs are psychedelic? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. 5-HT2A and 5-HT2C receptor agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. NMDA receptor antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Opioid k-receptor agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Non-clinical safety pharmacology evaluation of abuse and dependence risks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1. Chemical structure analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. In vitro receptor screening of drug-candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. Behavioural screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.3.1. The Irwin Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3.2. Head-twitch and wet dog shakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Specific regulatory behavioural tests to assess abuse and dependence potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1. Good Laboratory Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2. Drug exposure levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.3. Positive Controls and Reference Comparators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.4. Role of gender in abuse/dependence evaluations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.5. Coordination between non-clinical and clinical assessments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.6. Guidelines contain advice not instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.7. Drug-discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.7.1. Technical matters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.7.2. Key points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.8. Intravenous self-administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.8.1. Technical matters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.8.2. Conditioned place preference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.8.3. Key points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.9. Withdrawal-induced physical dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.9.1. Technical matters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.9.2. Key points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

The resurgent interest in developing psychedelic drugs to treatpsychiatric disorders is welcomed. There is unmet need in majordepressive disorder (including treatment-resistant depression andterminal illness depression), anxiety, substance use disorders, andpost-traumatic stress disorder (PTSD). Most psychedelic drugs areclassified as schedule 1 (C-I) controlled drugs (CDs), ie they have norecognised medical use, and carry a high risk for abuse and/or theinduction of psychological or physical dependence. For the existingpsychedelic drugs, regulatory approval will have to be accompaniedby a change from C-I to one of the CD schedules assigned to abuseddrugs that have an approved medical use, i.e., schedules 2 to 5 (C-IIto C-V) in the USA and UK. If the early promise of some psychedelicsin treating previously unmanageable psychiatric conditions isconfirmed in randomised, placebo-controlled, double-blind, pivotaltrials, there is every likelihood that they will receive regulatoryapproval and enter the formularies in the USA and Europe. The nextphase in pharmaceutical research and development will be todiscover and clinically evaluate novel psychedelic compounds thatcan be patent-protected. Patent protection is essential for phar-maceutical companies to justify the enormous cost of non-clinicaland clinical development to take a new drug to the market.

Evaluating the risks of abuse and withdrawal-induced physicaldependence is a mandatory part of the Safety Pharmacologyassessment for all new drugs for human use which have pharma-cological effects in the central nervous system (CNS). Guidancedocuments describing the procedures for the non-clinical evalua-tion of abuse/dependence potential have been issued by the

Please cite this article in press as: Heal, D.J., et al., Evaluating the abuseassessment for medical use in humans, Neuropharmacology (2018), http

Committee for Medical Products for Human use (CHMP) of Euro-pean Medicines Agency (EMA) in 2006 and by the Center for DrugEvaluation and Research (CDER) of the US Department of Healthand Human Services (DHHS) of Food and Drug Administration(FDA) in draft form in 2010 and very recently in final form in 2017(CHMP/EMA, 2006; CDER/FDA, 2010, 2017).

We will outline the non-clinical testing programme to deter-mine (or more correctly predict) the risks of abuse and/or depen-dence that novel psychedelic drugs need to undergo as part of theregulatory approval process. The wealth of information on thepsychoactive properties of existing psychedelic drugs provides asolid foundation to evaluate the established non-clinical abuse/dependence testing paradigms, including their strengths, weak-nesses and potential pitfalls. Finally, we offer some opinions onhow to address the unique challenges for the non-clinical abuse/dependence testing that are posed by the psychedelic drugs.

2. Which drugs are psychedelic?

According to the Oxford English Dictionary, the word “psyche-delic” “relates to, or denotes, drugs (especially lysergic acid dieth-ylamide [LSD]) that produce hallucinations and apparent expansion ofconsciousness”. The Meriam-Webster Medical Dictionary definespsychedelic as “relating to, or being, drugs (as LSD) capable of pro-ducing abnormal psychic effects (as hallucinations) and sometimespsychotic states”. However, the MediLexicon Medical Dictionarydefines psychedelic as “pertaining to a rather imprecise category ofdrugs with mainly central nervous system action, and with effects saidto be the expansion or heightening of consciousness, LSD, hashish,

potential of psychedelic drugs as part of the safety pharmacologys://doi.org/10.1016/j.neuropharm.2018.01.049

D.J. Heal et al. / Neuropharmacology xxx (2018) 1e27 3

mescaline, psilocybin”. From these three diverse definitions, it isclear that there is no precise or agreed terminology for defining theterm psychedelic. The ability of the drug to produce hallucinationsand mind expanding experiences is often regarded as a funda-mental property of the psychedelics. Some researchers, eg Glennon(1999), have proposed that the term “hallucinogen” should berestricted to drugs with affinity for the 5-HT2A receptor and whichproduce full generalisation to the hallucinogenic, 5-HT2 agonist,2,5-dimethoxy-4-methylamphetamine (DOM).

A search of the on-line illicit drug forums and chat rooms, e.g.,Bluelight Forum (http://www.bluelight.org/vb/forum.php), ERO-WID (https://www.erowid.org) and Drugs-Forum (https://drugs-forum.com), revealed a similar lack of consensus on what consti-tutes a psychedelic drug with many contributors including otherdrugs from other pharmacological classes that induce hallucina-tions and mind-expanding experiences as having psychedelicproperties, e.g., phencyclidine (PCP) and ketamine (NMDA antag-onists), 3,4-methylenedioxymethamphetamine (MDMA) (5-HT anddopamine releasing agent) and salvinorin A (k-opioid receptoragonist).

When assessing the risk of abuse posed by a known or novelpsychedelic drug, the definitive evidence will come not from pre-clinical or clinical experiments, but from events after the drug isapproved and available in the market. It is the drug abusing com-munity, which drives the process by self-experimentation, infor-mation sharing and creating demand that illicit suppliers seek tosatisfy. In turn, they generate the “real world” data on abuse lia-bility. For this reason, we have also taken the opinions of the drugabusing community into account and used a broad definition of“psychedelic” that includes the following pharmacological classesof drug.

1. 5-HT2 agonists including 5-HT2A agonists and 5-HT2C agonists,e.g., lorcaserin.

2. Indirect 5-HT2 agonists including MDMA (5-HT and dopaminereleasing agent) and fenfluramine and dexfenfluramine (5-HTreleasing agents).

3. NMDA antagonists, i.e., phencyclidine and ketamine.4. The k-opioid receptor agonists, i.e., salvinorin A and

(�)-pentazocine.

2.1. 5-HT2A and 5-HT2C receptor agonists

The first serotonergic hallucinogens to be discovered werenaturally occurring chemicals present in plants, e.g., mescalinefrom the peyote cactus (Lophophora williamsii), psilocybin, a psy-chedelic prodrug produced by more than 200 species of mush-rooms, collectively known as psilocybin mushrooms (most potentare members of the Psilocybe genus, e.g., P. azurescens, P. semi-lanceata, and P. cyanescens), and N,N-dimethyltryptamine (N,N-DMT) that is present in the plant derived concoction, “ayahuasca”.

The first serotonergic hallucinogen was synthesised by AlbertHofmann while working for Sandoz in Switzerland in 1938. Hesynthesised LSD while studying the chemistry of alkaloid com-pounds contained in ergot, a fungus which forms on rye. The lab-oratory name for the compound was the acronym for the German“Lyserg-s€aure-di€athylamid”, followed by a sequential number“LSD-25”. Albert Hofmann also became the first person to experi-ence the psychedelic properties of LSD when he accidentallyingested the drug in April, 1943. LSD was introduced as a com-mercial medication under the trade-name “Delysid” for variouspsychiatric uses in 1947.

These drugs can be chemically classified in two main structuralclasses, i.e., the phenylethylamines, e.g., mescaline, and


indoleamines, e.g., psilocybin and 5-methoxy-N,N-dimethyltryp-tamine (5-MeODMT), with the indoleamines also including theergolines, e.g., LSD, that possess the indoleamine moiety within thelarge multi-ring structure of the molecule.

Corne and Pickering (1967) proposed head-twitches in mice as abehavioural marker of hallucoinogenic properties with robust in-duction of this behaviour by LSD, mescaline and psilocybin. Afterthe discovery of the 5-HT receptor super-family and the division of5-HT2 receptors into three subtypes, 5-HT2A, 5-HT2B, and 5-HT2c(formerly classified as 5-HT1C) (Hoyer et al., 1994), it was soonestablished that head-twitches are mediated through activation ofthe 5-HT2A receptor subtype (for a more comprehensive descrip-tion of the evidence see later section on Head-Twitches and WetDog Shakes). In turn, this led to the hypothesis that the 5-HT2Areceptor is the hallucinogenic or psychedelic receptor. Although thehypothesis is supported by animal and human experimentationwith various novel, selective 5-HT2A receptor agonists, the story isnot quite that simple.

We have not included an extensive description of the humanpharmacology of the psychedelics because this topic is compre-hensively described by Sellers et al. (2017) in another review in thisSpecial Issue.

2.2. NMDA receptor antagonists

PCP is a non-competitive NMDA antagonist that was first syn-thesised at Parke-Davis in the 1950s and subsequently approved asa new and safe anaesthetic for human use (trade names: “Sernyl”and “Sernylan”). PCP's very long half-life did not make ideal as ananaesthetic. Its use was marred by serious side-effects includinghallucinations, sensory distortions, “out of body” experiences,mania, delirium and psychotic-like side-effects, which resulted inits withdrawal from formularies in 1965. PCP's use is currentlyrestricted to veterinary medicine. In the 1960s, PCP was abused forits hallucinogenic properties on the Californian drug scene where itwas either sold as “angel dust” or misrepresented as one of theother popular hallucinogens, e.g., LSD, mescaline or psilocybin.Anecdotally, the experiences of PCP were more likely to bedysphoric than enjoyable which meant it never achieved the samepopularity as the serotonergic hallucinogens. PCP abuse has beenassociated with violence, self-harm, suicide and florid psychosislasting for days (Lerner and Burns, 1978; Jacob et al., 1981; Isaacset al., 1986). In addition, deaths due to intoxication and drug-fuelled violence have also been reported (Burns and Lerner, 1978;Poklis et al., 1990). PCP abuse of reached its zenith in the 1970sand 1980s and then declined, but it has re-emerged with reports ofabuse of the PCP analogues, 3-methoxy-phencyclidine (3-MeO-PCP) and 4-methoxy-phencyclidine (4-MeO-PCP), in Sweden(B€ackberg et al., 2015).

Ketamine, another non-competitive NMDA antagonist, was firstsynthesized in 1962. Its ability to induce a trance-like state whileproviding pain relief, sedation and memory loss led to its devel-opment as a dissociative anaesthetic by Parke-Davis under thetrade name, “Ketelar”. Ketamine's short duration of action, reducedbehavioural toxicity combined with lack of cardiovascular andpulmonary effects made it preferable to PCP for clinical use. Keta-mine was widely used as an anaesthetic for US soldiers undergoingtrauma surgery during the VietnamWar. It is still widely used as ananaesthetic in many developing countries. Ketamine's utility intreating disorders including treatment-resistant depression(DiazGranados et al., 2010; Murrough et al., 2013; Singh et al.,2016), PTSD (Feder et al., 2014) and alcohol dependence(Krupitsky and Grinenko, 1997) is also under investigation. LikePCP, ketamine abuse started on the West Coast in the 1970s. Shramet al. (2011) studied the effects of orally administered ketamine in a


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group of recreational polydrug users. The subjects reported effectsof positive experience, e.g., drug liking, perceptual effects, e.g.,floating, detached, hallucinating, and sedation. An upsurge in theabuse of ketamine occurred with the growth of the “club” and“rave” scenes in the 1990s. Ketamine is abused by smoking, inha-lation or injection. Heavy ketamine abuse is associated withdysfunction and permanent damage to the bladder (Tsai et al.,2009; Mason et al., 2010; Gray and Dass, 2012), upper gastro-intestinal damage through ketamine inhalation (Poon et al.,2010), liver damage (Wong et al., 2014), and psychosis (Lim,2003). More recent trends that have accompanied the emergenceof “legal highs” include the abuse of the novel, non-competitiveNMDA antagonist, methoxetamine; being promoted as ketaminewithout the bladder damage (Corazza et al., 2013; Craig andLoeffler, 2014).

2.3. Opioid k-receptor agonists

For many years k-opioid agonists were considered to have nohuman abuse potential because their effects were dysphoric andthe accompanying perceptual distortions and hallucinations wereoften scary and discomforting.

Pentazocine was synthesised as part of a deliberate effort todevelop an effective analgesic with little or no abuse potential. Itcombines k-opioid agonist, very weak m-opioid partial agonist (orantagonist) and s receptor antagonist properties. Pentazocine isused clinically for the management of intense pain. Althoughpentazocine was not initially a CD, reports of pentazocine abuse inthe 1970s (e.g., Chambers et al., 1971) prompted the manufacturerto voluntarily accept C-IV in USA and UK. Pentazocine is a racematewith k- and m-opioid activity residing in the (�)-enantiomer and s-antagonist activity in the (þ)-enantiomer. Studies in human vol-unteers reveal that pentazocine produces subjective effects of“high”, “liking” and “good drug effects” at low doses of 7.5e45 mg,but at higher doses dysphoric effects, e.g., “nervous” and “bad ef-fects”, start to appear (Jasinski et al., 1970; Preston et al., 1987;Zacny et al., 1998).

Salvinorin A is a naturally occurring hallucinogen present in theresin secreted by the leaves of the sage, salvia divinorum. SalvinorinA is a selective, high potency, high efficacy, k-opioid receptoragonist (Roth et al., 2002; Chavkin et al., 2004). The antinociceptiveand hypothermic effects of salvinorin A are abolished in k-opioidreceptor-1 knockout mice (Ansonoff et al., 2006). The role of sal-vinorin A as the mediator of the pharmacological effect of salviadivinorum is also supported by the similarity between the phar-macokinetics of this potent hallucinogen in primates, i.e., braintmax¼ 4s and a very short half-life of 8min, and the rapid onset andshort duration of its effects in man (Hooker et al., 2008). Whensalvia divinorum leaves are smoked liberated salvinorin A producesa rapid and intense hallucinatory effect including a “highly modi-fied perception of external reality” that typically lasts between 10and 15min (Gonz�alez et al., 2006). Reports by salvinorin A abusersthat it produces positive after-effects (increased insight andimproved mood) which last >24 h (Baggott et al., 2010) haveincreased its popularity as a recreationally abused drug. Although itis legal in the USA and in other countries to purchase salviadivinorum from internet suppliers, there is concern about thehealth risks posed by its abuse. Twenty-three US states have passedlegislation controlling the use and sale of salvia divinorum (Siebert,2010), 15 of which have made possession or use of this plant illegal.Furthermore, the US Drug Enforcement Administration (DEA)regards salvia divinorum as a “drug of concern” (Brown, 2009).

Enadoline (CI-977) is a high affinity, selective, k-opioid receptoragonist. The Ki of enadoline for k-receptors is 0.11 nM and it shows~900 and ~9000-fold selectivity versus the m- and d-receptor


subtypes, respectively (Hunter et al., 1990). The compound alsolacks affinity for s and PCP binding sites (Hunter et al., 1990).Enadoline is a valuable source of information because it is highlyselective k-opioid agonist, and therefore, its effects cannot beascribed to actions at either s or PCP binding sites. Furthermore, itssubjective effects have been studied normal human volunteers anddrug-experienced subjects. Enadoline produces dose-related re-ports of “dizziness”, “emotional lability”, “feeling high” and“thinking abnormal” that peak at 30min (Reece et al., 1994). Vol-unteers reported perceptual changes in all five senses with thepossible exception of smell. Scores for the questions “experiencinga high?” and “are the drug effects bad?” increased with enadolinedose. Enadoline was compared with butorphanol and hydro-morphone in drug-experienced volunteers (Walsh et al., 2001a)and its influence on the pharmacodynamics and self-administration of cocaine have been investigated (Walsh et al.,2001b). The psychotomimetic effects of the highest dose of ena-dolinewere severe enough for it to be excluded from the remainderof the clinical trial. Lower doses of enadoline produced dose-dependent “highs”, but not “good effects” or “drug liking”; on thecontrary, enadoline dose-dependently induced “bad drug effects”.Volunteers also reported perceptual distortions and sedation. Thesubjective experience evoked by enadoline differed in almost allrespects from those evoked by the mixed m/k-opioid receptor par-tial agonist, butorphanol, and the m-opioid full agonist, hydro-morphone. The clinical development of enadoline was abandonedbecause of its unacceptable adverse event profile.

From this overview, it is evident that there are substantial dif-ferences between the subjective effects evoked by pentazocine,salvinorin A and enadoline. However, these k-opioid agonistsclearly have the ability to induce dissociative experiences andchanges in sensory perception, but the dose range for a positiveexperience appears to be limited. Even in drug-experienced sub-jects, excessive doses of these drugs produce psychotomimetic anddysphoric effects that are profoundly unpleasant and aversive.

3. Non-clinical safety pharmacology evaluation of abuse anddependence risks

The non-clinical abuse/dependence evaluation of novel drugcandidates explores 3 distinct aspects of risk.

1. The psychoactive profile of the drug-candidate. It answers thequestion whether the drug-candidate elicits an interoceptivecue that is similar or identical to those produced by knownrecreational substances of abuse. This is assessed by the drug-discrimination test.

2. A determination of whether the drug-candidate producesrewarding psychoactive effects (positive reinforcement) whichcould lead to drug-seeking and/or dose-escalation. This isevaluated by intravenous self-administration (IVSA) testing, oroccasionally by the conditioned place preference (CPP) test.

3. The propensity of the drug-candidate when given repeatedly toinduce physical dependence on withdrawal with or withoutpharmacological tolerance.

Non-clinical abuse/dependence testing predicts the risks posedto patients, who have been prescribed the drug (often referred to as“intended population”), and misuse or abuse by persons, who havenot been prescribed the drug, and use it for non-medical purposes(often referred to as “unintended population”).

The potential risks for abuse and/or dependence do not gener-ally impinge on the “benefit/risk” assessment performed by regu-latory agencies when deciding whether to approve a new drug forhuman use e rather it determines the level of additional



supervision and restriction needed to ensure that its abuse/dependence risks are appropriately managed. Hence, it is generallya topic for labelling rather than marketing approval. If a new CNS-active drug poses significant risks for abuse/dependence, they aremanaged by designating it as a CD. The risks are further managedby applying different levels of restriction on manufacture, distri-bution and prescribing via CD scheduling. FDA uses the ProductLabel and EMA the Summary of Product Characteristics to informpatients and healthcare professionals of the risks associated withthe misuse and abuse of product. FDA may impose risk manage-ment strategies to enhance the safe use of a drug (see Calderonet al., 2015; Calderon and Klein, 2017 and CDER/FDA, 2017 for amore in depth discussion of this topic).

The terms “assessment” and “evaluation” have been usedextensively in this Introduction. It should be emphasised that non-clinical and clinical assessments of abuse risks are predictions notoutcomes. As techniques and models in abuse/dependenceassessment have become more sophisticated, their predictive val-idity has improved substantially; however, the final arbiter ofwhether a drug poses a risk for abuse is what happens after it hasbeen introduced into the formulary and prescribed for severalyears. As described in the CDER/FDA Guidelines (CDER/FDA, 2017),the actual abuse risk of new CNS-active drugs can be determined inthe post-marketing phase through a wide variety of tools includingthe FDA's Adverse Events Reporting System (FAERS), the WorldHealth Organisation (WHO), VigiBase the National Survey of DrugUse and Health (NSDUH), the Treatment Episode Data Set (TEDS),the DEA's National Forensic Laboratory Information System (NFLIS)and the monitoring of internet forums and social media websiteswhere recreational drug abuse experiences are shared and dis-cussed. Similar resources are available in Europe, e.g., the EuropeanMonitoring Centre for Drugs and Drug Addiction (www.emcdda.europa.eu) and surveys conducted by the Home Office in the UK(www.gov.uk/government/collections/drug-misuse-declared).

Although the objectives and major principles of abuse/depen-dence evaluations are universally accepted, the requirements fordifferent regulatory agencies, e.g., FDA (USA), EMA (EuropeanUnion and affiliates) and Pharmaceuticals and Medical DevicesAgency (Japan), can vary markedly. Calderon et al. (2015) recentlypublished a comparative review on the similarities and differencesbetween non-clinical abuse/dependence evaluations for regulatoryagencies in USA and European Union. This review covers the topicin depth, but as it was published prior to the final FDAGuidelines onabuse/dependence evaluation in 2017; an updated synopsis of theFDA and EMA requirements is provided in Table 1.

For psychedelic drugs like psilocybin (C-I), it is well known thatthis drug has been subjected to abuse over many years, andtherefore, the risks it poses to abusers and society are established.This “real world” information provides a solid evidence base onwhich CSS can make a recommendation for the appropriate level ofCD restriction if the drug is approved for medical use in the USA. InEurope, the situation is more complex because CD scheduling isdetermined not by EMA, but in each individual European countrywhere the drug is registered. In the case of ketamine, which isalready clinically approved as an anaesthetic, its CD scheduling isalready established (C-III in USA) andwill remain unchanged unlessthere is evidence to suggest that the level of abuse is likely to in-crease. As an example, ketamine in the UKwas designated under C-IV, Part 1, but in 2015 it was upgraded to C-II because of an upturnin the level of diversion and abuse.

Novel psychedelic, drug-candidates for human use will have toundergo the standard package of non-clinical and clinical assess-ments of abuse and dependence risks with the evidence being usedto determine the appropriate CD scheduling (it is extremely un-likely that any psychedelic drug would not be classified as a CD).


3.1. Chemical structure analysis

The first level of evaluation is to determine if the drug-candi-date's chemical structure is related to that of a known substance ofabuse. For novel psychedelics, it is very probable that there will bestructural similarities to existing drugs, especially as these drugsmay have been used as chemical scaffolds to synthesise the newmolecule.

3.2. In vitro receptor screening of drug-candidates

As a precursor to experiments in animals, the drug-candidateshould be profiled in vitro for its affinity for a range of moleculartargets that mediate the actions of known substances of abuse.

� Dopamine receptors� Serotonin receptorse 5-HT2A and 5-HT2C receptors will be partof the screen.

� g-Aminobutyric acid (GABA)/benzodiazepine receptors� Opioid receptors e k-opioid receptors will be part of thescreen.

� Cannabinoid receptors� Nicotine receptors� N-methyl-D-aspartate (NMDA) receptors� Ion-channel complexes� Transporter sites e serotonin and dopamine reuptake trans-porters will be part of the screen.

For novel psychedelic drug-candidates, e.g., a novel 5-HT2Aagonist, NMDA antagonist or k-opioid receptor agonist, affinity andfunction at one of these molecular targets will be the pharmaco-logical mechanism responsible for its postulated therapeutic effect.However, it is also possible that the molecule may also havepharmacological affinity other abuse-related molecular target(s).The compound's affinity for these other abuse-related moleculartargets should be determined and it is also essential to characterisethe ligand-target functional interaction. As an obvious example, adrug-candidate that is a 5-HT2A receptor agonist poses verydifferent risks for abuse than one which is a 5-HT2A receptorantagonist.

3.3. Behavioural screening

3.3.1. The Irwin ProfileThe Irwin Profile is a broad assessment of CNS-mediated effects

through a series of behavioural and motor assessments in rats (fordetails see Roux et al., 2005; Fonck et al., 2015). The Irwin test isuseful because novel drug-candidates are tested across a wide doserange from pharmacologically relevant to toxicological. Specificbehavioural signals, e.g., the induction of head twitches or wet dogshakes (WDS) suggesting that it may have psychedelic properties,would likely be captured in the reporting of effects. The test willalso establish the drug-candidate's general characteristics, i.e.,sedative, stimulant or behaviourally neutral. Drug-candidates withnovel mechanisms or having multiple pharmacological actionspose particular problems when selecting Positive Controls orReference Comparators for intravenous self-administration (IVSA)or drug-discrimination experiments. Information from the IrwinProfile can be very helpful in making those decisions.

Drug-candidates are generally tested in isolation, i.e., there is avehicle group (Negative Control), but no Positive Control or Refer-ence Comparator, and therefore, no internal indices against whichto “bench mark” the findings. For this reason, the Irwin Profileprovides guidance for the non-clinical abuse potential assessmentrather than definitive information.


http://www.emcdda.europa.eu

http://www.emcdda.europa.eu

http://www.gov.uk/government/collections/drug-misuse-declared

Table 1A comparison of the European and American regulatory requirements for non-clinical evaluations of the abuse/dependence risks posed by novel CNS-active drug-candidates.


3.3.2. Head-twitch and wet dog shakesThe head-twitch response in mice (a rapid rotational flick of the

head and ears) and its homologue wet dog shakes (WDS) in rats (socalled because the rotational response of the head transitions downthe spine of the animal resembling the action of dogs when theyshake water from their coats) are now commonly regarded asfunctional models of 5-HT2A receptor agonism. The induction ofhead-twitches is being increasingly employed to predict whether5-HT2A agonists have hallucinogenic properties. The logical pro-gression is to consider whether head-twitches or WDS provide auseful behavioural screen for investigating the possible psychedelicproperties of CNS drug-candidates. Investigations on head-twitches and WDS started in the early 1960s and reached its firstpeak in the 1970s and 1980s (see reviews by Green and Heal, 1985;Heal et al., 1992b). The wealth of information in these early studiesprovides an important contribution to an assessment of the utilityof these behaviours as translational models of psychedelic activity.Although the first article by Corne et al. (1963) describes the in-duction of head-twitches by administration of the 5-HT precursor,5-hydroxytryptophan (5-HTP), in their following article entitled “Apossible correlation between drug-induced hallucinations in man anda behavioural response in mice” (Corne and Pickering, 1967) evi-dence was presented to show that head-twitches were not onlyinduced by 5-HT2A agonists, e.g., LSD, mescaline, psilocybin andN,N-DMT, but also by non-serotonergic hallucinogens, e.g., phen-cyclidine (PCP), and a range of non-hallucinogenic compounds, e.g.,atropine, hyoscine, yohimbine and ergometrine (see Table 2). Theexperiments also revealed that head-twitches and WDS can be


produced by a wide range of drugs that potentiate serotonergicfunction, e.g., 5-HTP, fenfluramine, quipazine, p-chlor-ophenylalanine (PCPA) (Corne et al., 1963; Bedard and Pycock,1977;see Green and Heal, 1985; Heal et al., 1992b) that have no psy-chedelic or hallucinogenic effects in man. Thus, the predictivevalidity of the head-twitch model for detecting serotonergic hal-lucinogens is somewhat compromised by several “false positives”in addition to the non-serotonergic anomalies reported by Corneand Pickering (1967). On the other hand, Corne and Pickering(1967) extended the scope of the model with their finding thatthis behaviour was induced by hallucinogenic agents from otherpharmacological classes (Table 2); an observation confirmed byothers (Yamaguchi et al., 1987; Nabeshima et al., 1987). Morerecently, Nakamura and Fukushima (1976) reported that head-twitches can be produced by some benzodiazepines (Table 2). Itis well recognised that benzodiazepines are abused by humans.

As shown in Table 2, a raft of naturally occurring and synthetic5-HT2A agonists produce head-twitches in mice and/orWDS in rats.This includes some novel synthetic 5-HT2A agonists which arerecreationally abused, e.g., 25I-NBOMe (Rose et al., 2013; Kueppersand Cooke, 2015) 25I-NBOH (Arantes et al., 2017). Head-twitchesand WDS induced by serotonergic agents are specifically medi-ated by 5-HT2 receptor activation (see Green and Heal, 1985; Healet al., 1992b). The sensitivity of head-twitches to inhibition byketanserin (Yap and Taylor, 1983; Lucki et al., 1984; Arnt et al., 1984;Niemegeers et al., 1983) indicated that this response was mediatedby the 5-HT2A receptor subtype. More recent antagonist experi-ments show that head-twitches induced by N,N-DMT (DPT) and


Table 2Drugs inducing head-twitch behaviour in rodents.

Reference Compound Species Head-twitches

5-HT2A agonist hallucinogensCorne and Pickering (1967) LSD Mouse ✓

Psilocin Mouse ✓

Psilocybin Mouse ✓

Mescaline Mouse ✓

a-Methyl-mescaline Mouse ✓

NN-DMT Mouse ✓

Bufotenine Mouse ✓

Silva and Calil (1975) LSD Mouse ✓

Mescaline Mouse ✓

Yamamoto and Ueki (1975) DOM Rat ✓

DOM Mouse ✓

Bedard and Pycock (1977) LSD Rat ✓

5MeODMT Rat ✓

Vetulani et al. (1980) LSD Rat ✓

Matthews and Smith (1980) 5MeODMT Rat ✓

Niemegeers et al. (1983) Mescaline Rat ✓

Yamamoto et al. (1983) Mescaline Rat ✓

DOM Rat ✓

Arnt and Hyttel (1989) DOI Rat ✓

Heal et al. (1985, 1986) 5MeODMT Mouse ✓

Darmani et al. (1992) DOI Mouse ✓

Fantegrossi et al. (2005a) 2C-T-7 Mouse ✓

(R)DOM Mouse ✓

Moya et al. (2007) DOI Rat ✓

DOM Rat ✓

DOB Rat ✓

2C-I Rat X2C-B Rat ?X2C-D Rat ?X2,5-DMA Rat ?X

Fantegrossi et al. (2008) DPT Mouse ✓

Fantegrossi et al. (2010) (R)DOI Mouse ✓

Ettrup et al. (2013) 25BN-BOMe Mouse ✓

[NBOMe-2C-B; BOM-2CB]Smith et al. (2014) DPT Mouse ✓

DIPT Mouse ✓

2C-T-7 Mouse ✓

(R)DOI Mouse ✓

Halberstadt and Geyer (2014) 2C-I Mouse ✓

25I-NBOME Mouse ✓

25I-NBD Mouse ✓

Fantegrossi et al. (2015) (R)DOI Mouse ✓

25-CN-NBOH Mouse ✓

5-HT2C preferring agonistsMaj and Lewandowska (1980) mCPP Mouse ✓

pCPP Mouse ✓

Yarosh et al. (2007) mCPP Mouse XTFMPP Mouse ✓

Vickers et al. (2001) Ro 60-0175 Rat ?X

Non-serotonergic hallucinogensCorne and Pickering (1967) Phencyclidine Mouse ✓

Yamaguchi et al. (1987) Phencyclidine Rat ✓

Nabeshima et al. (1987) Phencyclidine Rat ✓

AnomaliesCorne and Pickering (1967) Atropine Mouse ✓

Hyoscine Mouse ✓

Yohimbine Mouse ✓

Ergometrine Mouse ✓

Nakamura and Fukushima (1976) Clonazepam Mouse ✓

Nitrazepam Mouse ✓

Fludiazepam Mouse ✓

Balsara et al. (1986) Ergometrine Mouse ✓

✓¼Head-twitches reported. X¼ no head-twitches observed. ?X¼Numerical increase in head-twitches compared to vehicle control values. Failure to achieve significancemay have been due to a lack of statistical power.Full chemical names are provided in the Abbreviations list at the front of the article.


DOI are blocked by the highly selective 5-HT2A antagonist,M100907 (Fantegrossi et al., 2008, 2010; Fox et al., 2010; Doughertyand Aloyo, 2011; Serafine et al., 2015). In addition, it has been


shown that it is not possible to produce head-twitches in micelacking the 5-HT2A receptor (Gonz�alez-Maeso et al., 2007).Together, these findings show beyond reasonable doubt that head-



twitches induced by direct or indirect serotonergic agonists aremediated activation of the 5-HT2A receptor.

The head-twitch and WDS responses of rodents can be pro-foundly modulated by other neurotransmitter systems. The mostimportant modulatory neurotransmitters are noradrenaline andGABA. For example, head-twitches are inhibited by high doses ofa1-adrenoceptor agonists (Handley and Brown, 1982; Heal et al.,1985) and potentiated by a1-adrenoceptor antagonists (Handleyand Brown, 1982; Heal et al., 1986). Head-twitches and WDS arealso potently inhibited and potentiated by a2-adrenoceptor ago-nists and antagonists, respectively (Bednarczyk and Vetulani, 1978;Handley and Brown, 1982; Heal et al., 1986). Potentiating dopami-nergic activity in the CNS by administration of methamphetamine,amphetamine or methylphenidate attenuates head-twitches inmice (Corne et al., 1963) and apomorphine and D-amphetaminehave been reported to attenuate WDS in rats (Bedard and Pycock,1977; Arnt et al., 1984).

Head-twitches or WDS can serve as useful behavioural para-digms to demonstrate the functional effects of selective 5-HT2Aagonists (Table 2). In addition, the induction of head-twitches canalso detect the hallucinogenic properties of some non-serotonergicpsychedelics, e.g., phencyclidine (Corne and Pickering, 1967).However, the powerful modulatory influence of other neurotrans-mitter systems on head-twitches and WDS, particularly the cate-cholamines and GABA severely restricts the usefulness of thesebehavioural responses when evaluating psychedelic drugs withmultiple pharmacological effects. This point is amply illustrated byMDMA, which indirectly activates 5-HT2A receptors, but does notinduce head-twitches in mice (Fantegrossi et al., 2005b; data onfile, RenaSci Ltd). Paradoxically, however, Fantegrossi et al. (2004c,2005b) claim that head-twitches can be induced by the individualenantiomers of MDMA. MDMA has been used as a training cue indrug-discrimination experiments in our laboratory and we haveobserved no WDS in rats given MDMA by either the oral or intra-peritoneal route (data on file, RenaSci Ltd). Another caveat is head-twitches are elicited by some compounds that are not hallucino-genic (Table 2).

In summary, head-twitches and WDS are reasonably predictiveof hallucinogenic potential, but there are some “false positives”,and in addition, psychedelic drugs with complex pharmacology canshow up as “false negatives”. If these behaviours are used toinvestigate the psychedelic/hallucinogenic potential of novel, CNSdrug-candidates the findings need to be interpreted with caution.

4. Specific regulatory behavioural tests to assess abuse anddependence potential

An evaluation of abuse/dependence liability forms part of thesafety assessment of drug-candidates, and therefore, the testing issubject to practices and strictures that do not apply to “mode ofaction” experiments. The most important of them are described inthe following sections.

4.1. Good Laboratory Practice

The robustness, reproducibility, lack of bias, and traceability ofresults back the raw data in non-clinical findings are a source ofsome concern in drug development (e.g., see Macleod et al., 2009).Good Laboratory Practice (GLP) is a mechanism to address theseissues from experimental design through to the final reporting ofthe findings through a highly detailed prescriptive protocol,together with rigorous quality control and quality assuranceauditing conducted under the supervision of a regulatory agency,e.g., Medicines and Healthcare products Regulatory Agency(MHRA) or FDA. For a description of non-clinical GLP procedures,


see Adamo et al. (2012) and Andrade et al. (2016). As part of theSafety Pharmacology assessment of drugs for human use, non-clinical abuse/dependence studies should be conducted in accor-dance with Good Laboratory Practice (GLP) in line with the Inter-national Committee on Harmonisation (ICH) guidelines (S7A SafetyPharmacology Studies for Human Pharmaceuticals, 2001). The ICHGuidance (2001) describes the importance of ensuring the qualityand reliability of non-clinical safety studies “which is normallyaccomplished by the conduct of studies in compliance with GLP.”However, it not dogmatic on the point and also states “Due to theunique design of, and practical considerations for, some safety phar-macology studies, it may not be feasible to conduct these in compli-ance with GLP.” It further states “When studies are not conducted incompliance with GLP, study reconstruction should be ensured throughthe adequate documentation of study conduct and archiving of data.Any study or study component not conducted in compliance with GLPshould be adequately justified, and the potential impact on evaluationof the safety pharmacology endpoints should be explained.”

In the UK, GLP is conducted under Good Laboratory PracticeRegulations (Statutory Instrument 1999 No 3106 as amended byStatutory Instrument 2004 No. 994). These regulations are inaccordance with the Economic Co-operation and Development(OECD) Principles of Good Laboratory Practice (as revised in 1997).Because the CHMP/EMA (2006) and the CDER/FDA (2010) draftguidance were worded exactly in accordance with ICH (2001),neither was inflexible on the requirement for GLP for non-clinicalabuse/dependence studies. As a consequence, until recently manystudies were conducted either as non-GLP or GLP-like. The mainreasons are (i) the complex nature of drug-discrimination and IVSAexperiments which require a level of scientific expertise that oftendoes not exist outside of academic laboratories that are not GLP-compliant, and (ii) the requirement for a high degree of flexibilitywhen performing behavioural pharmacology experiments that canchange substantially based on a single day's results which does notsit well within rigid and highly specific GLP Study Plans.

The position of the CDER/FDA has shifted and the CDER/FDA(2017) guidance unequivocally states that the core abuse/depen-dence studies, i.e., drug-discrimination, IVSA and tolerance/dependence testing, must be conducted in accordance with GLP.Although this is a strongly worded recommendation in what is anon-binding advisory guideline, it relates to the FDA's ProposedRule in the Federal Register (8/24/16) mandating GLP for all non-clinical Safety Pharmacology studies [https://www.federalregister.gov/documents/2016/08/24/2016-19875/good-laboratory-practice-for-nonclinical-laboratory-studies].

With this change, another difference has emerged betweenCDER/FDA and CHMP/EMA on the conduct of abuse/dependencestudies (Table 1). It is too early to know whether CDER/FDA willadopt a hard line and refuse to accept abuse/dependence studiesthat have not been conducted to GLP, but have been accepted byother regulatory agencies, e.g., CHMP/EMA. What is certain is itadds to the complexity of planning abuse/dependence studies tosupport global drug approvals.

4.2. Drug exposure levels

Since abusers invariably self-administer prescription drugs atdoses several-fold higher than the maximum recommended ther-apeutic dose, it is essential to factor this inwhen conducting abuse/dependence experiments in animals. For drug-discrimination ex-periments, the doses of the test compound to be selected forevaluation should generate plasma Cmax concentrations �2� thehuman Cmax at the highest therapeutic dose. The same rulegenerally applies to tolerance/dependence experiments, butbecause they are repeat-dose experiments, the AUC is debatably a



more relevant pharmacokinetic parameter than Cmax for this typeof study. IVSA experiments are the exception because the outcomeis determined by the number of infusions of drug taken by theanimal. In this case, doses of the drug-candidate should be calcu-lated to be fractions of the clinical Cmax to prevent “false negative”results occurring because of an immediate over-dose or satiationfrom a single dose of drug-candidate (which is often administeredas a non-contingent infusion to start the test session).

4.3. Positive Controls and Reference Comparators

No distinction is made between Positive Controls and ReferenceComparators in either the CHMP/EMA (2006) or CDER/FDA (2017)guidances on non-clinical abuse/dependence studies. In the au-thors' opinion, there is a clear difference between the two e aPositive Control is used to validate the experimental test procedure,whereas Reference Comparators are selected to set the results forthe drug-candidate into context. As an example, D-amphetamine isoften used as a stimulant training drug in drug-discriminationstudies (Positive Control), while drugs like phentermine, methyl-phenidate, and bupropion are used as Reference Comparatorsbecause they represent a spectrum of drugs ranging from non-scheduled (bupropion) through C-IV (phentermine) up to C-II(methylphenidate).

4.4. Role of gender in abuse/dependence evaluations

It is generally accepted that males are almost exclusively used inlaboratory experiments with rodents because the results obtainedcannot be confounded by the estrous cycle. In view of the fact thatmost drugs used in clinical practice are not gender-specific andtoxicity studies are routinely conducted in both males and females,the tendency to use male rodents almost exclusively in SafetyPharmacology would appear to be a scientifically unsound.Certainly that is the view of the National Institute for Health (NIH)which has recently recommended that drugs for human use bepharmacologically evaluated in both males and females in animalexperiments (Clayton and Collins, 2014; Clayton, 2016). In turn,CDER/FDA (2017) has adopted the NIH recommendation and hasadvised in its guidance that non-clinical abuse/dependence ex-periments should be conducted in males and females. In contrast,the much earlier guidance from CHMP/EMA (2006) offers no rec-ommendations on this topic. Although at this time, it is unclearwhether or not it will be necessary to duplicate all abuse/depen-dence experiments in male and female rodents, it is clear thatproviding data only from males will not in the future be acceptableto FDA.

This discussion has focussed exclusively on rodent experimentsbecause drug-discrimination and IVSA experiments conductedusing primates is generally performed in a mixed population ofmales and females.

4.5. Coordination between non-clinical and clinical assessments

EMA does not rely on data from studies in drug-experiencedhuman volunteers when making its determination of the abuse/dependence risks posed by novel CNS-active drug-candidates(although it will take these findings into account if such studieshave been performed to support a US registration). Rather, EMAmakes its assessment based on the results from the key preclinicalabuse/dependence experiments, i.e., drug-discrimination, IVSA,and tolerance/dependence test, together with a careful analysis ofthe abuse-related adverse events in human volunteers (Phase 1)and in the treated patient population (Phases 2 and 3). In addition,the FDA relies extensively on evidence gathered from studies in


drug-experienced human volunteers. Consequently, it is importantto coordinate non-clinical and clinical assessments of the drug-candidate in drug-experienced human volunteers to provide con-tinuity and consistency between the studies performed in animalsand those in man. This applies particularly to the selection ofPositive Controls and Reference Comparators for use in the non-clinical and clinical protocols.

For a more comprehensive discussion of the differences be-tween the US and European approaches to abuse/dependenceevaluations see Calderon et al. (2015).

4.6. Guidelines contain advice not instructions

The CDER/FDA (2017) guidance contains the following state-ments which unequivocally inform reader that the guidance offersadvice and recommendations on abuse/dependence evaluations,but it is not an instruction manual.

“Contains non-binding recommendations”

And

“This guidance represents the current thinking of the Food andDrug Administration (FDA or Agency) on this topic. It does notestablish any rights for any person and is not binding on FDA or thepublic. You can use an alternative approach if it satisfies the re-quirements of the applicable statutes and regulations. To discuss analternative approach, contact the FDA office responsible for thisguidance as listed on the title page.”

This is critical because the CDER/FDA (2017) guidance includesdetailed descriptions of experimental procedures for non-clinicalabuse/dependence studies. In the authors' opinion, it is a poten-tial cause for confusion because there are many instances wherethe generic protocols in the CDER/FDA (2017) guidance will notdeliver viable results. In the final analysis, it is the Sponsor's re-sponsibility to provide viable and interpretable findings from theabuse/dependence evaluation to the regulatory agencies. TheCHMP/EMA (2006) does not explicitly state that the documentcontains advice rather than instructions; however, it is evidentfrom statements throughout the guidance that this is its intention.

4.7. Drug-discrimination

Drug-discrimination answers the question whether the psy-choactive effects of the drug-candidate are identical or similar tothose of known substances of abuse. A number of excellent reviewshave been written on the methodological aspects of drug-discrimination testing (Colpaert, 1995, 1999; Ator and Griffiths,2003; Stolerman et al., 2011). In addition, the use of this tech-nique as a tool for assessing abuse potential has also been thesubject of a number of good reviews (see Mori et al., 2012; Mead,2014; Swedberg, 2016) including those which discuss the relativestrengths and weaknesses of drug-discrimination for this purpose(Moser et al., 2011; Mead, 2014). Following the emergence of theCDER/FDA (2017) guidelines that include a recommendation toconduct drug-discrimination testing under extinction, i.e., no foodrewards for lever-pressing in testing sessions, Gauvin et al. (2016)have written a strong rebuttal of the FDA's thinking on the matterthat is well worth reading.

When selecting a drug for use as training cues in drug-discrimination to detect possible hallucinogenic/psychedelicproperties in either known or novel serotonergic drug-candidates,there are a number of factors to be taken into consideration. Sincethe objective is to detect the potential risk for abuse rather than



pharmacological specificity, i.e., 5-HT2A receptor agonist activity,the criteria in safety pharmacology testing may be different fromthose in academic research.

1. Does the discriminative cue elicited by the training druggeneralise to other abused drugs from the same pharmaco-logical class?

2. Does the discriminative cue elicited by the training druggeneralise to other abused drugs from the related pharmaco-logical classes?

3. Do drugs that lack hallucinogenic/psychedelic activity gener-alise to the training drug's discriminative cue (“false positives”)?

4. Do drugs with hallucinogenic/psychedelic activity fail togeneralise to the training drug's discriminative cue (“falsenegatives”).

Based on the knowledge that the LSD, mescaline and psilocybinare the prototypical, serotonergic psychedelics, these drugs wouldappear to be the ideal choices as training drugs because they are 5-HT2A receptor agonists and their discriminative cues are blocked bymoderately and highly selective 5-HT2A receptor antagonists. Thus,ketanserin and M100907 have been shown to block LSD cue in rats(Arnt, 1992; Gresch et al., 2007) and partially block the LSD cue inmice (Benneyworth et al., 2005). M100907 and ketanserin havesimilarly been reported to block the discriminative cues elicited byDOI (Glennon, 1986; Chojnacka-W�ojcik and Kłodzi�nska, 1997) andDOM (May et al., 2009).

Nothing is ever that simple and it has been reported that the 5-HT antagonists, methysergide and mianserin, generalise to LSD(Colpaert et al., 1982) and the 5-HT1A agonist, 8-OH-DPAT (2-(di-n-propylamino)-tetralin), generalised partially to LSD (Benneyworthet al., 2005; Reissig et al., 2005). Furthermore, MDMA does notgeneralise to LSD (Appel et al., 1982; Callahan and Appel, 1988;Baker and Taylor, 1997) even though its discriminative cue has a5-HT2A receptor agonist component (Fig. 1; Baker et al., 1997;Goodwin et al., 2003). In other drug-discrimination experiments,LSD and psilocin, but not mescaline, were observed to generalise tothe psilocybin (Koerner and Appel, 1982), and while MDMAgeneralised to the cue elicited by mescaline, it generalised to saline

Fig. 1. Profiles of various drugs in rats trained to discriminate MDMA from saline.Results are the mean percentage generalisation to MDMA± sem for various CNS-active serotrats were trained to discriminate MDMA (1.5 or 1.25mg/kg ip [result only for 1.5mg/kg shA An oral MDMA dose-response curve was performed to validate the model for detecting tB All other drugs were administered by intraperitoneal injection.


in LSD trained rats (Callahan and Appel, 1988). These psychedelicsare not highly selective 5-HT2A agonists and a failure to observecross-generalisation could be due to that fact. However, similardiscrepancies have been reported for more selective compoundswhere for example 25CN-NBOH only partially generalised to thediscriminative cue elicited by DOI (Fantegrossi et al., 2015). Inanother case, the selective 5-HT1A agonists, 8-hydroxy-2-dipropylaminotetralin (8OH-DPAT) and buspirone, and the antag-onist, ipsapirone, generalised to the cue elicited by 5-MeO-DMT(Spencer et al., 1987). For these reasons, we are of the view that 5-HT2A agonists are not the ideal choice as training drugs in drug-discrimination experiments where the objective is to identifywhether novel drug-candidates have (i) psychedelic propertiesand/or (ii) pose a risk of abuse.

We believe that MDMA is a better choice as a training cue inregulatory drug-discrimination studies. The MDMA discriminativecue has a strong serotonergic component because it is prevented bydepletion of 5-HT using PCPA (Schechter, 1991), lesioning by D-fenfluramine (Baker and Makhay, 1996) or preventing it fromentering serotonergic terminal by blockade of the reuptake trans-port by fluoxetine (Nichols et al., 1989). In addition, the discrimi-native cues elicited by racemic MDMA and (þ)-MDMA are partiallyblocked by 5-HT2 antagonist, pirenpirone (Baker et al., 1997), andthe selective 5-HT2A antagonist, MD100907, (Goodwin et al., 2003),respectively. In contrast, (þ)-MDMA's cue was not antagonised byeither the D2/a1 receptor antagonist, haloperidol, or the 5-HT1Aantagonist, WAY 100,135 (Baker et al., 1997).

A summary of the results for a wide range of CNS-active com-pounds using of MDMA as a cue is reported in Table 3. All of theserotonergic hallucinogens and entactogens investigated to datewith the exception of 2-CT-2 have been found to generalise toMDMA demonstrating its versatility for detecting drugs with abuseliability. In addition, its mixed serotonergic/dopaminergic phar-macology enables drugs with predominantly dopaminergic mech-anisms, e.g., D-amphetamine and cocaine, to generalise at leastpartially to MDMA thereby broadening the range of recreationallyabused drugs that can be detected. Webster et al. (2017) examinedthe difference between the discriminative cues elicited by low(1.75mg/kg) and high (3.0mg/kg) doses of MDMA and revealed

onergic drugs (n¼ 6e7 rats/group) in MDMA-cued drug-discrimination in rats. Femaleown for the sake of clarity]) from saline.he MDMA-like effects of compounds administered by this route.


Table 3Profiles of drugs against the MDMA discriminative cue.

Compound Discriminative cue Training dose(mg/kg)

Species Generalisation to cue Reference

Classical hallucinogensLSD MDMA 1.75 Rats Yes Oberlender and Nichols (1988)LSD MDMA ? Rats Yes Goodwin and Baker (2000)(±)-MDMA Mescaline 10 Rats Yes Callahan and Appel (1988)(þ)-MDMA Mescaline 10 Rats Yes Callahan and Appel (1988)(�)-MDMA Mescaline 10 Rats Yes Callahan and Appel (1988)

Novel hallucinogens and designer drugs5-APB MDMA 1.5 Mouse Yes Dolan et al. (2017)6-APDB MDMA 1.5 Mouse Yes Dolan et al. (2017)2C-C MDMA 1.5 Rats Yes Eshleman et al. (2014)2C-D MDMA 1.5 Rats Partial Eshleman et al. (2014)2C-E MDMA 1.5 Rats Yes Eshleman et al. (2014)2C-I MDMA 1.5 Rats Partial Eshleman et al. (2014)2C-T-2 MDMA 1.5 Rats No Eshleman et al. (2014)DOC MDMA 1.5 Rats Partial Eshleman et al. (2014)DOI MDMA 1.5 Rats Yes Gosden and Heal (1997)DOM MDMA 1.75 Rats Yes Oberlender and Nichols (1988)EDA MDMA 1.75 Rats Yes Nichols et al. (1989)4-FA MDMA 1.5 Mouse Yes Dolan et al. (2017)MBDB MDMA 1.75 Rats Yes Oberlender and Nichols (1988)Mephedrone MDMA 1.5 Rats Yes Harvey and Baker (2016)MDMA Mephedrone 1.0 Rats Yes Berquist et al. (2017a)MDA MDMA 1.75 Rats Yes Oberlender and Nichols (1988)MDE MDMA 1.5 Rats Yes Glennon and Misenheimer (1989)MDPV MDMA 1.5 Rats Yes Harvey and Baker (2016)MDMA MDPV 0.3 Mice Yes Fantegrossi et al. (2013)MMA MDMA 1.75 Rats Yes Johnson et al. (1991)MMA MDMA 1.75 Rats Yes Marona-Lewicka and Nichols (1994)(þ)-MMAP MDMA 1.75 Rats Yes Johnson et al. (1991)(�)-MMAP MDMA 1.75 Rats Yes Johnson et al. (1991)NN-DMT MDMA 1.5 Rats Yes Fantegrossi et al. (2008)N-OH-MDA MDMA 1.5 Rats Yes Glennon and Misenheimer (1989)IDA MDMA 1.75 Rats Yes Nichols et al. (1989)

StimulantsD-Amphetamine MDMA 1.75 Rats Yes Oberlender and Nichols (1988)D-Amphetamine MDMA 1.5 Rats Partial Harvey and Baker (2016)D-Amphetamine MDMA 1.5 Rats Partial Glennon and Misenheimer (1989)D-Amphetamine MDMA 1.5 Rats Partial Goodwin et al. (2003)D-Amphetamine MDMA 1.5 Rats No Webster et al. (2017)D-Amphetamine MDMA 3.0 Rats Yes Webster et al. (2017)MDMA D-Amphetamine 2.0 Pigeons Yes Evans and Johanson (1986)Cocaine MDMA 1.5 Rats Partial Harvey and Baker (2016)Cocaine MDMA 1.5 Rats Partial Kueh and Baker (2007)MDMA Cocaine 10 Mice Yes Gannon and Fantegrossi (2016)

Other CNS drugsBuspirone MDMA 1.25 Rats Partial Data on file, RenaSciClomipramine MDMA 1.5 Rats Yes Webster et al. (2017)Clomipramine MDMA 3.0 Rats No Webster et al. (2017)Fenfluramine MDMA 1.5 Rats Yes Goodwin et al. (2003)MDMA Fenfluramine 2.0 Rats Yes Schechter (1986)D-Fenfluramine MDMA 1.5 Rats Partial Gosden and Heal (1997)Fluoxetine MDMA 1.5 Rats Yes Webster et al. (2017)Fluoxetine MDMA 3.0 Rats No Webster et al. (2017)Lorcaserin MDMA 1.25 Rats No Data on file, RenaScimCPP MDMA 1.5 Rats Yes Webster et al. (2017)mCPP MDMA 3.0 Rats No Webster et al. (2017)Sibutramine MDMA 1.5 Rats No Gosden and Heal (1997)MDMA Ephedrine 4.0 Rats No Young and Glennon (1998)8-OH-DPAT MDMA 1.5 Rats Yes Webster et al. (2017)8-OH-DPAT MDMA 3.0 Rats No Webster et al. (2017)RU 24969 MDMA 1.5 Rats Yes Webster et al. (2017)RU 24969 MDMA 3.0 Rats No Webster et al. (2017)

Wherever possible data have been taken from experiments whereMDMAwas used as the training drug. In some cases (entries in italics) a different training drug was used andgeneralisation to this drug cue by MDMA was investigated.


that the dopaminergic contribution to MDMA's cue was muchgreater at the higher dose while the serotonergic component de-creases. Thus, the SRRIs, fluoxetine and chlorimipramine, gener-alise to the low, but not high, dose of MDMA, whereas the situation


is reversed for the dopaminergic stimulant, D-amphetamine. Theirfindings are not entirely consistent with reports from other labo-ratories (Oberlender and Nichols, 1988; Glennon and Misenheimer,1989; Goodwin et al., 2003; Harvey and Baker, 2016), but the main



thrust of their argument is probably correct. Using MDMA (1.25 or1.5mg/kg ip), we have observed that MDMA given by the intra-peritoneal or oral route generalise to the ip MDMA cue. 5-HT2Areceptor agonists like DOI generalised to MDMA, whereas the 5-HT2C agonist, lorcaserin, the 5-HT-preferring SNRI, venlafaxine,and the balanced SNRI, sibutramine, did not (Fig. 1; Table 3). The 5-HT releasing drug, D-fenfluramine, partially generalised to MDMA(Fig. 1), which is consistent with results from experiments per-formed using the racemate (Schechter, 1986; Goodwin et al., 2003).While it may appear to be a false positive in terms of predictingabuse, there have been reports that fenfluramine produceseuphoria, derealisation, and perceptual changes in humans and ithas been subjected to sporadic abuse (Levin, 1973; Rosenvinge,1975; Dare and Goldney, 1976).

The predictive validity of partial or full generalisation to MDMAfor abuse potential is very good but not perfect. In Table 3, The SSRIsand the 5-HT1A partial agonist, buspirone, have no history of abusein humans, but show up as false positives. Because the interocep-tive cue is pharmacologically driven, one of the weaknesses of thetechnique is drugs with similar, but not identical, pharmacologicalmechanisms generalise to the training cue even though they maynot mimic the psychoactive effects or abuse liability of the trainingdrug. The SSRIs enhance synaptic 5-HT concentrations makingthem a weak imitator of the actions of the 5-HT releasing agentslike MDMA and fenfluramine. The ability of buspirone and 8-OH-DPAT to generalise to the MDMA cue is probably related to the factthat the 5-HT releasing action of MDMA results activation of 5-HT1Aand 5-HT2A receptors and both contribute to its interoceptive cue.Moreover, it is known that the “serotonin syndrome” which ismediated by the activation of 5-HT1 and 5-HT2 receptors can beelicited by 5-HT releasing agents, e.g., fenfluramine and PCPA, 5-HT2A receptor agonists, e.g., 5-MeO-DMT, 5-methoxytryptamine,and LSD, and also 5-HT1A agonists (see Green and Heal, 1985; Healet al., 1992b).

PCP has been used extensively as a discriminative cue in pri-mates, rats and pigeons to detect the NMDA antagonist-like prop-erties of a wide range of compounds. In female, Lister hooded rats,PCP (5.0mg/kg ip) elicited a robust cue that was dose-dependentlyrecognised by the rats (Fig. 2). Ketamine, another recreationallyabused non-competitive NMDA antagonist, dose-dependentlygeneralised to the PCP cue (Fig. 2). These findings are consistentwith many other reports that ketamine, its enantiomers and dis-ocilpine plus a raft of other non-competitive NMDA antagonists

Fig. 2. Dose-response curves for PCP and ketamine in rats trained to discriminatePCP from saline.Results are the mean percentage generalisation to PCP± sem (n¼ 6e17 rats/group) inPCP-cued drug-discrimination in rats. Female rats were trained to discriminate thenon-competitive NMDA antagonist, PCP (5.0mg/kg ip), from saline. All drugs wereadministered by intraperitoneal injection.


generalise to PCP (Table 4). Methoxetamine, which has emerged asa “legal high” has also been reported to generalise to both PCP(Berquist et al., 2017b) and ketamine (Chiamulera et al., 2016).Competitive NMDA antagonists either do not generalise to PCP orgeneralise partially; similar results have been obtained with theglycine-site antagonists and polyamine sitemodulators (Table 4). Interms of specificity, the results reveal no overlap between the PCPdiscriminative cue and that elicited by the k-opioid agonists, butprobably some overlap with that of the 5-HT2A agonists and themephedrone, and the stimulants (Table 4). The m-opioid agonists,barbiturates do not generalise to PCP (Table 4).

(±)-Pentazocine has a mixture of pharmacological actionscomprising k-opioid agonist and m-opioid antagonist propertiesthat resides in the (�)-enantiomer (Chien and Pasternak, 1993;Remmers et al., 1999) and selective s1 antagonist activity that re-sides in the (þ)-enantiomer (Walker et al., 1992; Remmers et al.,1999). When selecting a known substance of abuse to use as aPositive Control for studying the psychoactive properties of k-opioid agonist drug-candidates, we selected pentazocine, but hadthe (�)-enantiomer custom synthesised to avoid any possiblecontribution of s1 antagonist activity to the cue. This precautionwas taken because it has been reported that the s antagonist, NE-100, substantially reduced the discriminative cue elicited by thek-opioid agonist, U-50488H (Mori et al., 2012).

Female non-food-restricted rats were trained to discriminatebetween (�)-pentazocine (5.0mg/kg ip) and saline on a FR5schedule for sweetenedmilk rewards using our standard procedure(Heal et al., 1992a, 2013). Intravenously injected (�)-pentazocinedose-dependently generalised to the cue elicited by intraperito-neally injected (�)-pentazocine with a 10-fold increase in potencythat would be expected when switching from the intraperitoneal tothe intravenous route (Heal et al., 2016, Fig. 3). The ReferenceComparator, butorphanol also dose-dependently generalised to the(�)-pentazocine cue (Heal et al., 2016, Fig. 3). These findings areconsistent with reports that (±)-pentazocine generalised to thediscriminative cue elicited by the selective k-agonist, U-50488H(Mori et al., 2012). Alazocine (s/k-agonist) generalised to(±)-pentazocine (Ukai et al., 1989) as did cyclazocine (k-agonist/m-partial agonist) and butorphanol (k-agonist/m-partial agonist)(White and Holtzman, 1982). The k-agonists, ketecyclazocine andethylketocyclazocine, partially generalised to (±)-pentazocine(White and Holtzman, 1982).

The discriminative profiles of various k-opioid agonists aredescribed in Table 5. It reveals that there is a high degree of cross-generalisation between different drugs from this pharmacologicalclass. Drugs with mixed k/m-opioid agonist activity usually gener-alise to the k-opioid agonists, as do the s1-agonists. As a rule, thediscriminative cue elicited by the k-opioid agonists does notgeneralise to m-opioid agonists, stimulants or cannabinoid CB1agonists. In terms of the various classes of psychedelics, Peet andBaker (2011) reported that LSD partially generalised to SalvinorinA and PCP and ketamine have been observed to generalise partiallyor fully to U-50488 and Salvinorin A (Mori et al., 2006; Peet andBaker, 2011), but not TRK-820 (Mori et al., 2006).

4.7.1. Technical mattersThere are numerous variants in drug-discrimination method-

ology, but for Safety Pharmacology assessments, they all rely on theoperant responses of animals in a 2-choice paradigm that issignalled either by lever-presses or nose-pokes.

Many laboratories food restrict rats to accelerate operanttraining and maintain a manageable body weights in studies thattake several months to complete. It's not an approach that wefavour because hunger could be a confounder that leads toperseverative responding when both levers are food rewarded.


Table 4Profiles of drugs against the discriminative cue elicited by non-competitive NMDA antagonists.



Non-competitive NMDA antagonists(±)-Ketamine PCP 5.0 Rat Yes Data on file, RenaSci(±)-Ketamine PCP 3.0 Rat Yes Brady and Balster (1982)(±)-Ketamine PCP 3.0 Rat Yes Tricklebank et al. (1987)(±)-Ketamine PCP 2.5 Rat Yes Koek et al. (1990)(±)-Ketamine PCP 4.0 Rat Yes Overton et al. (1989)(þ)-Ketamine PCP 3.0 Rat Yes Brady and Balster (1982)(�)-Ketamine PCP 3.0 Rat Yes Brady and Balster (1982)Memantine PCP 2.0 Rat Yes Nicholson et al. (1998)Amantadine PCP 2.0 Rat Partial Nicholson et al. (1998)Dizocilpine (MK-801) PCP 2.5 Rat Yes Koek et al. (1990)Dizocilpine PCP 3.0 Rat Yes Balster et al. (1994)Dizocilpine PCP 0.08 Monkey Yes Beardsley et al. (1990)Dizocilpine PCP 0.32/1.0 Pigeon Yes Baron and Woods (1995)Eticyclidine PCP 3.0 Rat Yes Berquist et al. (2017b)Tenocyclidine PCP 3.0 Rat Yes Berquist et al. (2017b)Methoxetamine PCP 3.0 Rat Yes Berquist et al. (2017b)Methoxetamine Ketamine 7.5 Rat Yes Chiamulera et al. (2016)PD-138289 PCP 2.0 Rat Yes Nicholson and Balster (2003)PD-137889 PCP 2.0 Rat Yes Nicholson and Balster (2003)FR-115427 PCP 2.0 Rat Yes Nicholson and Balster (2003)MRZ-2/579 PCP 2.0 Rat Yes Nicholson and Balster (2003)8-MPTIQ PCP 2.0 Rat Yes Nicholson and Balster (2003)1-MPTIQ PCP 2.0 Rat Partial Nicholson and Balster (2003)Alaproclate PCP 2.0 Rat Partial Nicholson and Balster (2003)1-PTIQ PCP 2.0 Rat No Nicholson and Balster (2003)Eliprodil PCP 3.0 Rat No Balster et al. (1994)Dextromethorphan PCP 1.25 Rat Yes Nicholson et al. (1999)Dextromethorphan PCP 0.1/0.08 Monkey Yes Nicholson et al. (1999)Dextrorphan PCP 1.25 Rat Yes Nicholson et al. (1999)Dextrorphan PCP 0.1/0.08 Monkey Yes Nicholson et al. (1999)

Competitive NMDA antagonists/glycine site antagonists/polyamine site modulatorsCGS-19755 PCP 2.5 Rat Weak Partial Koek et al. (1990)CGS-19755 PCP 0.32/1.0 Pigeon Partial Baron and Woods (1995)PCP CGS-19755 1.8 Pigeon No Baron and Woods (1995)Dizocilpine CGS-19755 1.8 Pigeon No Baron and Woods (1995)CPP PCP 2.5 Rat Partial Koek et al. (1990)(þ)-HA-966 PCP 0.32/1.0 Pigeon Partial Baron and Woods (1995)(þ)-HA-966 PCP 3.0 Rat No Singh et al. (1990)PCP (þ)-HA-966 30.0 Rat No Singh et al. (1990)(±)-HA-966 PCP 0.32/1.0 Pigeon No Baron and Woods (1995)(�)-HA-966 PCP 0.32/1.0 Pigeon No Baron and Woods (1995)L-701.324 PCP 2.0 Rat Partial Nicholson and Balster (2009)MDL-100,458 PCP 2.0 Rat No Nicholson and Balster (2009)MDL-100,748 PCP 2.0 Rat No Nicholson and Balster (2009)MDL-103,371 PCP 2.0 Rat Partial Nicholson and Balster (2009)MDL-104,472 PCP 2.0 Rat Partial Nicholson and Balster (2009)MDL-105,519 PCP 2.0 Rat Partial Nicholson and Balster (2009)MRZ 2/571 PCP 2.0 Rat No Nicholson and Balster (2009)MRZ-2/576 PCP 2.0 Rat Partial Nicholson and Balster (2009)ACEA-0762 PCP 2.0 Rat No Nicholson and Balster (2009)Spermine PCP 2.0 Rat No Nicholson and Balster (1998)Spermidine PCP 2.0 Rat No Nicholson and Balster (1998)Arcaine PCP 2.0 Rat No Nicholson and Balster (1998)PCP D-CPPene 1.0 Monkey Partial Wiley and Balster (1994)Ketamine D-CPPene 1.0 Monkey Partial Wiley and Balster (1994)

k-opioid agonistsCyclorphan PCP 4.0 Rat No Overton et al. (1989)PCP U-58488 5.6 Rat No Picker et al. (1990)PCP U-58488 4.2 Pigeon No Picker and Dykstra (1987)

m-opioid agonists/mixed opioid receptor ligandsMorphine PCP 0.32/1.0 Pigeon No Baron and Woods (1995)Fentanyl PCP 4.0 Rat No Overton et al. (1989)Cyclazocine PCP 4.0 Rat Partial Overton et al. (1989)

5-HT2A agonistsMescaline PCP 4.0 Rat No Overton et al. (1989)DPT PCP 3.0 Rat Partial Berquist et al. (2017b)LSD Ketamine 7.5 Rat Yes Chiamulera et al. (2016)

(continued on next page)


Please cite this article in press as: Heal, D.J., et al., Evaluating the abuse potential of psychedelic drugs as part of the safety pharmacologyassessment for medical use in humans, Neuropharmacology (2018), https://doi.org/10.1016/j.neuropharm.2018.01.049

Table 4 (continued )



s1 agonistsPCP (þ)-Pentazocine 2.0 Rat Partial Steinfels et al. (1988)

StimulantsD-Amphetamine PCP 4.0 Rat No Overton et al. (1989)PCP Mephadrone 3.2 Rat Partial DeLarge et al. (2017)Methamphetamine Ketamine 7.5 Rat Partial Chiamulera et al. (2016)Ketamine Mephadrone 3.2 Rat Partial DeLarge et al. (2017)

SedativesPentobarbital PCP 0.32/1.0 Pigeon No Baron and Woods (1995)Phenobarbital PCP 4.0 Rat No Overton et al. (1989)

Whenever possible data have been taken from experiments where selective non-competitive NMDA antagonists were used as the training drug. In some cases (entries initalics) a different training drug was used and generalisation to this drug cue by the k-opioid agonist was investigated.


Also, because we use female rats that are weight-stable as adultsthe problem of the rats becoming obese over the course of theexperiment is not an issue.

A related contentious issue is whether testing should take placeunder conditions of extinction (no food rewards for operantresponding), or food rewarded on both levers on the chosen FRschedule. CHMP/EMA, 2006 offers no opinion on the point, butCDER/FDA (2017) has stated that the analysis of discrimination tothe training cue should be based on the operant responses of ratsprior to receiving the first food reward to avoid lever selection bias.Some researchers in the field disagree strongly with FDA's thinkingon the matter and have recently published a rebuttal article(Gauvin et al., 2016). In our variant of the model, lever-presses inthe first 2.5min of test sessions are non-rewarded, i.e., underextinction. This design can be applied to the use of MDMA, PCP and(�)-pentazocine as training drugs, and therefore, poses no prob-lems when evaluating psychedelics. However, there are instanceswhen the orexigenic properties of the training drug, e.g., mid-azolam, are so strong that even non-food-restricted rats will switchlevers in the extinction part of the training session thereby makingthe food reinforcement an absolute necessity.

Regulatory agencies generally want drug-candidates evaluatedusing the clinically relevant route of administration (CHMP/EMA,2006; CDER/FDA, 2017). The intraperitoneal or subcutaneous in-jection route can still be used when training the rats to recognise

Fig. 3. Dose-response curves for intravenously administered (¡)-pentazocine and butofrom saline.Results are the mean percentage generalisation to (�)-pentazocine ± sem (n¼ 6e7 rats/grodiscriminate the k-opioid receptor agonist, (�)-pentazocine (1.5mg/kg ip) from saline. Intravalidate the model for detecting the k-opioid receptor agonist psychoactive effects of drug


the selected discriminative cue; all that needs to be done is todemonstrate that rats recognise the cue when the training drug isadministered by the route to be used to evaluate the drug-candidate, e.g., MDMA (oral) (Fig. 1) or (�)-pentazocine (intrave-nous) (Fig. 3).

One controversial subject is how to interpret “partial general-isation” results, i.e., responses that lie between 26 and 74% (or21e79%) generalisation to the Positive Control (training substanceof abuse) cue. The view of CDER/FDA (2017) is such outcomes revealsome similarity between the interoceptive cue elicited by the drug-candidate and the Positive Control. In view of the fact that all drugswhich generalise fully to the training cue show dose-dependentgeneralisation to the training cue (see Figs. 1e3) where partialgeneralisation is part of the dose-effect curve, the authors agreewith this interpretation.

CDER/FDA (2017) recommends that non-clinical abuse/depen-dence testing should be conducted in males and females. With angreater focus on gender differences in behavioural research, there isan increasing number of articles emerging highlighting differencesbetween males and females in response to various discriminativestimuli including cannabinoids (Wiley et al., 2017), nicotine (Junget al., 2000) and opiates (Craft et al., 1996), but interestingly notcocaine (Craft and Stratmann, 1996). It is important to note thatwhile these reports highlight differences in dose-responsiveness,acquisition and extinction rates, they do not cite a single instance

rphanol in rats trained to discriminate intraperitoneally injected (¡)-pentazocine

up) in (�)-pentazocine-cued drug-discrimination in rats. Female rats were trained tovenous dose-response curve were constructed for (�)-pentazocine and butorphanol to-candidate test compounds administered by this route.


Table 5Profiles of drugs against the discriminative cue elicited by selective k-opioid agonists.

Compound Discriminative cue Training dose (mg/kg) Species Generalisation to cue Reference

k-opioid agonistsSalvinorin A U-69593 0.56 Rat Yes Willmore-Fordham et al. (2007)Salvinorin A U-69593 0.13 Rat Yes Baker et al. (2009)Salvinorin A U-69593 0.56 or 0.013 Monkey Yes Butelman et al. (2004)Salvinorin A U-50488 3.0 Rat Yes Baker et al. (2009)U-50488 Salvinorin A 0.0015 Monkey Yes Butelman et al. (2010)U-50488 Enadoline 0.0017 Monkey Yes Carey and Bergman (2001)U-50488 Ethylketocyclazine 0.01 Monkey Yes Tang and Collins (1985)PD-117302 Enadoline 0.0017 Monkey Yes Carey and Bergman (2001)GR-89686A Enadoline 0.0017 Monkey Yes Carey and Bergman (2001)(�)-Spiradoline Enadoline 0.0017 Monkey Yes Carey and Bergman (2001)ICI-204448 Enadoline 0.0017 Monkey Yes Carey and Bergman (2001)Asimadoline Enadoline 0.0017 Monkey Yes Carey and Bergman (2001)U-9593 Salvinorin A 0.0015 Monkey Yes Butelman et al. (2010)Enadoline U-50488 3.0 Rat Yes Mori et al. (2004)KT-90 U-50488 3.0 Rat Yes Mori et al. (2004)ICI-199441 U-50488 3.0 Rat Yes Mori et al. (2004)Tifluadom U-50488 5.6 Rat Yes Negus et al. (1990)(�)-Pentazocine Butorphanol 0.1 Pigeon Yes Picker et al. (1996)

Mixed k/m-opioid ligandsBremazocine Enadoline 0.0017 Monkey Yes Carey and Bergman (2001)Bremazocine Salvinorin A 0.0015 Monkey Yes Butelman et al. (2010)Bremazocine U-50488 5.6 Rat Yes Negus et al. (1990)Bremazocine U-50588 5.6 Rat Yes Picker et al. (1990)Bremazocine U-50588 5.6 Pigeon Yes Picker and Dykstra (1987)Ethylketocyclazine Enadoline 0.0017 Monkey Yes Carey and Bergman (2001)Ethylketocyclazine U-50588 5.6 Rat Yes Picker et al. (1990)Ethylketocyclazine U-50588 5.6 Pigeon Partial Picker and Dykstra (1987)Ketocyclazine U-50588 5.6 Pigeon Partial Picker and Dykstra (1987)Nalbuphine Enadoline 0.0017 Monkey No Carey and Bergman (2001)(±)-Pentazocine U-50488 3.0 Rat Yes Mori et al. (2012)U-50488 Butorphanol 0.1 Pigeon Partial Picker et al. (1996)U-69593 Butorphanol 0.1 Pigeon Partial Picker et al. (1996)

m-opioid agonistsMorphine Salvinorin A 0.0015 Monkey No Butelman et al. (2010)Morphine Salvinorin A 2.0 Rat No Peet and Baker (2011)Morphine Enadoline 0.0017 Monkey No Carey and Bergman (2001)Morphine U-50488 5.6 Rat No Negus et al. (1990)Morphine U-50588 5.6 Rat No Picker et al. (1990)U-50488 Morphine 3.0 Rat No Craft et al. (1996)U-50488 Morphine 5.0 Rat No Broqua et al. (1998)U-50488 Morphine 2.0 Rat No Recker and Higgins (2004)U-50488 Morphine 10 Pigeon No France and Woods (1990)Fentanyl Salvinorin A 0.0015 Monkey No Butelman et al. (2010)Fentanyl U-50488 5.6 Rat No Negus et al. (1990)Fentanyl U-50588 5.6 Rat No Picker et al. (1990)Methadone U-50588 5.6 Rat No Picker et al. (1990)Buprenorphine U-50488 5.6 Rat No Negus et al. (1990)Nalorphine Enadoline 0.0017 Monkey Partial Carey and Bergman (2001)

s1 agonists(þ)-Pentazocine U-50488 3.0 Rat Yes Mori et al. (2012)SKF-10047 U-50488 3.0 Rat Yes Mori et al. (2012)

NMDA antagonistsPCP U-50488 3.0 Rat Yes Mori et al. (2006)PCP U-50588 5.6 Rat No Picker et al. (1990)PCP U-50588 4.2 Pigeon No Picker and Dykstra (1987)PCP TRK-820 3.0 Rat No Mori et al. (2006)Ketamine Salvinorin A 2.0 Rat Partial Peet and Baker (2011)Ketamine U-50488 3.0 Rat Yes Mori et al. (2006)Salvinorin A Ketamine 8.0 Rat No Killinger et al. (2010)MK-801 U-50488 3.0 Rat Yes Mori et al. (2006)MK-801 TRK-820 3.0 Rat No Mori et al. (2006)Ifendprodil U-50488 3.0 Rat Partial Mori et al. (2006)CPP U-50488 3.0 Rat Partial Mori et al. (2006)

StimulantsD-Amphetamine U-50588 5.6 Rat No Picker et al. (1990)D-Amphetamine U-50588 5.6 Pigeon No Picker and Dykstra (1987)U-50488 Cocaine 10 Rat No Mori et al. (2002)TRK-820 Cocaine 10 Rat No Ukai et al. (1997)

(continued on next page)


Please cite this article in press as: Heal, D.J., et al., Evaluating the abuse potential of psychedelic drugs as part of the safety pharmacologyassessment for medical use in humans, Neuropharmacology (2018), https://doi.org/10.1016/j.neuropharm.2018.01.049

Table 5 (continued )

Compound Discriminative cue Training dose (mg/kg) Species Generalisation to cue Reference

HallucinogensLSD Salvinorin A 2.0 Rat Partial Peet and Baker (2011)Salvinorin A LSD 0.08 Rat No Killinger et al. (2010)DOM Salvinorin A 2.0 Rat No Peet and Baker (2011)

CannabinoidsSalvinorin A D9-THC 10.0 Mouse No Walentiny et al. (2010)

SedativesPentobarbital U-50588 5.6 Rat No Picker et al. (1990)

Wherever possible data have been taken from experiments where selective k-opioid agonists were used as the training drug. In some cases (entries in italics) a differenttraining drug was used and generalisation to this drug cue by the k-opioid agonist was investigated.


of a substance of abuse being recognised by animals of one gender,but not the other. We have been running drug-discrimination ex-periments in female rats for more than 30 years using a wide arrayof substances of abuse from different pharmacological classes.When setting up a new model, we source information from thescientific literature which almost invariably means experimentsconducted using male rats. Our experience is apart from someminormodifications to dose every substance of abuse that has beenreported to serve as a discriminative cue in male rats can bereplicated in females. Furthermore, it also applies to every one ofthe Reference Comparator drugs that we have tested over the years.The implication is if a drug-candidate generalises to a discrimina-tive cue in male rats, it will generalise in female rats also. Althoughthis may not be the opinion of CDER/FDA, the authors' position onthis subject is there is nothing to be gained from conducting par-allel drug-discrimination experiments in separate cohorts of maleand female animals.

4.7.2. Key pointsRats or monkeys recognise a specific cue or cluster of cues

generated by the training drug of abuse; however, this is notnecessarily the same psychoactive effect (or effects) that makes thedrug attractive for abuse. On the contrary, the interoceptive cuesgenerated are generally very pharmacologically specific. Thus, theD-amphetamine cue is predominantly dopaminergic which meansthat although D-amphetamine-cued drug-discrimination is anexcellent model for detecting dopaminergic stimulants, e.g.,methylphenidate, phentermine, cocaine, methamphetamine etc.,many non-abused dopaminergic drugs, e.g., bupropion and variousdopamine agonists, show up as “false positive” in the model.

When evaluating results for drug-candidates from drug-discrimination experiments it is a gross over-interpretation of thedata to conclude that because a drug-candidate generalises to thecue elicited by a substance of abuse, e.g., cocaine, that the drug-candidate possesses cocaine-like psychoactive properties; evenmore so to conclude that generalisation to cocaine predicts that thedrug-candidate poses a real risk for abuse by stimulant abusers.

4.8. Intravenous self-administration

The second question to be answered when assessing the drug-candidate's risk for abuse is whether it produces a pleasurable,psychoactive experience that could lead to dose-escalation by pa-tients and/or drug-seeking by abusers. This question is generallyanswered by investigating whether the drug-candidate will sub-stitute for a substance of abuse in rats or primates in an intravenousself-administration (IVSA) test. We would draw the reader'sattention to other reviews on the general methodological aspects ofIVSA testing (Johanson, 1990; Balster, 1991; Ator and Griffiths,2003; Panlilio and Goldberg, 2007), IVSA testing in rats (O'Connoret al., 2011) and primates (Howell and Fantegrossi, 2009), and


also reviews proposing refinements to improve the sensitivity andpredictive validity of the model (Roberts et al., 2013; Mead, 2014).

Although many classes of recreational drugs that haverewarding properties in humans also produce similarly rewardingpsychoactive effects in animals, there are some notable gaps in thecase of the psychedelics, most notably with the 5-HT2A receptoragonists.

Yanagita (1986) studied the reinforcing potential of DOM inmonkeys using both restricted access and continuous access todrug protocols using a very low FR1 schedule of reinforcement.When DOMwas offered to naive and drug-experiencedmonkeys, itdid not serve as a reinforcer in his monkeys and the outcome wasthe same no matter which experimental design was employed.More recently Fantegrossi et al. (2004b) studied the self-administration of psilocybin, NN-DMT, mescaline and DOI in 4drug-experienced rhesus monkeys on a FR30 schedule of rein-forcement; none of the 5-HT2A receptor agonists served as positivereinforcers. The absence of articles from other researchers un-equivocally demonstrating that 5-HT2A receptor agonists serve aspositive reinforcers in animals confirms the view that Yanagita'soriginal findings were correct. Because IVSA relies on the consistentand sustained self-administration of drug at levels above saline, apotential confounder is the rapid development of pharmacologicaltolerance to 5-HT2A receptor agonists. Tolerance develops to thepharmacological effects of the 5-HT2A receptor agonists in bothanimal models (Darmani et al., 1992; Rangel-Barajas et al., 2014;Smith et al., 2014; Buchborn et al., 2015) and to their psychedeliceffects in humans (Abramson et al., 1956, 1958; Isbell et al., 1959,1961; Angrist et al., 1974). Smith et al. (2014) reported thatalthough head-twitches induced by phenylethylamine 5-HT2A ag-onists showed rapid tachyphylaxis, the response to tryptamine 5-HT2A agonists did not. However, the observation by Fantegrossiet al. (2004b) that DOI, psilocybin, mescaline and NN-DMT didnot serve as positive reinforcers in MDMA-experienced rhesusmonkeys argues strongly that monkeys at least do not find thepsychoactive effects of the 5-HT2A receptor agonists rewarding.CDER/FDA (2017) recognised the fact 5-HT2A receptor agonists donot serve as reinforcers in animals and has stated in its guidancethat Sponsors may approach the Agency to seek a waiver for con-ducting IVSA experiments with this type of drug.

MDMA also possesses psychedelic properties, and in contrast to5-HT2A receptor agonists, it has been shown to maintain self-administration in baboons (Lamb and Griffiths, 1987), rhesusmonkeys (Beardsley et al., 1986; Fantegrossi et al., 2002, 2004a,b),rats (Schenk et al., 2003), and mice (Trigo et al., 2006).

The experimental designs employed in most of these in-vestigations, particularly those conducted in rodents, use drug-naïve animals with extended access to MDMA during the acquisi-tion phase, they did not include a saline extinction phase todemonstrate the specificity of operant responding for MDMA, andresults were generally reported as operant responses rather than



infusions of drug taken. This type of protocol is not suitable for IVSAexperiments to be conducted as part of the Safety Pharmacology.

We have developed an IVSA protocol for evaluating the rein-forcing potential of CNS drug-candidates which may possessMDMA-like properties. The design is based on Smith et al. (2017)that we used previously to demonstrate that the benzodiaze-pines, midazolam and diazepam, served as positive reinforcers inrats. Mildly food-restricted male rats were initially trained to self-administer heroin (15 mg/kg/infusion) on a FR5 schedule of rein-forcement. After the acquisition of stable self-administration of

Table 6Comparison of the reinforcing effects of a range of recreationally abused drugs using obt

Test Compound Mode of action C

Strong reinforcerCocaine DA/NA stimulant CMethylphenidate DA/NA stimulant CHeroin Opioid m-agonist C

CRemifentanil Opioid m-agonist COxycodone Opioid m-agonist CNicotine Cholinergic agonist N

Weak reinforcer(�)Pentazocine Opioid k-agonist/m-antagonist CButorphanol Opioid k-agonist/m partial agonist N

CMDMA 5-HT/DA releasing drug CWIN 55,212 CB1 agonist CMorphine Opioid m-agonist CDiazepam Benzodiazepine CMidazolam Benzodiazepine C

Non-reinforcerNaltrexone Opioid antagonist NSaline N/A N

A: Data on file, RenaSci; B: Buckley et al. (2014); C: Heal et al. (2017); D: Smith et al. (20F: Smith et al. (2017).DA¼ dopamine; NA¼ noradrenaline; 5-HT¼ serotonin; N/A¼ not applicable.

Fig. 4. Evaluation of the reinforcing effects of MDMA by IVSA in heroin-maintained rats.Results are the mean± sem (n¼ 4e5 rats/MDMA dose; heroin and saline; n¼ 10) forthe number of infusions/session taken by the rats on a FR3 schedule with break-points(lever-presses/infusion) for various doses of MDMA determined on a PR schedule ofdrug reinforcement. Male rats were initially trained to self-administer heroin(0.015mg/kg/infusion) on FR5 in 2 h test sessions. After saline extinction, various dosesof MDMA were substituted for heroin on a FR3 schedule in 2 h test sessions. Break-points for MDMA reinforcement were determined by break-point determinations ona PR schedule in 4.0 h test sessions.Significantly different from saline by multiple t-test: yp< 0.05, yyp< 0.01.


heroin, the rats were extinguished on saline on the FR5 schedule.Those rats showing both stable self-administration of heroin(positive Control) and extinction on saline (Negative Control) werethen given access to various doses of MDMA. Although self-administration was acceptable on FR5, when the schedule wasdropped to FR3, MDMA served a robust reinforcer. The relativereinforcing effect of MDMA was determined by break-point deter-mination using a PR schedule of drug reinforcement. MDMA servedas a reinforcer over a 10-fold range of doses, i.e., 0.025e0.25mg/kg/infusion (Fig. 4). The break-point for self-administration of MDMAplaced it in the middle category of relative reinforcing efficacy, i.e.,lower than heroin, oxycodone, cocaine and methylphenidate buthigher than the benzodiazepines (Table 6). MDMA unequivocallyserved as a positive reinforcer in heroin-trained rats thereby vali-dating the model for detecting the reinforcing effects of drugs withMDMA-like properties. Using MDMA as Reference Comparator inheroin-trained rats rather than as a training reinforcer (PositiveControl) provides a much more practical approach for investigatingwhether drug-candidate with MDMA-like properties have rein-forcing properties; the reason being the attrition rate within thegroup of jugular catheterised rats. Some rats in a group fail to reachthe acceptance criterion for establishing IVSA even when usingpowerful reinforcers like heroin and cocaine, and of those rats, aproportion will fail to demonstrate extinction on saline. Ourexperience with the model predicts that using a low FR3 schedulecoupled with moderately rewarding drug like MDMA as thetraining reinforcer would lead to unacceptably high drop-out ratesat both the acquisition and extinction phases of the experiment.This approach also illustrates an important feature of the IVSAmodel that is often overlooked. We have shown that MDMA servesas a positive reinforcer in heroin-maintained rats. We have alsodemonstrated that heroin-maintained rats will also self-administerbenzodiazepines (Smith et al., 2017), k-opioid agonists (Heal et al.,2016) and cocaine (data on file, RenaSci). These drugs have verydifferent psychoactive properties, but are all reinforcing. The ratswork to self-administer CNS-active drugs because they arerewarding not because they are sedative or stimulant or psyche-delic. If a Sponsor has conducted an evaluation on a drug-candidatein a cocaine-maintained group of rats, regulatory agencies

ained by PR/break-point determinations in rat IVSA.

ontrolled Drug Schedule Break-point Source

-II 42 - 98 (range) A/B-II 41± 9 (6) A-II (UK) 60.2± 8.1 (31) C-I (USA) 73.4± 17.7 (7) D-II 48.1± 18.2 (5) D-II 69± 17 (7) Aot Scheduled 73.4± 14.8 (8) D

-IV 33.3± 6.7 (3) Aot Scheduled (UK) 26.9± 9.2 (4) A-IV (USA)-I 30.6± 7.4 (5) A-I 22.5± 4.0 (6) A-II 14.6± 2.5 (10) E-IV 18.9± 2.8 (4) F-IV 17.1± 2.8 (3) F

ot Scheduled 17.9± 3.8 (7) C/A 10.4± 0.8 (31) C

16); E: Heal et al. (2015b).


Fig. 5. Evaluation of the reinforcing effects of the k-opioid receptor agonist,(¡)-pentazocine by IVSA in heroin-maintained rats.Results are the mean ± sem (n¼ 8 rats/group) for the number of infusions/sessiontaken by the rats on a FR5 schedule. Male rats were initially trained to self-administerheroin (0.015mg/kg/infusion) on FR5 in 2 h test sessions. After saline extinction,various doses of (�)-pentazocine were substituted for heroin on a FR5 schedule in 2 htest sessions.Significantly different from saline by multiple t-test: yp< 0.05, yyyp< 0.001.


sometimes request it should conduct a second IVSA experiment inrats trained to self-administer a drug with sedative properties, e.g.,heroin or remifentanil. If the original IVSA study has used anappropriate design, a suitable FR schedule, and included appro-priate Reference Comparators, the likelihood of the second exper-iment replicating the findings for the drug-candidate obtained inthe original IVSA investigation are very high.

Monkeys will self-administer PCP when the drug is madeavailable by either the intravenous or oral route (Balster andWoolverton, 1979, 1980; Carroll and Meisch, 1980; Young andWoods, 1981; Carroll, 1987; Winger et al., 1989; Carroll et al.,2000, 2005; Campbell et al., 1998; Nicholson et al., 1999). Howev-er, there are very few articles describing PCP IVSA in rats. PCP(0.125mg/kg/infusion) made available to rats on a continuous 24hraccess maintained self-administration in 50% of freely-fed rats and6/6 rats when they were food-restricted (Carroll et al., 1981).Marquis et al. (1989a,b) demonstrated that PCP (0.125mg/kg/infusion) served as robust reinforcer on a FR10 schedule in rats thathad previously been established on cocaine, but the higher dose of0.25mg/kg/infusion was considerably more reinforcing. Veryrecently, Berquist et al. (2017b) reported that PCP (0.1mg/kg/infu-sion) maintained self-administration in rats on a FR1 reinforcementschedule.

Ketamine has also been shown to maintain self-administrationin monkeys when made available to drug-experienced subjects(Moreton et al., 1977; Broadbear et al., 2004), or when substitutedfor cocaine (Winger et al., 1989; Woolverton et al., 2001) or codeine(Young andWoods, 1981). It is a robust reinforcer maintaining self-administration at FR25 or FR30 in monkeys (Moreton et al., 1977;Winger et al., 1989; Woolverton et al., 2001; Broadbear et al.,2004) with a break-point for drug reinforcement of 200e250lever-presses/infusion (Moreton et al., 1977). In contrast to PCP,IVSA of ketamine has beenmore extensively studied in rat (Marquiset al., 1989b; Rocha et al., 1996; van der Kam et al., 2007; Huanget al., 2015; Venniro et al., 2015; Mutti et al., 2016; Wright et al.,2017). Ketamine will maintain self-administration in rats on FR3,but not FR10 (van der Kam et al., 2007) which is consistent with itbe a drugwith onlymoderate reinforcing efficacy (Table 6). Marquiset al. (1989b) maintained moderate levels of self-administration onFR10 when substituted for cocaine. Both observations are consis-tent with Huang et al. (2015) who determined that the break-pointfor reinforcement for ketamine (0.5mg/kg iv) was ~13 lever-presses/infusion which is low in comparison to powerful C-II re-inforcers, e.g., heroin, oxycodone, remifentanil, methylphenidate,cocaine or nicotine (Table 6). This point is also illustrated by theneed to use a minimal FR1 schedule for the acquisition and main-tenance of ketamine IVSA in many studies (Rocha et al., 1996;Huang et al., 2015; Venniro et al., 2015; Botanas et al., 2015;Wright et al., 2017).

Methoxetamine, a synthetic psychedelic derived from ketamine,has been evaluated in IVSA in rats where it has been shown tomaintain self-administration on FR1 across a range of doses(Botanas et al., 2015). In a less stringent experiment, with no salineextinction before giving the rats access to methoxetamine, it wasfound to substitute for ketamine on a FR1 schedule (Mutti et al.,2016). Similarly Berquist et al. (2017b) compared the reinforcingeffects of 3 doses of methoxetamine and PCP in rats on a FR1schedule of reinforcement and observed that each drug served as apositive reinforcer at a single dose and PCP supported far moreinfusions suggesting that it may be the more reinforcing of the 2compounds in rats (Berquist et al., 2017b).

Taking the findings for PCP, ketamine and methoxetaminetogether, they indicate that the reinforcing effects of non-competitive NMDA antagonists can be detected using IVSA inrats. However, the use of FR1 schedules is too low for Safety


Pharmacology determinations for drug-candidates. Based on theobservations of Marquis et al. (1989b) and van der Kam et al.(2007), we would establish rats on either cocaine or heroin, andsaline extinguish them before evaluating the NMDA antagonist-likeproperties of drug-candidates on either a FR3 or FR5 schedule ofreinforcement. The data also indicate that ketamine is the best ofthe non-competitive NMDA antagonists to be used as a ReferenceComparator in these studies.

Turning to the k-opioid agonists, racemic pentazocine has beenreported to maintain self-administration in rats (Lahti and Collins,1982; Yoshimura et al., 1984), in morphine-addicted rats(Steinfels et al., 1982) and to substitute for morphine in rat IVSA(Nishida et al., 1989). In primate IVSA experiments, (±)-pentazocinesubstituted for codeine (Hoffmeister,1988) and cocaine (Aigner andBalster, 1979) in monkeys and for cocaine in baboons (Lukas et al.,1986). (±)-Pentazocine has complex pharmacology with the(þ)-enantiomer being a potent, selective s1 ligand that is devoid ofk-opioid agonist activity (Walker et al., 1992; Remmers et al., 1999)while the (�)-enantiomer has k-opioid agonist and m-opioidantagonist properties (Chien and Pasternak, 1993; Remmers et al.,1999). It has long been known that s1 agonists serve as positivereinforcers (Slifer and Balster, 1983; Balster and Willetts, 1988) ands1 antagonists attenuate the self-administration of stimulants andother types of positive reinforcers (Martin-Fardon et al., 2007; Moriet al., 2014; Katz et al., 2016). To avoid potentially cofounding ef-fects of (þ)-pentazocine interfering with the possible reinforcingeffect of the k-opioid agonist actions of (�)-pentazocine, we hadthe latter custom synthesised and explored its reinforcing potentialin heroin-maintained rats (Heal et al., 2016). Male rats were initiallytrained to self-administer heroin (15 mg/kg/infusion) on a FR5schedule of reinforcement followed by saline extinction.(�)-pentazocine (0.03e0.245mg/kg infusion) maintained self-administration at levels significantly greater than saline (Fig. 5).Butorphanol, which has k-opioid agonist and m-opioid partialagonist properties also served as a positive reinforcer in heroin-maintained rats (Fig. 6).

Although the results of non-clinical experiments that haveinvestigated the reinforcing properties of salvinorin A appear to be


Fig. 6. Evaluation of the reinforcing effects of the mixed k/m-opioid receptoragonist, butorphanol, by IVSA in heroin-maintained rats.Results are the mean ± sem (n¼ 3e4 rats/group) for the number of infusions/sessiontaken by the rats on a FR5 schedule with break-points (lever-presses/infusion) forvarious doses of butorphanol determined on a PR schedule of drug reinforcement.Male rats were initially trained to self-administer heroin (0.015mg/kg/infusion) on FR5in 2 h test sessions. After saline extinction, various doses of butorphanol weresubstituted for heroin on a FR3 schedule in 2 h test sessions. Break-points for MDMAreinforcement were determined by break-point determinations on a PR schedule in4.0 h test sessions.Significantly different from saline by multiple t-test: yp< 0.05, yyp< 0.01.


contradictory, it is probable that the divergence is a dose-relatedphenomenon. Braida et al. (2008) reported that 0.5e40 mg/kg, s.c.produced conditioned place-preference in rats whilst doses of0.1e0.5 mg/infusion i.c.v. maintained self-administration. However,doses of 160 m/kg, s.c. and 1.0 mg/infusion, i.c.v. were aversive in theanimals. Salvinorin A (40 mg/kg, s.c.) increased dopamine efflux inthe nucleus accumbens which is consistent with its reinforcingeffects in the conditioned place preference test at this dose (Braidaet al., 2008). Salvinorin A (1.0 and 3.2mg/kg, i.p.) has been reportedto reduce dopamine efflux in the caudate putamen and induceconditioned place aversion in rats (Zhang et al., 2005). Thus, thereinforcing effects of salvinorin A exhibited an inverted U-shapeddose-response relationship with the drug being reinforcing in an-imals and humans over a relatively limited dose range. Severalselective k-opioid receptor agonists in addition to salvinorin A andenadoline (CI-177) are available as research compounds, e.g.,PD117302, GR89686A, (�)-spiradoline, TRK-820, bremazocine,tifluadom, ethylketocyclazocine and U50488H. The reinforcing ef-fect of some of these compounds has been studied in rats andprimates, but with the exception of enadoline, none has been intohuman subjects. U-50488H is a potent and selective k-opioid re-ceptor agonist (Ki values k¼ 0.69 nM; d¼ 9200 nM andm¼ 435 nM; data from Hunter et al., 1990). This compound isanalgesic against both mechanically and thermally induced pain(Hunter et al., 1990; Lunzer and Portoghese, 2007). Although thereinforcing effect of U-50488H has not been extensively investi-gated, this k-opioid receptor agonist did not support intravenousself-administration in rats (Tang and Collins, 1985) and it inducedplace aversion when administered to rats (Iwamoto, 1986).

Based on our non-clinical studies and those of others, thefindings predict that k-opioid agonists serve as positive reinforcers,but the dose-range for their positive effects is very limited. This


observation is consistent with clinical findings that have been ob-tained with salvinorin A and enadoline which evoke extremelydysphoric effects at high doses.

4.8.1. Technical mattersThe CDER/FDA (2017) guidance states that IVSA is usually per-

formed on a FR10 schedule of drug reinforcement and that has beenmisinterpreted as being a mandatory requirement; it is not. It is thehighest FR schedule in rats that CDER/FDA believes is sensitiveenough to detect the reinforcing properties of novel drug-candidates and this point was clarified by Controlled SubstancesStaff who presented at a workshop at the 2017 College on Problemsof Drug Dependence Meeting. For many moderately reinforcingdrugs, FR10 is too high and lower reinforcement schedules, e.g., FR3and FR5, are required when evaluating drugs of this type in IVSAexperiments. Since lower FR schedules increase the sensitivity ofthe paradigm their use is welcomed by CSS. However, FR ratios thatare lower than FR3 are not recommended because they can lead tonon-specific lever-pressing for drugs that have minimal reinforcingeffect, and very low FR schedules make the process of salineextinction much more difficult. The CHMP/EMA (2006) guidanceoffers no advice on FR schedules to be used in IVSA experiments.

The guidances produced by CHMP/EMA (2006) and CDER/FDA(2017) recommend that rats should be the species employed inabuse/dependence evaluations of drug-candidates and primatesshould be used only when there is a compelling scientific argumentfor the use of primates, Examples would be when the drug target isexpressed only in non-human and human primates, or when thereis a marked divergence in the potency and/or efficacy of the drug-candidate between humans and rodents. Nonetheless, there is awealth of information in the scientific literature on IVSA experi-ments in primates and they will no doubt be used in future ex-periments. That being the case, it is important to understand thedifferences that exist between IVSA results obtained in rats versusprimates. With IVSA experiments in rats, the animals are almostinvariably treatment naïve, they will be trained to a specific PositiveControl substance of abuse, employed to investigate a specific drug-candidate and set of Reference Comparators, and humanely killedat the end of the study. Primates are continuously used for manyyears in IVSA experiments, and will generally have experienced theself-administration of a wide range of known substances of abuseand other CNS-active compounds prior to exposure to the drug-candidate. Consequently, primates used to evaluate CNS-activedrug-candidates are rarely treatment-naïve. Rats are generallymildly food-restricted because it keeps male rats at a manageableweight in lengthy IVSA experiments, but more importantly, it en-hances the rewarding effects of drugs from several different phar-macological classes (Cabeza de Vaca and Carr, 1998; Carr et al.,2000). On the other hand, primates are fed to maintain a normal,healthy body weight. That being said, there is good congruencebetween results obtained using rats and primates. Also, responserequirements are generally much higher in primates than rats, e.g.,a response requirement for heroin in the rat would be FR5, but in arhesus monkey it is likely to be FR50.

A drug-candidate maintaining self-administration at levelssignificantly greater than vehicle on a FR schedule is the acceptedcriterion for a positive reinforcer. However, this result provides noinformation about how reinforcing the drug-candidate is relative toestablished recreationally abused drugs. CHMP/EMA (2006) rec-ommended that when drug-candidates are found to serve as pos-itive reinforcers, additional experiments should be conducted todetermine their relative reinforcing efficacy using a PR schedule ofreinforcement to determine the break-point for responding.Although CDER/FDA (2017) states that PR schedules should not beused to determine the reinforcing effect of drug-candidates, this



statement should not be misinterpreted. It implies is that PRschedules should not be employed in the first instance becausethey decrease the sensitivity of the IVSA procedure, which in turn,could lead to the false negative results for weak and moderatepositive reinforcing drug-candidates. However, there is no preclu-sion to performing a PR/break-point assessment to determine therelative reinforcing efficacy of a drug-candidate after it has beenfound to be a positive reinforcer on a FR schedule. On the contrary,the assessment of the reinforcing efficacy of a drug-candidaterelative to drugs of abuse with known abuse potential is impor-tant additional evidence to be taken into account in the regulatorydetermination of whether to classify a newmedication as a CD andif so how severely to schedule it.

In these experiments, the number of lever-presses required toobtain each subsequent drug infusion is incrementally increased todetermine the point at which the animal is no longer prepared towork to receive drug infusions. To this end we have been estab-lishing a database of the relative reinforcing effects of referencesubstances of abuse taken from different CD schedules anddifferent pharmacological classes (Buckley et al., 2014; Heal et al.,2015b, 2017; Smith et al., 2016, 2017; Table 6). From the data, it isevident that powerful reinforcers with high levels of reinforcingeffects, e.g., heroin, cocaine, oxycodone and nicotine, support highbreak-points, generally greater than 50 lever-presses/infusion,whereas moderate reinforcers, e.g., (�)-pentazocine, butorphanol,and MDMA, support break-points ~30 lever-presses/infusions,weak reinforcers, e.g., the benzodiazepines ~20 lever-presses/infusion, and saline ~10 lever-presses/infusion. While there is nota perfect correlation between CD schedule and the IVSA break-points, the paradigm clearly demonstrates that rats ascribe equalreinforcing value to powerful reinforcers irrespective of theirpharmacological mechanism, and sedative or stimulant actions.

When taking the relative reinforcing efficacy into account,(�)-pentazocine and butorphanol are moderate reinforcerscompared with m-opioid agonists like heroin and oxycodone. Theyalso show that there was no difference between the relative rein-forcing efficacy of the k-opioid agonist, (�)-pentazocine, and k-agonist/m-partial agonist, butorphanol. Thus, it can be concludedthat k-opioid agonism neither increases nor decreases the rein-forcing effect of butorphanol in the rat. That is in spite of the factthat drug-experienced human volunteers reported very differentsubjective experiences after receiving enadoline and butorphanol(Walsh et al., 2001a).

Based on CDER/FDA (2017) advice that non-clinical testingshould be in males and females, it's appropriate to address thistopic with regard to IVSA experiments. With respect to the psy-chedelics, a number of articles have appeared highlighting differ-ences between the responses males and females in IVSAexperiments. Carroll et al. (2005) reported differences betweenmale and female rhesus monkeys in the levels of oral self-administration of PCP on long-access (6 h), but not short access(3 h) access schedules. Newman et al. (2006) reported that self-administration of PCP by female rhesus monkeys was signifi-cantly higher in the luteal phase of the menstrual cycle than in thefollicular phase. However, the effect was observed only at the lowand middle doses of PCP and the difference was small, i.e., ~10%.Furthermore, all of the monkeys were experienced in PCP IVSA, andbecause testingwas 6e7 days/week, changes in themenstrual cyclehad clearly not prevented the initial acquisition of PCP self-administration. Wright et al. (2017) observed that rats in dies-trous did not acquire self-administration of low dose ketamine,whereas those in pro-estrous and male rats did. However, it shouldbe noted that rats were given access to drug on those specific daysin the cycle. Marquis et al. (1989a,b) observed acquisition andmaintenance of self-administration of both ketamine and PCP


when using a more conventional daily access schedule. Recently,Creehan et al. (2015) reported that female rats self-administeredMDMA, mephadrone and methylone which is consistent withdata obtained using males. In our laboratory, we have studied theself-administration of (�)-pentazocine in heroin-trained male andfemale rats and observed no gender-related differences in theirresponses to heroin, saline extinction, or the profile of(�)-pentazocine as a positive reinforcer. Therefore, although thereare reports of differences in rates of acquisition, rates of extinction,dose-responsiveness, and motivation for drug-seeking, they do notcite a single instance of a substance of abuse serving as a positivereinforcer in animals of one gender, but not the other. In trans-lational terms that also holds true for the abuse of prescription andillicit drugs by humans; there may be gender differences in therates of drug abuse, vulnerability, and descent into addiction, butno drug is exclusively abused either by men or by women. Whiledifferences in the responses of male and female animals can yieldvaluable insights about gender differences in vulnerability for drugdependence and addiction in humans, they are not relevant to thesimple question “Does the drug-candidate serve as a positivereinforcer in animals?” that is posed in a Safety Pharmacologyassessment. In the opinion of the authors, there is nothing to begained from conducting parallel IVSA experiments in separate co-horts of male and female animals.

4.8.2. Conditioned place preferenceConditioned place preference (CPP) is cited as an alternative

method for detecting the reinforcing properties of drug-candidatesin both the European and US guidances on abuse evaluation(CHMP/EMA, 2006; CDER/FDA, 2017). However, the technique doesnot yield the breadth of information on various classes of recrea-tionally abused drugs that derive from IVSA. Drugs are rarelyadministered by intravenous injection which is the preferred routefor evaluating rewarding effects of drug-candidates because it is aroute favoured by drug abusers. Because the test is performed in asingle session, there is also no possibility of determining whetherthe drug-candidate maintains reinforcement across days or weeks.For these reasons, CHMP/EMA and CDER/FDA do not consider thatthe information provided by CPP to be as robust as findings fromIVSA. However, there are situations, when a drug is insoluble in amedium suitable for intravenous administration, or it has a longhalf-life that would lead to drug accumulation in IVSAwhere CPP isthe only technique to evaluate a drug-candidate's reinforcingpotential.

4.8.3. Key pointsIVSA detects positive reinforcement (rewarding properties) of

compounds. This test does not reveal any information about thepharmacological class of the Positive Control, the Reference Com-parators or the drug-candidate itself. It should be recognised thatthe Positive Control used to train the animals maintains self-administration because its effects are rewarding not because it iseither a stimulant or a sedative. This point is demonstrated by theobservation that a group of rats trained in self-administration usingthe sedative euphoriant, heroin, readily self-administer MDMA,(�)-pentazocine, benzodiazepines or cocaine when substituted forheroin. Moreover, the break-points for the powerful reinforcers,heroin and cocaine, responding determined on a PR schedule ofreinforcement were not different (Table 6).

Sponsors are often requested by regulatory agencies to performmultiple IVSA experiments when assessing the reinforcing poten-tial of novel drug-candidates. On the basis of cross-substitutionbetween stimulant and sedative euphoriants, the scientific ratio-nale to support such requests is questionable.

A common misconception about the interpretation of IVSA



results is to assume that the number of infusions/session taken bythe animals is an indicator of the drug's relative reinforcing effect.Since abused drugs show inverted U-shaped dose-response func-tions, it is evident that the number of infusions taken is not onlydictated by the relative reinforcing effect of the drug, but also theunit dose under investigation.

Outcomes from FR testing in IVSA experiments provide verylimited information about the risk for abuse posed by novel CNS-active drug-candidates. It is well established that some recrea-tionally abused drugs have more powerful rewarding effects thanothers, and therefore, pose substantially greater risks for abuse. Byapplying PR/break-point analyses in addition to FR testing in IVSAexperiments, important additional information about the relativereinforcing efficacy of the drug-candidate can be obtainedproviding a more discriminating assessment of its predicted abusepotential.

4.9. Withdrawal-induced physical dependence

The final part of the Safety Pharmacology assessment is toinvestigate whether the drug-candidate produces pharmacologicaltolerance when given for a prolonged period and/or a syndrome ofphysical dependence on withdrawal. This evaluation is alwaysperformed in rats; the use of primates in tolerance/dependenceexperiments may be justifiable in drug-discrimination and IVSAexperiments in some situations, but it is not ethically acceptable fortolerance/dependence determinations. The drug-candidate is usu-ally are tested at two doses (one in the pharmacological rangerelevant to the therapeutic mechanism, and one that is supra-therapeutic [a dose that produces plasma exposure in rats that isat least 2 to 3� the maximum clinical exposure in humans][CHMP/EMA, 2006; CDER/FDA, 2017]). Positive Controls for tolerance/dependence experiments are generally an opiate, e.g., morphine, ora benzodiazepine, e.g., diazepam. The vehicle for the drug-candidate is the Negative Control. Depending on the biologicalhalf-life of the drug-candidate and Positive Control, the dosingregimens for these compounds will be tailored to optimise drugexposure over each day, e.g., once-daily or twice-daily dosing. Thepharmacological effects of these treatments are monitored in ratsusing a comprehensive panel of behavioural and physical symp-toms (we use a battery of 50 signs) and physiological measures, e.g.,food consumption, water intake, body weight and core tempera-ture. These signs are monitored on a regular basis, e.g., severaltimes/week or daily, to demonstrate that the Positive Control anddrug-candidate are producing clear evidence of pharmacologicaleffect in the rats.

During the 28-day on-dose phase of the experiment, theemergence of pharmacological tolerance can often be detected. Asan example, sedation that is initially observed after administeringopiates to rats rapidly disappears and is replaced by signs of hy-peractivity, e.g., increased locomotor activity, jumping, explosivemovements and attempts to escape from the home cage. At the endof the dosing phase, administration of the Positive Control, drug-candidate and vehicle are abruptly terminated and signs of phys-ical dependence are then monitored in the 7-day recovery periodusing the same panel of behavioural, physical and physiologicalsigns.

In the case of the serotonergic psychedelics, it is well knownthat pharmacological tolerance develops rapidly to the pharma-cological effects of the 5-HT2A agonists in animals (Darmani et al.,1992; Rangel-Barajas et al., 2014; Smith et al., 2014; Buchbornet al., 2015) and in humans (Abramson et al., 1956,1958; Isbell et al.,1959, 1961, Angrist et al., 1974). However, the consensus view is thedevelopment of pharmacological tolerance does not result in asyndrome of physical dependence onwithdrawal. A PubMed search


using the terms “5-HT2A” and “dependence” yielded no articlesdescribing physical dependence in either animals or humans. In thecase of the indirect 5-HT2A receptor agonists, e.g., MDMA, we haveobserved no tolerance to its pharmacological effects when using itas a discriminative cue in rats evenwhen given the 4e5 times/weekover a period of several months, and in addition, no signs ofphysical dependence when MDMA dosing is halted for a period ofdays or weeks. A similar lack of tolerance to MDMA's effects wasobserved by LeSage et al. (1993) when studying the acute andchronic effects of MDMA in pigeons responding using a delayed-matching-to-sample procedure. A review of the clinical literaturerevealed clear evidence that excessive MDMA use can lead to apsychologically dependent state in humans (e.g., Jansen, 1999; Wuet al., 2008; Degenhardt et al., 2010; Uosukainen et al., 2015), butnone of the research points to a syndrome of physical dependence.

Stafford et al. (1983) administered PCP to rats on an escalating3� daily dosing regimen up to a maximum of 72mg/kg/day for 4weeks. Two rats died on the regime showing it was well into thetoxicological range. When dosing was abruptly terminated, the ratsshowed unequivocal signs of physical dependence including facialtwitches, bruxism, increased susceptibility to audiogenic seizures,ptosis, WDS and aggressiveness. The withdrawal syndromeappeared ~24hr after termination of dosing and lasted for ~2 days.Similar withdrawal states have been observed in rats and monkeysafter prolonged access to self-administration of oral or intravenousPCP (Spain and Klingman, 1985; Balster and Woolverton, 1980;Carroll, 1987). The withdrawal syndrome and the time-course ofits duration are similar to those induced by opiates or benzodiaz-epines. However, a PubMed search yielded no reports of physicaldependence in human subjects after prolonged abuse of PCP. Therewere also no reports that prolonged administration of ketamineinduces physical dependence in either animals or humans. On thecontrary, there are several reports of human subjects ingestinglarge quantities of ketamine over prolonged periods, and whilethere is clear evidence of psychological dependence (the “k-hole”),no signs of physical dependence were observed (Jansen andDarracot-Cankovic, 2001; Pal et al., 2002; Muetzelfeldt et al.,2008). These findings raise some important points relating to as-sessments physical dependence liability for drug-candidates. It isevident that if the dose of PCP is pushed to extremis, it will inducephysical dependence; however, that syndrome has not beenobserved in human subjects. The safety pharmacology assessmentrequired by CHMP/EMA (2006) and CDER/FDA (2017) is notfocussed on toxicological phenomena, but on a determination ofthe actual risks posed to patients and drug abusers. Hence, thedoses used in these early studies are too high for that specificpurpose.

Gmerek and Woods (1986) administered U-50488, which is abrain-penetrant, potent and selective k-opioid receptor agonist, tomonkeys for a period of 135 days using an escalating dosingregimen (starting at 0.3mg/kg 4� daily finishing at 17.5mg/kg 6�daily). Over this period monkeys were deprived of drug for periods�15hr. Over the dosing period, complete tolerance developed to U-50488's sedative effect, but in the withdrawal periods only a verymild behavioural syndrome consisting of excessive scratching,grooming of cage mates, picking at fingers and toes, and yawningwas observed; therewere no incidences of aggression or abdominaldefence behaviour (a reaction to cramps seen with m-opioiddependence). Mello and Negus (1998) reported similarly mildsyndromes after terminating administration of 2 other k-opioidreceptor agonists, i.e., enadoline and Mr-2033, to monkeys after 28days on-dose. Recently, we evaluated whether CR-845, a potent,selective, k-opioid agonist with restricted brain entry would pro-duce a syndrome of dependence in rats after 28 days administra-tion at 5.0mg/kg iv (a dose that yielded plasma concentrations in



the rats that were greater than the targeted human clinical expo-sures by 10� and 50� for Cmax and AUC, respectively) (Heal et al.,2015a). On termination of dosing, clear evidence of physicaldependence was seen with the Reference Comparator, morphine,but nothing of note with CR-845. These findings are consistent withearlier reports cited in the section showing that although phar-macological tolerance to k-opioid receptor agonist occurs withrepeated dosing, it does not result in physical dependence onwithdrawal.

When the results are viewed overall they indicate that none ofthe psychedelic drugs, i.e., directly and indirectly acting 5-HT2Aagonists, non-competitive NMDA antagonists and k-opioid receptoragonists, produces withdrawal induced physical dependence.

4.9.1. Technical mattersCDER/FDA (2017) stipulates that drug-candidates should be

investigated for physical dependence liability in rats of both gen-ders. Since dosing is prolonged and neuroadaptation may be hor-monally influenced, there is the possibility of differences inwithdrawal syndromes between males and females. For thatreason, it is the only one of the Safety Pharmacology abuse/dependence evaluations where we believe it is scientifically andethically justifiable to conduct parallel studies in males and fe-males. However, having run several tolerance/dependence studiesin both genders, we have seen no difference between males andfemales in the studies that we have performed.

4.9.2. Key pointsThe role of the Positive Control, e.g., an opiate or benzodiaze-

pine, is as an internal standard to validate the abuse/dependencetesting methodology. Unless the drug-candidate is from the samepharmacological class as the Positive Control, the latter is notintended to serve as a direct comparator, or to represent a partic-ular pharmacological class of substance of abuse.

The potential of the drug-candidate to induce tolerance or tocause physical dependence is specific to the compound itself. It isrelated to many factors, e.g., rate of brain entry, duration of targetoccupancy, half-life, the presence of active metabolites, and mul-tiple pharmacological actions; it is not simply dependent on thepharmacological class of the drug.

Tolerance/dependence assessments are designed to detect andmeasure the severity of withdrawal-induced physical dependence.The behavioural parameters used in these evaluations lack thediscrimination to detect signs of psychological dependence, e.g.,anxiety, anhedonia, and drug-craving. Some limited information onpsychological dependence is provided by results from the IVSAexperiments which measure the ability of drug-candidates toinduce drug-seeking behaviour.

5. Conclusions

As set out in the introduction, there is clear evidence that allpsychedelic drugs pose risks for human abuse and for each specificclass there are indisputable incidences of harm to individuals andsociety. As described by other contributors to the Special Issue onPsychedelics, data are emerging from clinical trials to show thatcertain psychedelic drugs have the potential to be effective treat-ments in areas of high unmet clinical need, e.g., alcohol abuse,treatment-resistant depression, terminal depression/anxiety, andPTSD. In that regard, they are no different frommany other types ofdrug in clinical use, e.g., opiates, barbiturates, benzodiazepines, andstimulants. For the known psychedelic drugs undergoing clinicalevaluation and novel drug-candidates with psychedelic properties,there needs to be a systematic non-clinical and clinical assessmentof their potential for abuse and dependence to replace the


patchwork of information that we have currently. It is also clearthat psychedelic drugs pose specific challenges for non-clinicalabuse/dependence assessments, but in that regard those chal-lenges are no more or less difficult than those applying to manyother CNS-active drug-candidates that are undergoing clinicaldevelopment.

References

Abramson, H.A., Jarvik, M.E., Gorin, M.H., Hirsch, M.W., 1956. Lysergic acid dieth-ylamide (LSD-25): XVII. Tolerance development and its relationship to a theoryof psychosis. J. Psychol. 41, 81e105.

Abramson, H.A., Sklarofsky, B., Baron, M.O., Fremont-Smith, N., 1958. Lysergic aciddiethylamide (LSD-25) antagonists. II. Development of tolerance in man to LSD-25 by prior administration of MLD-41 (1-methyl-d-lysergic acid diethylamide).Arch. Neurol. Psychiatry 79, 201e207.

Adamo, J.E., Bauer, G., Berro, M., Burnett, B.K., Hartman, K.A., Masiello, L.M.,Moorman-White, D., Rubinstein, E.P., Schuff, K.G., 2012. A roadmap for aca-demic health centers to establish good laboratory practice-compliant infra-structure. Acad. Med. 87, 279e284.

Aigner, T.G., Balster, R.L., 1979. Rapid substitution procedure for intravenous drugself-administration studies in rhesus monkeys. Pharmacol. Biochem. Behav. 10,105e112.

Andrade, E.L., Bento, A.F., Cavalli, J., Oliveira, S.K., Schwanke, R.C., Siqueira, J.M.,Freitas, C.S., Marcon, R., Calixto, J.B., 2016. Non-clinical studies in the process ofnew drug development - Part II: good laboratory practice, metabolism, phar-macokinetics, safety and dose translation to clinical studies. Braz. J. Med. Biol.Res. 49 e5646.

Angrist, B., Rotrosen, J., Gershon, S., 1974. Assessment of tolerance to the halluci-nogenic effects of DOM. Psychopharmacology 36, 203e207.

Ansonoff, M.A., Zhang, J., Czyzyk, T., Rothman, R.B., Stewart, J., Xu, H., Zjwiony, J.,Siebert, D.J., Yang, F., Roth, B.L., Pintar, J.E., 2006. Antinociceptive and hypo-thermic effects of salvinorin A are abolished in a novel strain of k-opioidreceptor-1 knockout mice. J. Pharmacol. Exp. Ther. 318, 641e648.

Appel, J.B., White, F.J., Holohean, A.M., 1982. Analyzing mechanism(s) of halluci-nogenic drug action with drug discrimination procedures. Neurosci. Biobehav.Rev. 6, 529e536.

Arantes, L.C., Júnior, E.F., de Souza, L.F., Cardoso, A.C., Alcantara, T.L.F., Li~ao, L.M.,Machado, Y., Lordeiro, R.A., Neto, J.C., Andrade, A.F.B., 2017. 25I-NBOH: a newpotent serotonin 5-HT2A receptor agonist identified in blotter paper seizures inBrazil. Forensic Toxicol. 35, 408e414.

Arnt, J., 1992. Sertindole and several antipsychotic drugs differentially inhibit thediscriminative stimulus effects of amphetamine, LSD and St 587 in rats. Behav.Pharmacol. 3, 11e18.

Arnt, J., Hyttel, J., 1989. Facilitation of 8-OHDPAT-induced forepaw treading of ratsby the 5-HT2 agonist doi. Eur. J. Pharmacol. 161, 45e51.

Arnt, J., Hyttel, J., Larsen, J.J., 1984. The citalopram/5-HTP-induced head shakesyndrome is correlated to 5-HT2 receptor affinity ad also influenced by othertransmitters. Acta Pharmacol. Toxicol. 55, 363e372.

Ator, N.A., Griffiths, R.R., 2003. Principles of drug abuse liability assessment inlaboratory animals. Drug Alcohol Depend. 70 (3 Suppl. l), S55eS72.

B€ackberg, M., Beck, O., Helander, A., 2015. Phencyclidine analog use in Sweden -intoxication cases involving 3-MeO-PCP and 4-MeO-PCP from the STRIDAproject. Clin. Toxicol. 53, 856e864.

Baggott, M.J., Erowid, E., Erowid, F., Galloway, G.P., Mendelson, J., 2010. Use patternsand self-reported effects of Salvia divinorum: an internet-based survey. DrugAlcohol Depend. 111, 250e256.

Baker, L.E., Makhay, M.M., 1996. Effects of (þ)-fenfluramine on 3,4-methylenedioxymethamphetamine (MDMA) discrimination in rats. Pharma-col. Biochem. Behav. 53, 455e461.

Baker, L.E., Panos, J.J., Killinger, B.A., Peet, M.M., Bell, L.M., Haliw, L.A., Walker, S.L.,2009. Comparison of the discriminative stimulus effects of salvinorin A and itsderivatives to U69,593 and U50,488 in rats. Psychopharmacology 203, 203e211.

Baker, L.E., Taylor, M.M., 1997. Assessment of the MDA and MDMA optical isomers ina stimulant-hallucinogen discrimination. Pharmacol. Biochem. Behav. 57,737e748.

Baker, L.E., Virden, T.B., Miller, M.E., Sullivan, C.L., 1997. Time-course analysis of thediscriminative stimulus effects of the optical isomers of 3,4-methylenedioxymethamphetamine (MDMA). Pharmacol. Biochem. Behav. 58,505e516.

Balsara, J.J., Bapat, T.R., Nandal, N.V., Gada, V.P., Chandorkar, A.G., 1986. Head-twitchresponse induced by ergometrine in mice: behavioural evidence for directstimulation of central 5-hydroxytryptamine receptors by ergometrine. Psy-chopharmacology 88, 275e278.

Balster, R.L., 1991. Drug abuse potential evaluation in animals. Br. J. Addict. 86,1549e1558.

Balster, R.L., Nicholson, K.L., Sanger, D.J., 1994. Evaluation of the reinforcing effects ofeliprodil in rhesus monkeys and its discriminative stimulus effects in rats. DrugAlcohol Depend. 35, 211e216.

Balster, R.L., Willetts, J., 1988. Receptor mediation of the discriminative stimulusproperties of phencyclidine and sigma-opioid agonists. Psychopharmacol. Ser.4, 122e135.


http://refhub.elsevier.com/S0028-3908(18)30057-1/sref1







































































































Balster, R.L., Woolverton, W.L., 1979. Intravenous phencyclidine self-administrationby rhesus monkeys leading to physical dependence. NIDA Res. Monogr. 27,205e211.

Balster, R.L., Woolverton, W.L., 1980. Continuous-access phencyclidine self-administration by rhesus monkeys leading to physical dependence. Psycho-pharmacology 70, 5e10.

Baron, S.P., Woods, J.H., 1995. Competitive and uncompetitive N-methyl-D-aspartateantagonist discriminations in pigeons: CGS 19755 and phencyclidine. Psycho-pharmacology 118, 42e51.

Beardsley, P.M., Balster, R.L., Harris, L.S., 1986. Self-administration of methyl-enedioxymethamphetamine (MDMA) by rhesus monkeys. Drug AlcoholDepend. 18, 149e157.

Beardsley, P.M., Hayes, B.A., Balster, R.L., 1990. The self-administration of MK-801can depend upon drug-reinforcement history, and its discriminative stimulusproperties are phencyclidine-like in rhesus monkeys. J. Pharmacol. Exp. Ther.252, 953e959.

Bedard, P., Pycock, C.J., 1977. “Wet-dog” shake behaviour in the rat: a possiblequantitative model of central 5-hydroxytryptamine activity. Neuropharma-cology 16, 663e670.

Bednarczyk, B., Vetulani, J., 1978. Antagonism of clonidine to shaking behaviour inmorphine abstinence and to head twitches produced by serotonergic agents inthe rat. Pol. J. Pharmacol. Pharm. 30, 307e322.

Benneyworth, M.A., Smith, R.L., Barrett, R.J., Sanders-Bush, E., 2005. Complexdiscriminative stimulus properties of (þ)lysergic acid diethylamide (LSD) inC57Bl/6J mice. Psychopharmacology 179, 854e862.

Berquist, M.D., Hyatt, W.S., Bauer-Erickson, J., Gannon, B.M., Norwood, A.P.,Fantegrossi, W.E., 2017b. Phencyclidine-like in vivo effects of methoxetamine inmice and rats. Neuropharmacology, 2017 Aug 19. pii: S0028e3908(17)30395-7.Epub ahead of print.

Berquist, M.D., Thompson, N.A., Baker, L.E., 2017a. Evaluation of training dose inmale Sprague-Dawley rats trained to discriminate 4-methylmethcathinone.Psychopharmacology 234, 3271e3278.

Botanas, C.J., de la Pe~na, J.B., Dela Pe~na, I.J., Tampus, R., Yoon, R., Kim, H.J., Lee, Y.S.,Jang, C.G., Cheong, J.H., 2015. Methoxetamine, a ketamine derivative, producedconditioned place preference and was self-administered by rats: evidence of itsabuse potential. Pharmacol. Biochem. Behav. 133, 31e36.

Brady, K.T., Balster, R.L., 1982. Discriminative stimulus properties of ketamine ste-reoisomers in phencyclidine-trained rats. Pharmacol. Biochem. Behav. 17,291e295.

Braida, D., Limonta, V., Capurro, V., Fadda, P., Rubino, T., Mascia, P., Zani, A., Gori, E.,Fratta, W., Parolaro, D., Sala, M., 2008. Involvement of kappa-opioid andendocannabinoid system on Salvinorin A-induced reward. Biol. Psychiatry 63,286e292.

Broadbear, J.H., Winger, G., Woods, J.H., 2004. Self-administration of fentanyl,cocaine and ketamine: effects on the pituitary-adrenal axis in rhesus monkeys.Psychopharmacology 176, 398e406.

Broqua, P., Wettstein, J.G., Rocher, M.N., Rivi�ere, P.J., Dahl, S.G., 1998. The discrimi-native stimulus properties of U50,488 and morphine are not shared by fedo-tozine. Eur. Neuropsychopharmacol. 8, 261e266.

Brown, D.J., 2009. Neuroscience. Salvia on schedule. Sci. Am. 301, 20e21.Buchborn, T., Schr€oder, H., Dieterich, D.C., Grecksch, G., H€ollt, V., 2015. Tolerance to

LSD and DOB induced shaking behaviour: differential adaptations of fronto-cortical 5-HT2A and glutamate receptor binding sites. Behav. Brain Res. 281,62e68.

Buckley, N.W., Gosden, J., Johnson, E.L., Smith, S.L., Heal, D.J., 2014. A progressiveratio determination of the relative reinforcing effect of methylphenidate versuscocaine by intravenous self-administration testing in rats. In: Proceedings ofCollege on Problems of Drug Dependence Meeting, Puerto Rico, June 14-19,2017, p. 89.

Burns, R.S., Lerner, S.E., 1978. Phencyclidine deaths. J. Am. Coll. Emerg. Phys. 7,135e141.

Butelman, E.R., Harris, T.J., Kreek, M.J., 2004. The plant-derived hallucinogen, sal-vinorin A, produces kappa-opioid agonist-like discriminative effects in rhesusmonkeys. Psychopharmacology 172, 220e224.

Butelman, E.R., Rus, S., Prisinzano, T.E., Kreek, M.J., 2010. The discriminative effectsof the kappa-opioid hallucinogen salvinorin A in nonhuman primates: disso-ciation from classic hallucinogen effects. Psychopharmacology 210, 253e262.

Cabeza de Vaca, S., Carr, K.D., 1998. Food restriction enhances the central rewardingeffect of abused drugs. J. Neurosci. 18, 7502e7510.

Calderon, S., Giarola, A., Heal, D., 2015. Regulatory framework and guidance to theevaluation of the abuse liability of drugs in the United States and Europe. In:Markgraf, C.G., Hudzik, T.J., Compton, D.R. (Eds.), Non-clinical Assessment ofAbuse Potential for New Pharmaceuticals. Academic Press, pp. 245e268.

Calderon, S.N., Klein, M., 2017. A regulatory perspective on the evaluation ofhallucinogen drugs for human use. Neuropharmacology. Nov 24. pii:S0028e3908(17)30537-3. E-pub ahead of print.

Callahan, P.M., Appel, J.B., 1988. Differences in the stimulus properties of 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine inanimals trained to discriminate hallucinogens from saline. J. Pharmacol. Exp.Ther. 246, 866e870.

Campbell, U.C., Thompson, S.S., Carroll, M.E., 1998. Acquisition of oral phencyclidine(PCP) self-administration in rhesus monkeys: effects of dose and an alternativenon-drug reinforcer. Psychopharmacology 137, 132e138.

Carey, G.J., Bergman, J., 2001. Enadoline discrimination in squirrel monkeys: effectsof opioid agonists and antagonists. J. Pharmacol. Exp. Ther. 297, 215e223.


Carr, K.D., Kim, G.Y., Cabeza de Vaca, S., 2000. Chronic food restriction in ratsaugments the central rewarding effect of cocaine and the delta1 opioid agonist,DPDPE, but not the delta2 agonist, deltorphin-II. Psychopharmacology 152,200e207.

Carroll, M.E., 1987. A quantitative assessment of phencyclidine dependence pro-duced by oral self-administration in rhesus monkeys. J. Pharmacol. Exp. Ther.242, 405e412.

Carroll, M.E., Batulis, D.K., Landry, K.L., Morgan, A.D., 2005. Sex differences in theescalation of oral phencyclidine (PCP) self-administration under FR and PRschedules in rhesus monkeys. Psychopharmacology 180, 414e426.

Carroll, M.E., France, C.P., Meisch, R.A., 1981. Intravenous self-administration ofetonitazene, cocaine and phencyclidine in rats during food deprivation andsatiation. J. Pharmacol. Exp. Ther. 217, 241e247.

Carroll, M.E., Meisch, R.A., 1980. Oral phencyclidine (PCP) self-administration inrhesus monkeys: effects of feeding conditions. J. Pharmacol. Exp. Ther. 214,339e346.

Carroll, M.E., Roth, M.E., Voeller, R.K., Nguyen, P.D., 2000. Acquisition of oralphencyclidine self-administration in rhesus monkeys: effect of sex. Psycho-pharmacology 149, 401e408.

CDER/FDA, U.S. Department of Health and Human Services, Food and DrugAdministration, Centre for Drug Evaluation and Research, 2010. Draft Guidancefor Industry on the Assessment of Abuse Potential of Drugs. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM198650.pdf.

CDER/FDA, U.S. Department of Health and Human Services, Food and DrugAdministration, Centre for Drug Evaluation and Research, 2017. Assessment ofAbuse Potential of Drugs. Guidance for Industry. www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm334743.pdf.

Chambers, C.D., Inciardi, J.A., Stephens, R.C., 1971. A critical review of pentazocineabuse. HSMHA Health Rep. 86, 627e636.

Chavkin, C., Sud, S., Jin, W., Stewart, J., Zjawiony, J.K., Siebert, D.J., Toth, B.A.,Hufeisen, S.J., Roth, J.K., 2004. Salvinorin A, an active component of the hallu-cinogen sage salvia divinorum is a highly efficacious k-opioid receptor agonist:structural and functional considerations. J. Pharmacol. Exp. Ther. 308,1197e1203.

Chiamulera, C., Armani, F., Mutti, A., Fattore, L., 2016. The ketamine analoguemethoxetamine generalizes to ketamine discriminative stimulus in rats. Behav.Pharmacol. 27 (2e3 Spec Issue), 204e210.

Chien, C.C., Pasternak, G.W., 1993. Functional antagonism of morphine analgesia by(þ)-pentazocine: evidence for an anti-opioid sigma 1 system. Eur. J. Pharmacol.250, R7eR8.

CHMP/EMA, European Medicine Agency, Committee for Medicinal Products forHuman Use, 2006. Guidance of the Non-clinical Investigation of the Depen-dence Potential of Medicinal Products. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003360.pdf.

Chojnacka-W�ojcik, E., Kłodzi�nska, A., 1997. Involvement of 5-hydroxytryptamine 2A(5-HT2A) receptors in the mediation of the discriminative stimulus properties of(þ/-)DOI in rats. Pol. J. Pharmacol. 49, 299e304.

Clayton, J.A., 2016. Studying both sexes: a guiding principle for biomedicine. FASEBJ. 30, 519e524.

Clayton, J.A., Collins, F.S., 2014. NIH to balance sex in cell and animal studies. Nature509, 282e283.

Colpaert, F.C., 1995. Drug discrimination: no evidence for tolerance to opiates.Pharmacol. Rev. 1995 (47), 605e629.

Colpaert, F.C., 1999. Drug discrimination in neurobiology. Pharmacol. Biochem.Behav. 64, 337e345.

Colpaert, F.C., Niemegeers, C.J., Janssen, P.A., 1982. A drug discrimination analysis oflysergic acid diethylamide (LSD): in vivo agonist and antagonist effects ofpurported 5-hydroxytryptamine antagonists and of pirenperone, a LSD-antag-onist. J. Pharmacol. Exp. Ther. 221, 206e214.

Corazza, O., Assi, S., Schifano, F., 2013. The evolution of the recreational use of ke-tamine and methoxetamine. From “Special K” to “Special M” CNS Neurosci.Ther. 19, 454e460.

Corne, S.J., Pickering, R.W., Warner, B.T., 1963. A method for assessing the effects ofdrugs on the central actions of 5-hydroxytryptamine. Br. J. Pharmacol. Che-mother. 20, 106e120.

Corne, S.J., Pickering, R.W., 1967. A possible correlation between drug-inducedhallucinations in man and a behavioural response in mice. Psychopharmacol-ogy 11, 65e78.

Craft, R.M., Kalivas, P.W., Stratmann, J.A., 1996. Sex differences in discriminativestimulus effects of morphine in the rat. Behav. Pharmacol. 7, 764e778.

Craft, R.M., Stratmann, J.A., 1996. Discriminative stimulus effects of cocaine in fe-male versus male rats. Drug Alcohol Depend. 42, 27e37.

Craig, C.L., Loeffler, G.H., 2014. The ketamine analog methoxetamine: a newdesigner drug to threaten military readiness. Mil. Med. 179, 1149e1157.

Creehan, K.M., Vandewater, S.A., Taffe, M.A., 2015. Intravenous self-administrationof mephedrone, methylone and MDMA in female rats. Neuropharmacology92, 90e97.

Dare, G.L., Goldney, R.D., 1976. Fenfluramine abuse. Med. J. Aust. 2, 537e540.Darmani, N.A., Martin, B.R., Glennon, R.A., 1992. Behavioral evidence for differential

adaptation of the serotonergic system after acute and chronic treatment with(þ/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) or ketanserin.J. Pharmacol. Exp. Ther. 262, 692e698.

Degenhardt, L., Bruno, R., Topp, L., 2010. Is ecstasy a drug of dependence? DrugAlcohol Depend. 107, 1e10.

















































































































































http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM198650.pdf



http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm334743.pdf

http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm334743.pdf




















http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003360.pdf

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003360.pdf































































DeLarge, A.F., Erwin, L.L., Winsauer, P.J., 2017. Atypical binding at dopamine andserotonin transporters contribute to the discriminative stimulus effects ofmephedrone. Neuropharmacology 119, 62e75.

DiazGranados, N., Ibrahim, L.A., Brutsche, N.E., Ameli, R., Henter, I.D.,Luckenbaugh, D.A., Machado-Vieira, R., Zarate Jr., C.A., 2010. Rapid resolution ofsuicidal ideation after a single infusion of an N-methyl-D-aspartate antagonistin patients with treatment-resistant major depressive disorder. J. Clin. Psychi-atry 71, 1605e1611.

Dougherty, J.P., Aloyo, V.J., 2011. Pharmacological and behavioral characterization ofthe 5-HT2A receptor in C57BL/6N mice. Psychopharmacology 215, 581e593.

Dolan, S.B., Forster, M.J., Gatch, M.B., 2017. Discriminative stimulus and locomotoreffects of para-substituted and benzofuran analogs of amphetamine. DrugAlcohol Depend. 180, 39e45.

Eshleman, A.J., Forster, M.J., Wolfrum, K.M., Johnson, R.A., Janowsky, A., Gatch, M.B.,2014. Behavioral and neurochemical pharmacology of six psychoactivesubstituted phenethylamines: mouse locomotion, rat drug discrimination andin vitro receptor and transporter binding and function. Psychopharmacology231, 875e888.

Ettrup, A., Holm, S., Hansen, M., Wasim, M., Santini, M.A., Palner, M., Madsen, J.,Svarer, C., Kristensen, J.L., Knudsen, G.M., 2013. Preclinical safety assessment ofthe 5-HT2A receptor agonist PET radioligand [11C]Cimbi-36. Mol. Imag. Biol. 15,376e383.

Evans, S.M., Johanson, C.E., 1986. Discriminative stimulus properties of (þ/-)-3,4-methylenedioxymethamphetamine and (þ/-)-3,4-methylenedioxyamphetamine in pigeons. Drug Alcohol Depend. 18, 159e164.

Fantegrossi, W.E., Harrington, A.W., Eckler, J.R., Arshad, S., Rabin, R.A., Winter, J.C.,Coop, A., Rice, K.C., Woods, J.H., 2005a. Hallucinogen-like actions of 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7) in mice and rats. Psy-chopharmacology 181, 496e503.

Fantegrossi, W.E., Gannon, B.M., Zimmerman, S.M., Rice, K.C., 2013. In vivo effects ofabused 'bath salt' constituent 3,4-methylenedioxypyrovalerone (MDPV) inmice: drug discrimination, thermoregulation, and locomotor activity. Neuro-psychopharmacology 38, 563e573.

Fantegrossi, W.E., Gray, B.W., Bailey, J.M., Smith, D.A., Hansen, M., Kristensen, J.L.,2015. Hallucinogen-like effects of 2-([2-(4-cyano-2,5-dimethoxyphenyl)ethyl-amino]methyl)phenol (25CN-NBOH), a novel N-benzylphenethylamine with100-fold selectivity for 5-HT2A receptors, in mice. Psychopharmacology 232,1039e1047.

Fantegrossi, W.E., Kiessel, C.L., De la Garza, R., Woods, J.H., 2005b. Serotonin syn-thesis inhibition reveals distinct mechanisms of action for MDMA and its en-antiomers in the mouse. Psychopharmacology 181, 529e536.

Fantegrossi, W.E., Kiessel, C.L., Leach, P.T., Van Martin, C., Karabenick, R.L., Chen, X.,Ohizumi, Y., Ullrich, T., Rice, K.C., Woods, J.H., 2004c. Nantenine: an antagonistof the behavioral and physiological effects of MDMA in mice. Psychopharma-cology 173, 270e277.

Fantegrossi, W.E., Reissig, C.J., Katz, E.B., Yarosh, H.L., Rice, K.C., Winter, J.C., 2008.Hallucinogen-like effects of N,N-dipropyltryptamine (DPT): possible mediationby serotonin 5-HT1A and 5-HT2A receptors in rodents. Pharmacol. Biochem.Behav. 88, 358e365.

Fantegrossi, W.E., Simoneau, J., Cohen, M.S., Zimmerman, S.M., Henson, C.M.,Rice, K.C., Woods, J.H., 2010. Interaction of 5-HT2A and 5-HT2C receptors in R(-)-2,5-dimethoxy-4-iodoamphetamine-elicited head twitch behavior in mice.J. Pharmacol. Exp. Ther. 335, 728e734.

Fantegrossi, W.E., Ullrich, T., Rice, K.C., Woods, J.H., Winger, G., 2002. 3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) and its stereoisomersas reinforcers in rhesus monkeys: serotonergic involvement. Psychopharma-cology 161, 356e364.

Fantegrossi, W.E., Woods, J.H., Winger, G., 2004b. Transient reinforcing effects ofphenylisopropylamine and indolealkylamine hallucinogens in rhesus monkeys.Behav. Pharmacol. 15, 149e157.

Fantegrossi, W.E., Woolverton, W.L., Kilbourn, M., Sherman, P., Yuan, J.,Hatzidimitriou, G., Ricaurte, G.A., Woods, J.H., Winger, G., 2004a. Behavioral andneurochemical consequences of long-term intravenous self-administration ofMDMA and its enantiomers by rhesus monkeys. Neuropsychopharmacology 29,1270e1281.

FDA, Department of Health and Human Services, Food and Drug Administration,August 24, 2016. Good Laboratory Practice for Nonclinical Laboratory Studies.Proposed Rule. 81 FR 58342. https://www.federalregister.gov/documents/2016/08/24/2016-19875/good-laboratory-practice-for-nonclinical-laboratory-studies. (Accessed 5 January 2018).

Feder, A., Parides, M.K., Murrough, J.W., Perez, A.M., Morgan, J.E., Saxena, S.,Kirkwood, K., Aan Het Rot, M., Lapidus, K.A., Wan, L.B., Iosifescu, D.,Charney, D.S., 2014. Efficacy of intravenous ketamine for treatment of chronicpost-traumatic stress disorder: a randomized clinical trial. JAMA Psychiatry 71,681e688.

Fonck, C., Easter, A., Pietras, M.R., Bialecki, R.A., 2015. CNS adverse effects: fromfunctional observation battery/Irwin tests to electrophysiology. Handb. Exp.Pharmacol. 229, 83e113.

Fox, M.A., French, H.T., LaPorte, J.L., Blackler, A.R., Murphy, D.L., 2010. The serotonin5-HT2A receptor agonist TCB-2: a behavioral and neurophysiological analysis.Psychopharmacology 212, 13e23.

France, C.P., Woods, J.H., 1990. Discriminative stimulus effects of opioid agonists inmorphine-dependent pigeons. J. Pharmacol. Exp. Ther. 254, 626e632.

Gannon, B.M., Fantegrossi, W.E., 2016. Cocaine-like discriminative stimulus effectsof mephedrone and naphyrone in mice. J. Drug Alcohol Res. 5 pii: 236009.


Gauvin, D.V., Zimmermann, Z.J., Kallman, M.J., 2016. Current FDA regulatory guid-ance on the conduct of drug discrimination studies for NDA review: does thescientific literature support recent recommendations? Drug Alcohol Depend.168, 307e319.

Glennon, R.A., 1999. Arylalkylamine drugs of abuse: an overview of drug discrim-ination studies. Pharmacol. Biochem. Behav. 64, 251e256.

Glennon, R.A., 1986. Discriminative stimulus properties of the serotonergic agent 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Life Sci. 39, 825e830.

Glennon, R.A., Misenheimer, B.R., 1989. Stimulus effects of N-monoethyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDE) and N-hydroxy-1-(3,4-methylenedioxyphenyl)-2-aminopropane (N-OH-MDA) in rats trained todiscriminate MDMA from saline. Pharmacol. Biochem. Behav. 33, 909e912.

Gmerek, D.E., Woods, J.H., 1986. Kappa receptor mediated opioid dependence inrhesus monkeys. Life Sci. 39, 987e992.

Gonz�alez, D., Riba, J., Bouso, J.C., G�omez-Jarabo, G., Barbanoj, M.J., 2006. Pattern ofuse and subjective effects of Salvia divinorum among recreational users. DrugAlcohol Depend. 85, 157e162.

Gonz�alez-Maeso, J., Weisstaub, N.V., Zhou, M., Chan, P., Ivic, L., Ang, R., Lira, A.,Bradley-Moore, M., Ge, Y., Zhou, Q., Sealfon, S.C., Gingrich, J.A., 2007. Halluci-nogens recruit specific cortical 5-HT2A receptor-mediated signaling pathways toaffect behavior. Neuron 53, 439e452.

Goodwin, A.K., Baker, L.E., 2000. A three-choice discrimination procedure dissoci-ates the discriminative stimulus effects of d-amphetamine and (þ/-)-MDMA inrats. Exp. Clin. Psychopharmacol. 8, 415e423. PubMed PMID: 10975633.

Goodwin, A.K., Pynnonen, D.M., Baker, L.E., 2003. Serotonergic-dopaminergicmediation of MDMA's discriminative stimulus effects in a three-choicediscrimination. Pharmacol. Biochem. Behav. 74, 987e995.

Gosden, J., Heal, D.J., 1997. Evaluation of sibutramine, its active metabolites anddexfenfluramine in the MDMA-cued drug-discrimination test. Obes. Res. 5(Suppl. 1), P49, 56S.

Gray, T., Dass, M., 2012. Ketamine cystitis: an emerging diagnostic and therapeuticchallenge. Br. J. Hosp. Med. 73, 576e579.

Green, A.R., Heal, D.J., 1985. The effects of drugs on serotonin-mediated behaviouralmodels. In: Green, A.R. (Ed.), Neuropharmacology of Serotonin. Oxford Uni-versity Pres, Oxford, p. 326365.

Gresch, P.J., Barrett, R.J., Sanders-Bush, E., Smith, R.L., 2007. 5-Hydroxytryptamine(serotonin) 2A receptors in rat anterior cingulate cortex mediate the discrimi-native stimulus properties of d-lysergic acid diethylamide. J. Pharmacol. Exp.Ther. 320, 662e669.

Halberstadt, A.L., Geyer, M.A., 2014. Effects of the hallucinogen 2,5-dimethoxy-4-iodophenethylamine (2C-I) and superpotent N-benzyl derivatives on the headtwitch response. Neuropharmacology 77, 200e207.

Handley, S.L., Brown, J., 1982. Effects of 5-hydroxytryptamine-induced head-twitchof drugs with selective actions on a1- and a2-adrenoceptors. Neuropharma-cology 21, 507e510.

Harvey, E.L., Baker, L.E., 2016. Differential effects of 3,4-methylenedioxypyrovalerone (MDPV) and 4-methylmethcathinone (mephe-drone) in rats trained to discriminate MDMA or a d-amphetamine þ MDMAmixture. Psychopharmacology 233, 673e680.

Heal, D.J., Buckley, N.W., Gosden, J., Slater, N., France, C.P., Hackett, D., 2013.A preclinical evaluation of the subjective and reinforcing properties of lisdex-amfetamine in comparison to d-amfetamine, methylphenidate and modafinil.Neuropharmacology 73, 348e358.

Heal, D.J., Frankland, A.T.J., Gosden, J., Hutchins, L.J., Prow, M.R., Luscombe, G.P.,Buckett, W.R., 1992a. A comparison of the effects of sibutramine hydrochloride,bupropion and methamphetamine on dopaminergic function: evidence thatdopamine is not a pharmacological target for sibutramine. Psychopharmacol-ogy 107, 303e309.

Heal, D.J., Gosden, J., Slater, N., Holland, S.J., Slade, J., Spencer, R., Menzaghi, F.,Smith, S.L., 2016. Investigation of the discriminative and reinforcing propertiesof the k-opioid receptor agonist CR845. In: Proceedings of Society for Neuro-sciences Meeting, San Diego, Nov 12-16, 2016. Abst 819.04.

Heal, D.J., Goddard, S., Gosden, J., Dykes, S., Brammer, R., Spencer, R., Menzaghi, F.,2015a. A preclinical evaluation of the potential of the k-opioid receptor agonistCR845 to induce tolerance and a syndrome of dependence on withdrawal inrats. In: Proceedings of American College of NeuropsychopharmacologyMeeting, Dec 6-10, Hollywood FL, 2015. M129.

Heal, D.J., Luscombe, G.P., Martin, K.F., 1992b. Pharmacological identification of 5-HTreceptor subtypes using behavioural models. In: Marsden, C.A., Heal, D.J. (Eds.),Central Serotonin Receptors and Psychotropic Drugs. Blackwell Scientific, Ox-ford, pp. 56e99, 1992.

Heal, D.J., Philpot, J., Molyneux, S.G., Metz, A., 1985. Intracerebroventricularadministration of 5,7-dihydroxytryptamine to mice increases both head-twitchresponse and the number of cortical 5-HT2 receptors. Neuropharmacology 24,1201e1205.

Heal, D.J., Philpot, J., O'Shaughnessy, K.M., Davies, C.L., 1986. The influence of centralnoradrenergic function on 5-HT2-mediated head-twitch responses in mice:possible implications for the actions of antidepressant drugs. Psychopharma-cology 89, 414e420.

Heal, D.J., Smith, S.L., Dean, R.L., Todtenkopf, M.S., 2017. Investigation of the possiblereinforcing effects of samidorphan and naltrexone by fixed and progressiveratio intravenous self-administration testing in rats. In: Proceedings of Amer-ican College of Neuropsychopharmacology, Palm Springs, Dec 3-7, 2017.

Heal, D.J., Smith, S.L., Gosden, J., Johnson, E.L., Hallam, M., Dean, R.L.,Todtenkopf, M.S., 2015b. Evaluation of remifentanil and morphine as reference






























































































https://www.federalregister.gov/documents/2016/08/24/2016-19875/good-laboratory-practice-for-nonclinical-laboratory-studies








































































































































reinforcers in a rat intravenous self-administration procedure. In: Proceedingsof College on Problems of Drug Dependence Meeting, Phoenix, June 13-18,2015, p. 266.

Hoffmeister, F., 1988. A comparison of the stimulus effects of codeine in rhesusmonkeys under the contingencies of a two lever discrimination task and a crossself-administration paradigm: tests of generalization to pentazocine, bupre-norphine, tilidine, and different doses of codeine. Psychopharmacology 94,315e320.

Hooker, J.M., Xu, Y., Schiffer, W., Shea, C., Carter, P., Fowler, J.S., 2008. Pharmacoki-netics of the potent hallucinogen, salvinorin A in primates parallels the rapidonset, short duration of effects in humans. Neuroimage 41, 1044e1050.

Howell, L.L., Fantegrossi, W.E., 2009. Intravenous drug self-administration in non-human primates. In: Buccafusco, J.J. (Ed.), Methods of Behavior Analysis inNeuroscience, second ed. CRC Press/Taylor & Francis, Boca Raton (FL). Chapter 9.

Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J.,Saxena, P.R., Humphrey, P.P., 1994. International Union of Pharmacology clas-sification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol. Rev. 46,157e203.

Huang, X., Huang, K., Zheng, W., Beveridge, T.J., Yang, S., Li, X., Li, P., Zhou, W., Liu, Y.,2015. The effects of GSK-3b blockade on ketamine self-administration andrelapse to drug-seeking behavior in rats. Drug Alcohol Depend. 147, 257e265.

Hunter, J.C., Leighton, G.E., Meecham, K.G., Boyle, S.J., Horwell, D.C., Rees, D.C.,Hughes, J., 1990. CI-977, a novel and selective agonist for the k-opioid receptor.Br. J. Pharmacol. 101, 183e189.

ICH Guidance for Industry, 2001. S7A Safety Pharmacology Studies for HumanPharmaceuticals. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm074959.pdf.

Isaacs, S.O., Martin, P., Washington, J.A., 1986. Phencyclidine (PCP) abuse. A close-uplook at a growing problem. Oral Surg. Oral Med. Oral Pathol. 61, 126e129.

Isbell, H., Miner, E.J., Logan, C.R., 1959. Cross tolerance between D-2-brom-lysergicacid diethylamide (BOL-148) and the D-diethylamide of lysergic acid (LSD-25).Psychopharmacology 1, 109e116.

Isbell, H., Wolbach, A.B., Wikler, A., Miner, E.J., 1961. Cross tolerance between LSDand psilocybin. Psychopharmacology 2, 147e159.

Iwamoto, E.T., 1986. Place-conditioning properties of m, k and s-opioid agonists.Alcohol Drug Res. 6, 327e339.

Jacob, M.S., Carlen, P.L., Marshman, J.A., Sellers, E.M., 1981. Phencyclidine ingestion:drug abuse and psychosis. Int. J. Addict. 16, 749e758.

Jansen, K.L., 1999. Ecstasy (MDMA) dependence. Drug Alcohol Depend. 53, 121e124.Jansen, K.L., Darracot-Cankovic, R., 2001. The nonmedical use of ketamine, part two:

a review of problem use and dependence. J. Psychoact. Drugs 33, 151e158.Jasinski, D.R., Martin, W.R., Hoeldtke, R.D., 1970. Effects of short- and long-term

administration of pentazocine in man. Clin. Pharmacol. Ther. 11, 385e403.Johanson, C.E., 1990. The evaluation of the abuse liability of drugs. Drug Saf. 5

(Suppl. 1), 46e57.Johnson, M.P., Frescas, S.P., Oberlender, R., Nichols, D.E., 1991. Synthesis and phar-

macological examination of 1-(3-methoxy-4-methylphenyl)-2-aminopropaneand 5-methoxy-6-methyl-2-aminoindan: similarities to 3,4-(methylenedioxy)methamphetamine (MDMA). J. Med. Chem. 34, 1662e1668.

Jung, M.E., Wallis, C.J., Gatch, M.B., Lal, H., 2000. Sex differences in nicotine sub-stitution to a pentylenetetrazol discriminative stimulus during ethanol with-drawal in rats. Psychopharmacology 149, 235e240.

Katz, J.L., Hiranita, T., Kopajtic, T.A., Rice, K.C., Mesangeau, C., Narayanan, S.,Abdelazeem, A.H., McCurdy, C.R., 2016. Blockade of cocaine or s receptoragonist self-administration by subtype-selective s receptor antagonists.J. Pharmacol. Exp. Ther. 358, 109e124.

Killinger, B.A., Peet, M.M., Baker, L.E., 2010. Salvinorin A fails to substitute for thediscriminative stimulus effects of LSD or ketamine in Sprague-Dawley rats.Pharmacol. Biochem. Behav. 96, 260e265.

Koek, W., Woods, J.H., Colpaert, F.C., 1990. N-methyl-D-aspartate antagonism andphencyclidine-like activity: a drug discrimination analysis. J. Pharmacol. Exp.Ther. 253, 1017e1025.

Koerner, J., Appel, J.B., 1982. Psilocybin as a discriminative stimulus: lack of speci-ficity in an animal behavior model for 'hallucinogens'. Psychopharmacology 76,130e135.

Krupitsky, E.M., Grinenko, A.Y., 1997. Ketamine psychedelic therapy (KPT): a reviewof the results of ten years of research. J. Psychoact. Drugs 29, 165e183.

Kueh, D., Baker, L.E., 2007. Reinforcement schedule effects in rats trained todiscriminate 3,4-methylenedioxymethamphetamine (MDMA) or cocaine. Psy-chopharmacology 189, 447e457.

Kueppers, V.B., Cooke, C.T., 2015. 25I-NBOMe related death in Australia: a casereport. Forensic Sci. Int. 249, e15ee18.

Lahti, R.A., Collins, R.J., 1982. Opiate effects on plasma corticosteroids: relationshipto dysphoria and self-administration. Pharmacol. Biochem. Behav. 17, 107e109.

Lamb, R.J., Griffiths, R.R., 1987. Self-injection of d,1-3,4-methylenedioxymethamphetamine (MDMA) in the baboon. Psychopharma-cology 91, 268e272.

Lerner, S.E., Burns, R.S., 1978. Phencyclidine use among youth: history, epidemi-ology, and acute and chronic intoxication. NIDA Res. Monogr. Aug (21), 66e118.

LeSage, M., Clark, R., Poling, A., 1993. MDMA and memory: the acute and chroniceffects of MDMA in pigeons performing under a delayed-matching-to-sampleprocedure. Psychopharmacology 110, 327e332.

Levin, A., 1973. Abuse of fenfluramine. Br. Med. J. 2 (5857), 49, 1973 Apr 7.Lim, D.K., 2003. Ketamine associated psychedelic effects and dependence. Singap.

Med. J. 44, 31e34.


Lukas, S.E., Brady, J.V., Griffiths, R.R., 1986. Comparison of opioid self-injection anddisruption of schedule-controlled performance in the baboon. J. Pharmacol.Exp. Ther. 238, 924e931.

Lucki, I., Nobler, M.S., Frazer, A., 1984. Differential actions of serotonin antagonistson two behavioral models of serotonin receptor activation in the rat.J. Pharmacol. Exp. Ther. 228, 133e139.

Lunzer, M.M., Portoghese, P.S., 2007. Selectivity of d- and k-opioid ligands dependson the route of central administration in mice. J. Pharmacol. Exp. Ther. 322,166e171.

Macleod, M.R., Fisher, M., O'Collins, V., Sena, E.S., Dirnagl, U., Bath, P.M., Buchan, A.,van der Worp, H.B., Traystman, R., Minematsu, K., Donnan, G.A., Howells, D.W.,2009. Good laboratory practice: preventing introduction of bias at the bench.Stroke 40, e50e52.

Maj, J., Lewandowska, A., 1980. Central serotoninmimetic action of phenyl-piperazines. Pol. J. Pharmacol. Pharm. 32, 495e504.

Marona-Lewicka, D., Nichols, D.E., 1994. Behavioral effects of the highly selectiveserotonin releasing agent 5-methoxy-6-methyl-2-aminoindan. Eur. J. Pharma-col. 258, 1e13.

Marquis, K.L., Gussio, R., Webb, M.G., Moreton, J.E., 1989a. Cortical EEG changesduring the self-administration of phencyclinoids. Neuropharmacology 28,1193e1198.

Marquis, K.L., Webb, M.G., Moreton, J.E., 1989b. Effects of fixed ratio size and doseon phencyclidine self-administration by rats. Psychopharmacology 97, 179e182.

Martin-Fardon, R., Maurice, T., Aujla, H., Bowen, W.D., Weiss, F., 2007. Differentialeffects of sigma1 receptor blockade on self-administration and conditionedreinstatement motivated by cocaine vs natural reward. Neuro-psychopharmacology 321, 967e973.

Mason, K., Cottrell, A.M., Corrigan, A.G., Gillatt, D.A., Mitchelmore, A.E., 2010. Ke-tamine-associated lower urinary tract destruction: a new radiological chal-lenge. Clin. Radiol. 65, 795e800.

Matthews, W.D., Smith, C.D., 1980. Pharmacological profile of a model for centralserotonin receptor activation. Life Sci. 26.

May, J.A., Sharif, N.A., Chen, H.H., Liao, J.C., Kelly, C.R., Glennon, R.A., Young, R.,Li, J.X., Rice, K.C., France, C.P., 2009. Pharmacological properties and discrimi-native stimulus effects of a novel and selective 5-HT2 receptor agonist AL-38022A [(S)-2-(8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl)-1-methylethylamine]. Pharmacol. Biochem. Behav. 91, 307e314.

Mead, A.N., 2014. Appropriate experimental approaches for predicting abuse po-tential and addictive qualities in preclinical drug discovery. Expet Opin. DrugDiscov. 9, 1281e1291.

Mello, N.K., Negus, S.S., 1998. Effects of kappa opioid agonists on cocaine- and food-maintained responding by rhesus monkeys. J. Pharmacol. Exp. Ther. 286,812e824.

Moreton, J.E., Meisch, R.A., Stark, L., Thompson, T., 1977. Ketamine self-administration by the rhesus monkey. J. Pharmacol. Exp. Ther. 203, 303e309.

Mori, T., Nomura, M., Nagase, H., Narita, M., Suzuki, T., 2002. Effects of a newlysynthesized k-opioid receptor agonist, TRK-820, on the discriminative stimulusand rewarding effects of cocaine in rats. Psychopharmacology 161, 17e22.

Mori, T., Nomura, M., Yoshizawa, K., Nagase, H., Narita, M., Suzuki, T., 2004. Dif-ferential properties between TRK-820 and U-50,488H on the discriminativestimulus effects in rats. Life Sci. 75, 2473e2482.

Mori, T., Nomura, M., Yoshizawa, K., Nagase, H., Sawaguchi, T., Narita, M., Suzuki, T.,2006. Generalization of NMDA-receptor antagonists to the discriminativestimulus effects of kappa-opioid receptor agonists U-50,488H, but not TRK-820in rats. J. Pharmacol. Sci. 100, 157e161.

Mori, T., Rahmadi, M., Yoshizawa, K., Itoh, T., Shibasaki, M., Suzuki, T., 2014. Inhib-itory effects of SA4503 on the rewarding effects of abused drugs. Addict. Biol. 19,362e369.

Mori, T., Yoshizawa, K., Nomura, M., Isotani, K., Torigoe, K., Tsukiyama, Y., Narita, M.,Suzuki, T., 2012. Sigma-1 receptor function is critical for both the discriminativestimulus and aversive effects of the kappa-opioid receptor agonist U-50488H.Addict. Biol. 17, 717e724.

Moser, P., Wolinsky, T., Duxon, M., Porsolt, R.D., 2011. How good are current ap-proaches to non-clinical evaluation of abuse and dependence? J. Pharmacol.Exp. Ther. 336, 588e595.

Moya, P.R., Berg, K.A., Guti�errez-Hernandez, M.A., S�aez-Briones, P., Reyes-Parada, M.,Cassels, B.K., Clarke, W.P., 2007. Functional selectivity of hallucinogenic phe-nethylamine and phenylisopropylamine derivatives at human 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors. J. Pharmacol. Exp. Ther.321, 1054e1061.

Muetzelfeldt, L., Kamboj, S.K., Rees, H., Taylor, J., Morgan, C.J., Curran, H.V., 2008.Journey through the K-hole: phenomenological aspects of ketamine use. DrugAlcohol Depend. 95, 219e229.

Murrough, J.W., Iosifescu, D.V., Chang, L.C., Al Jurdi, R.K., Green, C.E., Perez, A.M.,Iqbal, S., Pillemer, S., Foulkes, A., Shah, A., Charney, D.S., Mathew, S.J., 2013.Antidepressant efficacy of ketamine in treatment-resistant major depression: atwo-site randomized controlled trial. Am. J. Psychiatry 170, 1134e1142.

Mutti, A., Aroni, S., Fadda, P., Padovani, L., Mancini, L., Collu, R., Muntoni, A.L.,Fattore, L., Chiamulera, C., 2016. The ketamine-like compound methoxetaminesubstitutes for ketamine in the self-administration paradigm and enhancesmesolimbic dopaminergic transmission. Psychopharmacology 233, 2241e2251.

Nabeshima, T., Ishikawa, K., Yamaguchi, K., Furukawa, H., Kameyama, T., 1987.Phencyclidine-induced head-twitch response in rats treated chronically withmethysergide. Eur. J. Pharmacol. 133, 319e328.

Nakamura, M., Fukushima, H., 1976. Head twitches induced by benzodiazepines and
































http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm074959.pdf

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm074959.pdf




































































































































































































the role of biogenic amines. Psychopharmacology 49, 259e261.Negus, S.S., Picker, M.J., Dykstra, L.A., 1990. Interactions between m and k opioid

agonists in the rat drug discrimination procedure. Psychopharmacology 102,465e473.

Newman, J.L., Thorne, J.J., Batulis, D.K., Carroll, M.E., 2006. Effects of menstrual cyclephase on the reinforcing effects of phencyclidine (PCP) in rhesus monkeys.Pharmacol. Biochem. Behav. 85, 584e591.

Nichols, D.E., Oberlender, R., Burris, K., Hoffman, A.J., Johnson, M.P., 1989. Studies ofdioxole ring substituted 3,4-methylenedioxyamphetamine (MDA) analogues.Pharmacol. Biochem. Behav. 34, 571e576.

Nicholson, K.L., Balster, R.L., 1998. Phencyclidine-like discriminative stimulus effectsof polyamine modulators of N-methyl-D-aspartate receptor activity in rats.Neurosci. Lett. 253, 53e56.

Nicholson, K.L., Balster, R.L., 2003. Evaluation of the phencyclidine-like discrimi-native stimulus effects of novel NMDA channel blockers in rats. Psychophar-macology 170, 215e224.

Nicholson, K.L., Balster, R.L., 2009. The discriminative stimulus effects of N-methyl-D-aspartate glycine-site ligands in NMDA antagonist-trained rats. Psycho-pharmacology 203, 441e451.

Nicholson, K.L., Hayes, B.A., Balster, R.L., 1999. Evaluation of the reinforcing prop-erties and phencyclidine-like discriminative stimulus effects of dextromethor-phan and dextrorphan in rats and rhesus monkeys. Psychopharmacology 146,49e59.

Nicholson, K.L., Jones, H.E., Balster, R.L., 1998. Evaluation of the reinforcing anddiscriminative stimulus properties of the low-affinity N-methyl-D-aspartatechannel blocker memantine. Behav. Pharmacol. 9, 231e243.

Niemegeers, C.J.E., Colpaert, F.C., Leysen, J.E., Awouters, F., Janssen, P.A.J., 1983.Mescaline-induced head-twitches in the rat; an in vivo method to evaluate S2antagonists. Drug Dev. Res. 3, 123e135.

Nishida, N., Hasegawa, Y., Chiba, S., Wakimasu, M., Fujino, M., 1989. Reinforcingeffects of the enkephalin analogs, EK-209 and EK-399, in rats. Eur. J. Pharmacol.166, 453e458.

Oberlender, R., Nichols, D.E., 1988. Drug discrimination studies with MDMA andamphetamine. Psychopharmacology 95, 71e76.

O'Connor, E.C., Chapman, K., Butler, P., Mead, A.N., 2011. The predictive validity ofthe rat self-administration model for abuse liability. Neurosci. Biobehav. Rev. 35,912e938.

OECD Principles of Good Laboratory Practice (as revised in 1997). http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote¼env/mc/chem(98)17&doclanguage¼en.

Overton, D.A., Shen, C.F., Ke, G.Y., Gazdick, L.P., 1989. Discriminable effects ofphencyclidine analogs evaluated by multiple drug (PCP versus OTHER)discrimination training. Psychopharmacology 97, 514e520.

Pal, H.R., Berry, N., Kumar, R., Ray, R., 2002. Ketamine dependence. Anaesth.Intensive Care 30, 382e384.

Panlilio, L.V., Goldberg, S.R., 2007. Self-administration of drugs in animals andhumans as a model and an investigative tool. Addiction 102, 1863e1870.

Peet, M.M., Baker, L.E., 2011. Salvinorin B derivatives, EOM-Sal B and MOM-Sal B,produce stimulus generalization in male Sprague-Dawley rats trained todiscriminate salvinorin A. Behav. Pharmacol. 22, 450e457.

Picker, M.J., Benyas, S., Horwitz, J.A., Thompson, K., Mathewson, C., Smith, M.A.,1996. Discriminative stimulus effects of butorphanol: influence of training doseon the substitution patterns produced by m-, k- and d-opioid agonists.J. Pharmacol. Exp. Ther. 279, 1130e1141.

Picker, M.J., Doty, P., Negus, S.S., Mattox, S.R., Dykstra, L.A., 1990. Discriminativestimulus properties of U50,488 and morphine: effects of training dose onstimulus substitution patterns produced by mu and kappa opioid agonists.J. Pharmacol. Exp. Ther. 254, 13e22.

Picker, M., Dykstra, L.A., 1987. Comparison of the discriminative stimulus propertiesof U50,488 and morphine in pigeons. J. Pharmacol. Exp. Ther. 243, 938e945.

Poklis, A., Graham, M., Maginn, D., Branch, C.A., Gantner, G.E., 1990. Phencyclidineand violent deaths in St. Louis, Missouri: a survey of medical examiners' casesfrom 1977 through 1986. Am. J. Drug Alcohol Abuse 16, 265e274.

Poon, T.L., Wong, K.F., Chan, M.Y., Fung, K.W., Chu, S.K., Man, C.W., Yiu, M.K.,Leung, S.K., 2010. Upper gastrointestinal problems in inhalational ketamineabusers. J. Dig. Dis. 11, 106e110.

Preston, K.L., Bigelow, G.E., Liebson, I.A., 1987. Comparative evaluation of morphine,pentazocine and ciramadol in postaddicts. J. Pharmacol. Exp. Ther. 240,900e910.

Rangel-Barajas, C., Malik, M., Vangveravong, S., Mach, R.H., Luedtke, R.R., 2014.Pharmacological modulation of abnormal involuntary DOI-induced head twitchresponse in male DBA/2J mice: I. Effects of D2/D3 and D2 dopamine receptorselective compounds. Neuropharmacology 83, 18e27.

Recker, M.D., Higgins, G.A., 2004. The opioid receptor like-1 receptor agonist Ro 64-6198 (1S,3aS-8-2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one) produces a discriminative stimulus in ratsdistinct from that of a m-, k-, and d-opioid receptor agonist cue. J. Pharmacol.Exp. Ther. 311, 652e658.

Reece, P.A., Sedman, A.J., Rose, S., Wright, D.S., Dawkins, R., Rajagopalan, R., 1994.Diuretic effects, pharmacokinetics, and safety of a new centrally acting kappa-opioid agonist (CI-977) in humans. J. Clin. Pharmacol. 34, 1126e1132.

Reissig, C.J., Eckler, J.R., Rabin, R.A., Winter, J.C., 2005. The 5-HT1A receptor and thestimulus effects of LSD in the rat. Psychopharmacology 182, 197e204.

Remmers, A.E., Clark, M.J., Mansour, A., Akil, H., Woods, J.H., Medzihradsky, F., 1999.Opioid efficacy in a C6 glioma cell line stably expressing the human kappa


opioid receptor. J. Pharmacol. Exp. Ther. 288, 827e833.Roberts, D.C., Gabriele, A., Zimmer, B.A., 2013. Conflation of cocaine seeking and

cocaine taking responses in IV self-administration experiments in rats: meth-odological and interpretational considerations. Neurosci. Biobehav. Rev. 37,2026e2036.

Rocha, B.A., Ward, A.S., Egilmez, Y., Lytle, D.A., Emmett-Oglesby, M.W., 1996.Tolerance to the discriminative stimulus and reinforcing effects of ketamine.Behav. Pharmacol. 7, 160e168.

Rose, S.R., Poklis, J.L., Poklis, A., 2013. A case of 25I-NBOMe (25-I) intoxication: anew potent 5-HT2A agonist designer drug. Clin. Toxicol. 51, 174e177.

Rosenvinge, H.P., 1975. Letter: abuse of fenfluramine. Br. Med. J. 1 (5960), 735. Mar29.

Roth, B.L., Baner, K., Westkaemper, R., Siebert, D., Rice, K.C., Steinberg, S.,Ernsberger, P., Rothman, R.B., 2002. Salvinorin A: a potent naturally occurringnon-nitrogenous k-opioid selective agonist. Proc. Natl. Acad. Sci. 99,11934e11939.

Roux, S., Sabl�e, E., Porsolt, R.D., 2005. Primary observation (Irwin) test in rodents forassessing acute toxicity of a test agent and its effects on behavior and physio-logical function. Curr. Protoc. Pharmacol. 27, 10.10.1e10.10.23. Chapter 10:Unit10.10.

Schechter, M.D., 1986. Discriminative profile of MDMA. Pharmacol. Biochem. Behav.24, 1533e1537.

Schechter, M.D., 1991. Effect of serotonin depletion by p-chlorophenylalanine upondiscriminative behaviours. Gen. Pharmacol. 22, 889e893.

Schenk, S., Gittings, D., Johnstone, M., Daniela, E., 2003. Development, maintenanceand temporal pattern of self-administration maintained by ecstasy (MDMA) inrats. Psychopharmacology 169, 21e27.

Sellers, E.M., Romach, M.K., Leiderman, D.B., 2017 Nov 21. Studies with psychedelicdrugs in human volunteers. Neuropharmacology. https://doi.org/10.1016/j.neuropharm.2017.11.029 pii: S0028e3908(17)30546-4. [Epub ahead of print].

Serafine, K.M., Rice, K.C., France, C.P., 2015. Directly observable behavioral effects oflorcaserin in rats. J. Pharmacol. Exp. Ther. 355, 381e385.

Shram, M.J., Sellers, E.M., Romach, M.K., 2011. Oral ketamine as a positive control inhuman abuse potential studies. Drug Alcohol Depend. 114, 185e193.

Siebert, D., 2010. The Legal Status of Salvia divinorum. The Salvia divinorum Researchand Information Center. http://www.sagewisdom.org/legislation.html.

Silva, M.T., Calil, H.M., 1975. Screening hallucinogenic drugs: systematic study ofthree behavioral tests. Psychopharmacology 42, 163e171.

Singh, J.B., Fedgchin, M., Daly, E.J., De Boer, P., Cooper, K., Lim, P., Pinter, C.,Murrough, J.W., Sanacora, G., Shelton, R.C., Kurian, B., Winokur, A., Fava, M.,Manji, H., Drevets, W.C., Van Nueten, L., 2016. A double-blind, randomized,placebo-controlled, dose-frequency study of intravenous ketamine in patientswith treatment-resistant depression. Am. J. Psychiatry 173, 816e826.

Singh, L., Menzies, R., Tricklebank, M.D., 1990. The discriminative stimulus prop-erties of (þ)-HA-966, an antagonist at the glycine/N-methyl-D-aspartate re-ceptor. Eur. J. Pharmacol. 186, 129e132.

Slifer, B.L., Balster, R.L., 1983. Reinforcing properties of stereoisomers of the putativesigma agonists N-allylnormetazocine and cyclazocine in rhesus monkeys.J. Pharmacol. Exp. Ther. 225, 522e528.

Smith, D.A., Bailey, J.M., Williams, D., Fantegrossi, W.E., 2014. Tolerance and cross-tolerance to head twitch behavior elicited by phenethylamine- andtryptamine-derived hallucinogens in mice. J. Pharmacol. Exp. Ther. 351,485e491.

Smith, S.L., Holland, S., Gray, R.A., Heal, D.J., 2017. An investigation of the reinforcingeffects of diazepam and midazolam in rats trained to self-administer heroin. In:Proceedings of College on Problems of Drug Dependence Meeting, Montreal,June 17-22, 2017, p. 57.

Smith, S.L., Johnson, E., Slade, J., Holland, S., Hallam, M., Heal, D.J., 2016. Comparisonof the reinforcing effects of nicotine with three Schedule II listed drugs using ratintravenous self-administration. In: Proceedings of Society for NeurosciencesMeeting, San Diego, November 12-16, 2016, p 549.10.

Spain, J.W., Klingman, G.I., 1985. Continuous intravenous infusion of phencyclidinein unrestrained rats results in the rapid induction of tolerance and physicaldependence. J. Pharmacol. Exp. Ther. 234, 415e424.

Spencer Jr., D.G., Glaser, T., Traber, J., 1987. Serotonin receptor subtype mediation ofthe interoceptive discriminative stimuli induced by 5-methoxy-N,N-dimethyl-tryptamine. Psychopharmacology 93, 158e166.

Stafford, I., Tomie, A., Wagner, G.C., 1983. Effects of SKF-10047 in the phencyclidine-dependent rat: evidence for common receptor mechanisms. Drug AlcoholDepend. 12, 151e156.

Steinfels, G.F., Alberici, G.P., Tam, S.W., Cook, L., 1988. Biochemical, behavioral, andelectrophysiologic actions of the selective sigma receptor ligand (þ)-pentazo-cine. Neuropsychopharmacology 1, 321e327.

Steinfels, G.F., Young, G.A., Khazan, N., 1982. Self-administration of nalbuphine,butorphanol and pentazocine by morphine post-addict rats. Pharmacol. Bio-chem. Behav. 16, 167e171.

Stolerman, I.P., Childs, E., Ford, M.M., Grant, K.A., 2011. Role of training dose in drugdiscrimination: a review. Behav. Pharmacol. 22, 415e429.

Swedberg, M.D., 2016. Drug discrimination: a versatile tool for characterization ofCNS safety pharmacology and potential for drug abuse. J. Pharmacol. Toxicol.Meth. 81, 295e305.

Tang, A.H., Collins, R.J., 1985. Behavioural effects of a novel k-opioid analgesic, U-50488, in rats and rhesus monkeys. Psychopharmacology 85, 309e314.

Tricklebank, M.D., Singh, L., Oles, R.J., Wong, E.H., Iversen, S.D., 1987. A role for re-ceptors of N-methyl-D-aspartic acid in the discriminative stimulus properties of




















































http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/mc/chem(98)17&doclanguage=en



















































































































http://www.sagewisdom.org/legislation.html


































































phencyclidine. Eur. J. Pharmacol. 141, 497e501.Trigo, J.M., Panayi, F., Soria, G., Maldonado, R., Robledo, P., 2006. A reliable model of

intravenous MDMA self-administration in naïve mice. Psychopharmacology184, 212e220.

Tsai, T.H., Cha, T.L., Lin, C.M., Tsao, C.W., Tang, S.H., Chuang, F.P., Wu, S.T., Sun, G.H.,Yu, D.S., Chang, S.Y., 2009. Ketamine-associated bladder dysfunction. Int. J. Urol.16, 826e829.

Ukai, M., Mori, E., Kameyama, T., 1997. Modulatory effects of morphine, U-50488Hand 1,3-di-(2-tolyl)guanidine on cocaine-like discriminative stimulus in the ratusing two-choice discrete-trial avoidance paradigm. Methods Find. Exp. Clin.Pharmacol. 19, 541e546.

Ukai, M., Nakayama, S., Hamada, T., Kameyama, T., 1989. Discriminative stimulusproperties of the mixed agonist-antagonist pentazocine in the rat using two-choice discrete-trial avoidance paradigm. Pharmacol. Biochem. Behav. 33,355e359.

Uosukainen, H., Tacke, U., Winstock, A.R., 2015. Self-reported prevalence ofdependence of MDMA compared to cocaine, mephedrone and ketamine amonga sample of recreational poly-drug users. Int. J. Drug Pol. 26, 78e83.

van der Kam, E.L., de Vry, J., Tzschentke, T.M., 2007. Effect of 2-methyl-6-(phenyl-ethynyl) pyridine on intravenous self-administration of ketamine and heroin inthe rat. Behav. Pharmacol. 18, 717e724.

Venniro, M., Mutti, A., Chiamulera, C., 2015. Pharmacological and non-pharmacological factors that regulate the acquisition of ketamine self-administration in rats. Psychopharmacology 232, 4505e4514.

Vetulani, J., Bednarczyk, B., Reichenberg, K., Rokosz, A., 1980. Head twitches inducedby LSD and quipazine: similarities and differences. Neuropharmacology 19,155e158.

Vickers, S.P., Easton, N., Malcolm, C.S., Allen, N.H., Porter, R.H., Bickerdike, M.J.,Kennett, G.A., 2001. Modulation of 5-HT2A receptor-mediated head-twitchbehaviour in the rat by 5-HT2C receptor agonists. Pharmacol. Biochem. Behav.69, 643e652.

Walentiny, D.M., Vann, R.E., Warner, J.A., King, L.S., Seltzman, H.H., Navarro, H.A.,Twine, C.E., Thomas, B.F., Gilliam, A.F., Gilmour, B.P., Carroll, F.I., Wiley, J.L., 2010.Kappa opioid mediation of cannabinoid effects of the potent hallucinogen,salvinorin A, in rodents. Psychopharmacology 210, 275e284.

Walker, J.M., Bowen, W.D., Goldstein, S.R., Roberts, A.H., Patrick, S.L., Hohmann, A.G.,DeCosta, B., 1992. Autoradiographic distribution of [3H](þ)-pentazocine and[3H]1,3-di-o-tolylguanidine (DTG) binding sites in Guinea pig brain: acomparative study. Brain Res. 581, 33e38.

Walsh, S.L., Strain, E.C., Abreu, M.E., Bigelow, G.E., 2001a. Enadoline, a selective k-opioid agonist: comparison with butorphanol and hydromorphone in humans.Psychopharmacology 157, 151e162.

Walsh, S.L., Geter-Douglas, B., Strain, E.C., Bigelow, G.E., 2001b. Enadoline andbutorphanol: evaluation of k-agonists on cocaine pharmacodynamics andcocaine self-administration in humans. J. Pharmacol. Exp. Ther. 299, 147e158.

Webster, J.I., Harper, D.N., Schenk, S., 2017. Generalization of serotonin and dopa-mine ligands to the discriminative stimulus effects of different doses of ±3,4-methylenedioxymethamphetamine. Behav. Pharmacol. 28, 245e254.

White, J.M., Holtzman, S.G., 1982. Properties of pentazocine as a discriminativestimulus in the squirrel monkey. J. Pharmacol. Exp. Ther. 223, 396e401.

Wiley, J.L., Balster, R.L., 1994. Effects of competitive and non-competitive N-methyl-D-aspartate (NMDA) antagonists in squirrel monkeys trained to discriminate D-CPPene (SDZ EAA 494) from vehicle. Psychopharmacology 116, 266e272.

Wiley, J.L., Lefever, T.W., Marusich, J.A., Craft, R.M., 2017. Comparison of thediscriminative stimulus and response rate effects of D9-tetrahydrocannabinol


and synthetic cannabinoids in female and male rats. Drug Alcohol Depend. 172,51e59.

Willmore-Fordham, C.B., Krall, D.M., McCurdy, C.R., Kinder, D.H., 2007. The hallu-cinogen derived from Salvia divinorum, salvinorin A, has kappa-opioid agonistdiscriminative stimulus effects in rats. Neuropharmacology 53, 481e486.

Winger, G., Palmer, R.K., Woods, J.H., 1989. Drug-reinforced responding: rapiddetermination of dose-response functions. Drug Alcohol Depend. 24, 135e142.

Wong, G.L., Tam, Y.H., Ng, C.F., Chan, A.W., Choi, P.C., Chu, W.C., Lai, P.B., Chan, H.L.,Wong, V.W., 2014. Liver injury is common among chronic abusers of ketamine.Clin. Gastroenterol. Hepatol. 12, 1759e1762 e1.

Woolverton, W.L., Hecht, G.S., Agoston, G.E., Katz, J.L., Newman, A.H., 2001. Furtherstudies of the reinforcing effects of benztropine analogs in rhesus monkeys.Psychopharmacology 154, 375e382.

Wright, K.N., Strong, C.E., Addonizio, M.N., Brownstein, N.C., Kabbaj, M., 2017.Reinforcing properties of an intermittent, low dose of ketamine in rats: effectsof sex and cycle. Psychopharmacology 234, 393e401.

Wu, L.T., Ringwalt, C.L., Mannelli, P., Patkar, A.A., 2008. Hallucinogen use disordersamong adult users of MDMA and other hallucinogens. Am. J. Addict. 17,354e363.

Yamaguchi, K., Nabeshima, T., Ishikawa, K., Yoshida, S., Kameyama, T., 1987. Phen-cyclidine-induced head-weaving and head-twitch through interaction with 5-HT1 and 5-HT2 receptors in reserpinized rats. Neuropharmacology 26,1489e1497.

Yamamoto, T., Ueki, S., 1975. Behavioral effects of 2,5-dimethoxy-4-methylamphetamine (DOM) in rats and mice. Eur. J. Pharmacol. 32, 156e162.

Yamamoto, T., Tazoe, N., Ueki, S., Shimomura, K., Satoh, H., Mori, J., 1983. Effect ofzotepine on head-twitch induced by L-5-hydroxytryptophan, mescaline and2,5-dimethoxy-4-methylamphetamine in mice and rats. Jpn. J. Pharmacol. 33,319e325.

Yanagita, T., 1986. Intravenous self-administration of (-)-cathinone and 2-amino-1-(2,5-dimethoxy-4-methyl)phenylpropane in rhesus monkeys. Drug AlcoholDepend. 17, 135e141.

Yap, C.Y., Taylor, D.A., 1983. Involvement of 5-HT2 receptors in the wet-dog shakebehaviour induced by 5-hydroxytryptophan in the rat. Neuropharmacology 22,801e804.

Yarosh, H.L., Katz, E.B., Coop, A., Fantegrossi, W.E., 2007. MDMA-like behavioraleffects of N-substituted piperazines in the mouse. Pharmacol. Biochem. Behav.88, 18e27.

Yoshimura, K., Horiuchi, M., Inoue, Y., Yamamoto, K., 1984. Pharmacological studieson drug dependence. (III): intravenous self-administration of some CNS-affecting drugs and a new sleep-inducer, 1H-1, 2, 4-triazolyl benzophenonederivative (450191-S), in rats. Nihon Yakurigaku Zasshi 83, 39e67.

Young, A.M., Woods, J.H., 1981. Maintenance of behavior by ketamine and relatedcompounds in rhesus monkeys with different self-administration histories.J. Pharmacol. Exp. Ther. 218, 720e727.

Young, R., Glennon, R.A., 1998. Discriminative stimulus properties of (-)ephedrine.Pharmacol. Biochem. Behav. 60, 771e775.

Zacny, J.P., Hill, J.L., Black, M.L., Sadeghi, P., 1998. Comparing the subjective, psy-chomotor and physiological effects of intravenous pentazocine and morphine innormal volunteers. J. Pharmacol. Exp. Ther. 286, 1197e1207.

Zhang, Y., Butelman, E.R., Schlussman, S.D., Ho, A., Kreek, M.J., 2005. Effects of theplant-derived hallucinogen salvinorin A on basal dopamine levels in thecaudate putamen and in a conditioned place aversion assay in mice: agonistactions at kappa opioid receptors. Psychopharmacology 179, 551e558.


























































































































































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