Cannabis and the Use of Amphetamine-Like Substances A

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CHAPTER e10 Cannabis and the Use of Amphetamine-Like Substances A. Porcu , M.P. Castelli Department of Biomedical Sciences, Division of Neuroscience and Clinical Pharmacology, Cittadella Universitaria, Monserrato, CA, Italy

SUMMARY POINTS KEY FACTS ON THE • This chapter focuses on coabuse of cannabis and MONOAMINERGIC TARGET SYSTEM amphetamine-like substances (ALS). • Monoamines refer to the particular neurotransmitters • Cannabis (main psychoactive compound dopamine, norepinephrine and serotonin. Δ 9 -Tetrahydrocannabinol, Δ 9 -THC) is consumed • These neurotransmitters are involved in mediating a by 2.9–4.3% of the global population aged 16–64 wide range of physiological and homeostatic functions in the world. such as cognition, memory, learning, mood, and behavior. • Cannabis consumption is frequently associated • These neurotransmitters are synthesized within with simultaneous use of other psychoactive particular neurons, and exert an effect when they are compounds such as alcohol, cocaine, and ALS released into the synapses, where they are able to act (ie, amphetamine, methamphetamine, and on specifi c receptors. 3,4-methylenedioxymethamphetamine). • The dopamine transporter (DAT), serotonin • New psychoactive substances (NPS), such as transporter (SERT), and the norepinephrine transporter cathinones and its derivatives, are frequently (NET) transport monoamines in or out of a cell. coabused with cannabis. • ALS may increase the activity of dopamine, • Cannabis + ASL coabuse enhances the subjective norepinephrine, or serotonin by either increasing effects of the drugs in humans, counteracts release, blocking reuptake, inhibiting metabolism, or the dysphoric symptoms of “coming down” acting directly on a receptor. from 3,4-methylenedioxymethamphetamine or methamphetamine (MDMA and METH), and/or improves and mellows the drug experience. KEY FACTS ON NEW PSYCHOACTIVE • Both pharmacological and genotype animal SUBSTANCES (NPS) studies support the role of the cannabinoid type • NPS are known in the drug market under the names of 1 receptor (CB1R) in the rewarding and addictive “designer drugs,” “legal highs,” “herbal highs,” “bath properties of MDMA. salts,” or “research chemicals.” • Preclinical and clinical studies investigating the • NPS have become a global phenomenon, and they effects of cannabis + ASL and their interaction have affected all regions of the world; 70 (out of 80) in METH/MDMA-induced neurotoxicity have countries and territories (88%) examined by UNODC reported confl icting fi ndings: cannabis might (2014) have reported the emergence of NPS. exacerbate or attenuate METH/MDMA induced • The number of NPS on the global market more than neurotoxicity. doubled over the period 2009–13. Legislation has not

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RNS Reactive nitrogen species been established, making their sale easy and offi cially ROS Reactive oxygen species legal in almost all countries. SERT Serotonin transporter • The most popular of these drugs are based on the TH Tyrosine hydroxylase substance cathinone (2-amino-1-phenylpropanone) TTAR1 Activate trace amine associated receptor 1 and its synthetic analogs. TUNEL Terminal deoxynucleotidyl transferase • The chemical structure of NPS is similar to that of VMAT-2 Vesicular monoamine transporter-2 amphetamine, as well as their physiological and psychological actions. INTRODUCTION KEY FACTS ON POLYDRUG USE • Research on polydrugs use is typically done on Cannabis is the most widely consumed illicit drug relatively small convenient samples (eg, street- worldwide, with an estimated global use by 2.9– based, emergency rooms, and club patrons), where 4.3% of the population aged 16–64 (UNODC, 2010) multiple drugs are frequently used consecutively or (Fig. e10.1). simultaneously, often for their perceived counteracting While the prevalence of illicit drugs such as cocaine, or complementary effects. amphetamine-like substances (ALS) [ie, amphetamine • It is associated with a higher risk of developing (AMPH), methamphetamine (METH), and 3,4-methy- psychiatric and health problems. lenedioxymethamphetamine (MDMA)] appeared to remain stable between 2009 and 2011, since 2009 the extent of cannabis and opioids use has increased. Cannabis LIST OF ABBREVIATIONS consumption is frequently associated with simultaneous and/or concurrent use of other psychoactive compounds Δ 9 -THC Δ 9 -Tetrahydrocannabinol such as alcohol, cocaine, and ALS (Gouzoulis-Mayfrank 5HT Serotonin & Daumann, 2006 ). ADHD Attention-defi cit/hyperactivity disorder A growing body of clinical and preclinical studies has AMPH Amphetamine highlighted the simultaneous use of MDMA, also called ec- ALS Amphetamine-like substances stasy, and cannabis ( Parrott, Milani, Gouzoulis-Mayfrank, BDNF Brain-derived neurotrophic factor & Daumann, 2007). Strote, Lee, & Wechsler (2002), survey- CB1-KO Cannabinoid type 1 receptor knockout mice ing a sample of over 10,000 US college students, reported a CB1R Cannabinoid type 1 receptor prevalence of ecstasy use of 4.7%, with 92% of consumers CB2-KO Cannabinoid type 2 receptors knockout mice also consuming cannabis. Wish, Fitzelle, O’Grady, & Hsu CB2R Cannabinoid type 2 receptor (2006) confi rmed that 98% of recreational MDMA users CPP Conditioned place preference in a sample of East Coast College students also consume DA Dopamine cannabis. Moreover, the presence of a metabolite of the DAT Dopamine transporter main psychoactive compound Δ 9-tetrahydrocannabinol DEA Drug Enforcement Administration ( Δ9 -THC), 11-nor-9 carboxy-THC, was found in urine sam- dUTP 2 Ј -deoxyuridine 5Ј -triphosphate ples collected from 198 ecstasy users between 2005 and ECS Endocannabinoid system 2008 (Black, Cawthon, Robert, & Moser, 2009). FDA Food and Drug Administration In Europe, the prevalence of couse of MDMA + can- fMRI Functional magnetic resonance technique nabis ranges from 73% to 100% in different countries GFAP Glial fi brillary acidic protein (Sala & Braida, 2005), and is higher in males and in MAO Monoamine oxidase regular cannabis users than in women and occasional MDMA 3,4-Methylenedioxymethamphetamine METH Methamphetamine MJ Marijuana NE Norepinephrine NET Norepinephrine transporter nNOS Neuronal nitric oxide synthase NO Nitric oxide NPS New psychoactive substances pCREB Phosphorylated cAMP response element– binding protein FIGURE e10.1 Estimated number of people who used cannabis at PKA Protein kinase A least once in the past year, and its prevalence among the population PKC Protein kinase C aged 15–64, by region. Source: UNODC (2010) .

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cannabis consumers (ESPAD, 2000). Due to its low cost, are complex, and data regarding their coadministra- wide availability, and high potential for abuse, METH tion in human and animal studies are contradictory is the second most frequently abused recreational drug (Mohamed, Ben Hamida, Cassel, & de Vasconcelos, 2011; among adolescents, and is often coabused with cannabis Schulz, 2011 ). ( Gonzalez, Rippeth, Carey, & Heaton, 2004 ). Δ 9-THC and other cannabis derivatives act via canna- Several psychosocial and functional reasons can ac- binoid type 1 receptors (CB1R) on the endocannabinoid count for the high percentage of concurrent use of Δ 9-THC system (ECS), while the ALS, including MDMA, METH and METH or MDMA. Cannabis is often considered a and the cathinones, act by increasing synaptic levels of “gateway drug,” and its consumption might predict a the monoamines dopamine (DA), serotonin (5HT), and signifi cantly higher risk for subsequent use of heavy il- norepinephrine (NE). licit drugs such as ALS. However, numerous reports have For a detailed description of the ECS and the mecha- failed to support this theory, while other studies point to nism of action of Δ9 -THC and other cannabinoids, see social, environmental, and genetic factors to explain the designated Chapters of this book. couse of cannabis and other drugs (Parrott et al., 2007). In the following paragraphs, we will briefl y describe MDMA and METH are psychostimulant drugs that the ALS, including cathinones, their molecular pharma- induce euphoria, increased sociability, and empathy cology, and the pharmacological/neurobiological effects ( Parrott, 2013 ), while cannabis produces relaxation and induced by ALS + cannabis. feelings of happiness ( Green, Kavanagh, & Young, 2003 ). One of the main reasons for using cannabis in association with MDMA or METH is that the combination of AMPHETAMINE-LIKE SUBSTANCES: the two drugs enhances the subjective drug effects, and AMPH, METH, MDMA, counteracts the dysphoric symptoms of the ecstasy or AND CATHINONES METH “coming down” ( Schulz, 2011 ). Indeed, MDMA abusers often consume cannabis in the immediate post- Amphetamine (AMPH) is the parent compound of ecstasy period as a symptomatic relief against the nega- a class of molecules with similar chemical structures tive physiological and emotional states (eg, anhedonia, and biological properties, referred to as amphetamines depression) that follow ecstasy’s “high.” Many ecstasy ( Fig. e10.2 ) ( Fleckenstein, Volz, Riddle, & Gibb, 2007 ). users also report taking cannabis in the initial acute In 1935, AMPH was introduced to the market by phase of MDMA to “improve” and “mellow” the experi- the pharmaceutical company Smith, Kline and French, ence ( Parrott et al., 2007 ). with the brand name of “Benzedrine.” for the treat- In the last few years, many recreational drugs have ment of narcolepsy, mild depression, postencephalitic been synthesized as legal alternatives to scheduled can- Parkinsonism, and other disorders. Since 1971, AMPH nabis and AMPH/METH stimulants. New psychoactive and METH, a substituted amphetamine, due to their substances (NPS) have been found mostly in Europe and addictive potential, have been inserted in Schedule North America, but also in Oceania, South America, and II of the Controlled Substances Act, and are Food and Africa. Because they are new, legislation has not been es- Drug Administration (FDA)-approved for treatment of tablished yet, making their sale easy and offi cially legal. attention-defi cit/hyperactivity disorder (ADHD), nar- Their global market has increased dramatically in recent colepsy, and obesity (DEA Congressional Testimony by years: by December 2013, the number of NPS reported to Terrance Woodworth; 2000, May 10). UNODC had reached 348, up from 251 as of July 2012, METH is one of the most common ALS; also known and 166 in 2009 ( UNODC, 2014 ). as “crystal,” “chalk,” or “ice,” it is taken orally, smoked, The most popular of these drugs are based on the substance cathinone (2-amino-1-phenylpropanone), an analogue of amphetamine, a natural compound of the khat plant ( Paillet-Loilier, Cesbron, Le Boisselier, Bourgine, & Debruyne, 2014 ), which acts on the central dopamine system. Dopaminergic stimulation of the reward system could explain the development of dependence after frequent consumption of cathinone derivatives. Due to the recent discovery of the NPS, only a few survey studies are currently available on their simultaneous use with cannabis, but no preclinical or clinical studies. On the other hand, despite the high prevalence FIGURE e10.2 Chemical structures of amphetamines. Amphet- of cannabis use among MDMA and METH users, the amine, methamphetamine, and MDMA are the amphetamines most neurobiological interactions between these two drugs frequently coabused with cannabis.

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snorted, or dissolved in water or alcohol, and injected. The annual prevalence of METH in the United States was 0.5% of the population aged 15–64 in 2012, similar to that estimated between 2009 and 2011 ( SAMHSA, 2013 ). MDMA, developed and patented in 1918 by Merck for use in synthesis of styptic drugs, was used in the United States by psychotherapists from 1978 to 1985, when, due to its high abuse potential, the DEA (Drug Enforcement Administration) included it in Schedule I. After cannabis, the second most widely used illegal drugs worldwide are ALS, particularly MDMA ( Parrott, 2013 ). Also known as “ecstasy,” “Molly,” “lover’s speed,” or “the FIGURE e10.4 Chemical structures of cathinones. R1, R2, R3, R4 love drug,” MDMA is used by 10.5–25.8 million people are the various groups that can be substituted to obtain other synthetic worldwide, corresponding to 0.2–0.6% of the adult pop- derivatives. ulation (16–64 years) (UNODC, 2010). MDMA is mostly consumed at late-night parties (“raves,” “dance clubs,” “techno parties”), where the percentage of users can be nitrogen atom (R2 and R3), and on the phenyl group (R4) up to 95%. The 2009 National Survey on Drug Use and ( Fig. e10.4 ). Health reported that the percent consuming MDMA was 1.4% among adolescents (12–17 years old), 3.9% among young adults (18–25 years old) and 0.3% for adults (old- ALS Pharmacology er than 26 years) ( NSDUH, 2010 ). The psychostimulant and rewarding properties of Among the NPS, the most abused are derivatives of AMPH, METH, and MDMA are due to their ability to cathinone, which is a β -cheto analogue of AMPH used enhance neuronal excitability in the brain and, in par- as a structural template for the discovery of compounds ticular, to increase dopaminergic neuronal activity in the with a wide range of pharmacological activities (Carroll, mesolimbic pathway. Blough, Mascarella, & Navarro, 2010 ). Indeed, ALS are historically considered potent indi- NPS are usually marketed as tablets of various colors rect monoaminergic agonists, raising the synaptic levels and shapes, or capsules, powders, and crystals, and sold of DA, NE, and, to a lesser extent, 5HT through different as “bath salts” or “plant fertilizers.” In recent years, sev- mechanisms such as inhibition of: (1) the transporters of eral synthetic analogs of cathinone have emerged on the DA (DAT), NE (NET), and 5HT (SERT); (2) the vesicular market, and circulate on the Internet or are sold in smart monoamine transporter-2 (VMAT-2); and (3) monoamine shops with the misleading indication that they are “not oxidase (MAO) activity. Specifi cally, although with dif- for human consumption” ( Fig. e10.3 ). ferent affi nities, AMPH and its derivatives are substrates The general structure of these substituted compounds of plasmalemma DAT, NET, and SERT, with METH and shows substitution patterns at four locations on the AMPH having greater affi nity for DAT, and MDMA hav- cathinone molecule, for example, on the carbon atom ing greater affi nity for SERT (Sulzer, Sonders, Poulsen, & linked to the carbon in the alpha position (R1), on the Galli, 2005 ). According to the “exchange-diffusion model,” the lipophilic AMPH, METH, and MDMA at high concentrations can diffuse directly across the plasmalemma membrane, while at low concentrations they are trans- ported into the cell by competing with synaptic DA or 5HT at the extracellular site of DAT or SERT. Then, AMPH can disrupt the proton gradient between the in- side and outside of the storage vesicle, resulting in an increased release of DA or 5HT from vesicular compart- ments. At physiological concentrations, AMPH binds to VMAT2, thus inhibiting vesicular monoamine uptake and enhancing cytoplasmic DA levels. As cytoplasmic DA levels rise, DA exits the neuron via reverse transport and/or channel like transport (Fleckenstein et al., 2007 ). FIGURE e10.3 Representative packaging of NPS products. The AMPH can also interact indirectly with DAT. Once indication that this substance is “not for human consumption” makes entering the neuron, it can bind to and activate trace it automatically legal. amine associated receptor 1 (TTAR1), a G-protein

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coupled receptor that is important for the regulation of substrates for NET, DAT, and SERT at nanomolar concen- monoamine transporter function and METH’s action on trations, but are nonselective substrates for monoamine DAT activity. In particular, AMPH-induced TTAR1 acti- transporters in vitro, exhibiting a potency and selectivity vation via protein kinase A (PKA) and protein kinase C comparable to those of MDMA. Mephedrone and meth- (PKC) signaling determines DAT phosphorylation and ylone produce concurrent elevations in extracellular DA internalization, thereby decreasing DAT DA uptake. and 5HT in vivo, and high-dose administration produc- METH and MDMA also increase glutamate release in es hyperthermia and motor stimulation, but no lasting the striatum and hippocampus, respectively (Panenka, changes in brain tissue monoamines ( Baumann, Ayestas, Procyshyn, Lecomte, & MacEwan, 2013 ). Partilla, & Sink, 2012 ). Besides its acute actions, moderate to high doses of ALS administered in a “binge pattern” (repeated ad- ministrations at short time intervals) induce long-term ALS + CANNABIS effects such as dopaminergic and serotoninergic defi cits, including reductions in expression of the enzymes ty- Animal Studies rosine and tryptophan hydroxylase and DAT and SERT density, as well as decreases in DA and 5HT tissue con- Pharmacological and genotype studies in animals tent (Marshall & O’Dell, 2012). In addition to damage support the existence of interactions between cannabi- to DA and 5HT terminals, METH and MDMA cause ac- noid and MDMA/METH related to their rewarding ef- tivation of microglia, and a signifi cant increase in glial fects, and the role played by CB1Rs in these effects (Sala fi brillary acidic protein (GFAP), an index of gliosis and & Braida, 2005 ). The reinforcing properties elicited by central nervous system (CNS) toxicity and cell death. the simultaneous use of MDMA and Δ9 -THC have been METH increases terminal deoxynucleotidyl transferase investigated by means of several animal paradigms, dUTP nick end labeling (TUNEL), a marker of apoptotic such as conditioned place preference (CPP) and intracra- cell death and DNA damage, and alters expression of the nial and intravenous self-administration. In particular, Bcl2 gene and causes apoptosis by increasing caspase-3 in rats trained to self-administer MDMA intracerebro- activity, and the Fas/Fasl cell death pathways. MDMA ventricularly, the CB1R agonist CP55940 reduced the exposure in vitro causes an increase in reactive nitrogen amount of lever-pressing to obtain MDMA, while the species (RNS) and caspase-3 and, in vivo, an enhance- CB1R antagonist SR141617A (SR) increased MDMA- ment in protein levels associated with apoptosis (eg, associated lever-pressing, suggesting an increase and BAX, cytochrome c, caspase-3) ( Capela, Carmo, Remião, a decrease in MDMA’s reinforcing effects, respectively. & Bastos, 2009 , Krasnova & Cadet, 2009 ). SR decreases MDMA-induced CPP in rats, further sup- Moreover, the increased extracellular glutamate lev- porting the role of the CB1R in reward processes (Sala els induced by METH-binging activate N-methyl-D- & Braida, 2005 ). Accordingly, CB1-KO mice fail to self- aspartate (NMDA) receptors, promoting an increase in administer MDMA, while MDMA induces CPP and intracellular Ca2+ that leads to activation of neuronal ni- enhances extracellular DA levels in the nucleus ac- tric oxide synthase (nNOS) and subsequent production cumbens of both wild type and CB1-KO mice ( Touriño of nitric oxide (NO), and activation of apoptotic path- et al., 2008 ). This discrepancy might be attributed to the ways ( Halpin, Collins, & Yamamoto, 2014 ). different animal species (rats in the pharmacological Increasing evidence now suggests the involvement of studies, and mice in the genetic studies), or to the differ- several interacting mechanisms underlying ALS-induced ent behavioral paradigms utilized (self-administration neurotoxicity, such as hyperthermia, increased produc- vs CPP). Indeed, several brain circuits are involved in tion of reactive oxygen (ROS) and RNS, induction of in- the self-administration paradigm, such as those regulat- fl ammatory responses, apoptotic pathways, DNA dam- ing motivation, consciousness, arousal, and learning, age, and excitotoxic injury. Indeed, ALS-neurotoxicity is while CPP is mostly based on conditioning processes. associated with long-lasting impairments in mood and Intriguingly, subthreshold doses of Δ 9 -THC coadmin- cognitive functions in humans and animals ( Marshall & istered with a subthreshold dose of MDMA produced O’Dell, 2012 ). CPP in mice, while administration of the same dose of As concerns synthetic cathinones, data concern- Δ 9-THC with a rewarding dose of MDMA decreased CPP ing their pharmacological and toxicological proper- (Robledo et al., 2007). At low doses, the CB1R agonist ties, mechanisms of action and toxic effects are still WIN55212-2 (WIN) increased the rewarding properties inconclusive. of low doses of MDMA, while MDMA reinstated WIN- Just as AMPH and cocaine, cathinones in the CNS in- induced CPP, although WIN was not able to reinstate crease synaptic concentrations of catecholamines by in- MDMA-induced CPP in animals with no prior exposure hibiting their uptake transporters. The meth-cathinone to cannabinoids (Manzanedo, Rodríguez-Arias, Daza- analogs mephedrone and methylone function as Losada, & Maldonado, 2010 ).

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Accumulating evidence supports the role of the ECS regulation of emotional and cognitive responses, includ- in a crucial phase of the addiction cycle, that is, relapse to ing the medial prefrontal cortex, striatum, basolateral drug-seeking after a period of abstinence. Animal stud- amygdala, and hippocampal formation ( Bortolato, Frau, ies have shown that a priming with CB1R agonists re- Bini, & Luesu, 2010 ). instates not only the extinguished cannabinoid-seeking, MDMA-induced neurotoxicity is associated with but also cocaine-, heroin-, and alcohol-seeking, while functional defi cits, such as the loss of the ability to ther- reinstatement of cannabinoid-, heroin-, nicotine-, and moregulate, and alterations in locomotor activity in rats methamphetamine-seeking is prevented by blockade of ( Capela et al., 2009 ). CB1Rs ( Fattore, Fadda, & Fratta, 2007 ). Notably, a recent The ECS exerts either protective or toxic properties in study reported that SR, either alone or coadministered several in vitro and in vivo models of neuronal injury with a priming dose of any of the MDMA, reinstated and neurodegenerative pathologies, including gluta- MDMA-induced CPP. Conversely, WIN had no effect mate excitotoxicity, hypoxia, ischemic stroke, brain trau- on CPP reinstatement, but potentiated the effects of a ma, oxidative stress, Alzheimer’s disease, Parkinson’s subthreshold priming dose of MDMA (Daza-Losada, disease, and Huntington’s disease (Fernández-Ruiz, Miñarro, Aguilar, & Valverde, 2011). Collectively, all García, Sagredo, & Gómez-Ruiz, 2010 ). these fi ndings supported interactions between the ECS Given the frequent coabuse of Δ 9 -THC and MDMA in and MDMA regulating either drug-taking or drug-seek- humans (see Section “Introduction”), their interaction in ing behaviors. MDMA-induced neurotoxicity has also been investigat- The rewarding effects of Δ 9 -THC and MDMA/METH ed in terms of neurotoxicity. are likely mediated through a common neurochemical Coadministration of Δ 9 -THC and CP55940 prevented system, that is, the mesocorticolimbic pathway. In fact, MDMA-induced hyperthermia, hyperlocomotion, and MDMA raises DA levels by different mechanisms, while long-term increase in anxiety measured in the emergence cannabinoids infl uence the synthesis, release, and turn- test, but not in the social interaction test in rats (Morley, over of DA. In addition, cannabinoid and dopamine re- Li, Hunt, & Mallet, 2004 ). Accordingly, pretreatment ceptor expression overlaps in some brain areas, includ- with 3 mg/kg of Δ 9 -THC, considered a dose equivalent ing the nucleus accumbens. Finally, the ECS modulates to that consumed by moderate cannabis users, prevented DAergic transmission by controlling excitatory and in- MDMA-induced hyperthermia in mice receiving a neu- hibitory inputs on dopaminergic neurons. Both cannabi- rotoxic regimen of MDMA (Touriño et al., 2010). This noids and MDMA also modify the glutamatergic system hyperthermic effect was blocked by the CB1R antago- in areas involved in reward and addiction (Maldonado, nist AM251 and absent in CB1-KO mice, while neither a Valverde, & Berrendero, 2006 ). CB2R antagonist nor deletion of the CB2R gene abolished A plethora of data obtained from animal models has Δ9 -THC’s preventive effect on hyperthermia. Moreover, shown that administration of high or repeated doses of MDMA-induced microglial and astrocyte activation was MDMA and METH results in neurotoxicity to the sero- abolished by Δ 9 -THC in wild-type mice, and partially at- toninergic or dopaminergic system. MDMA neurotoxic- tenuated in CB2-KO mice, while Δ 9 -THC had no effect in ity is species-dependent, as in rats and monkeys MDMA CB1-KO and double CB1/CB2-KO mice. Recently, it was mostly affects the serotoninergic pathway, while in mice demonstrated that an ultra-low dose of Δ 9 -THC protects it targets the dopaminergic one. As revealed by biochem- mice from various insults, including ecstasy-induced ical, histological and immunohistochemical analyses neurotoxicity and cognitive defi cits. This protective effect (see the Section “ALS Pharmacology”), administration was long-lasting, and accompanied by increased levels of high or repeated doses of MDMA or METH causes of pCREB (phosphorylated cAMP response element- long-lasting depletion of 5HT and DA in several brain binding protein) and BDNF (brain-derived neurotroph- regions. ic factor) in some brain areas (Fishbein, Gov, Assaf, & In association with these neurotoxic effects on the Gafni, 2012 ). Altogether, these fi ndings suggest that DAergic and 5HTergic systems, binge METH regimens Δ 9-THC protects an animal from MDMA-neurotoxicity also cause cognitive and behavioral alterations similar by lowering body temperature and neuroinfl ammation to those observed in METH abusers (Marshall, Belcher, through both CB1 and CB2Rs. Feinstein, & O’Dell, 2007 ). Recently, our group demon- Chronic administration of Δ 9-THC during adolescence strated long-term memory defi cits and startle respon- attenuated MDMA-induced hyperthermia, and blunted siveness (index of pathological anxiety) in adult rats, as some behavioral MDMA-induced effects (Shen, Ali, & well as loss of DAergic and 5HTergic brain terminals, Meyer, 2011 ). Additionally, Δ 9 -THC prevented MDMA- following a METH binge neurotoxic regimen (Bortolato, induced decreases in exploratory behavior, and reduc- Frau, Piras, & Luesu, 2009 ). Moreover, the same neu- tions in 5HT and SERT levels in the striatum, frontal, and rotoxic METH treatment upregulated CB1R expres- parietal cortex, suggesting that chronic coadministra- sion in brain regions that play an important role in the tion during adolescence might provide some protection

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against MDMA-neurotoxicity. Indeed, chronic treatment Stephens, Roffman, & Babor, 2002 ), and when taken to- with Δ 9 -THC and MDMA during adolescence produced gether can determine neurocognitive defi cits in several sex-dependent long-lasting molecular, behavioral, and domains. Noteworthy, while in animal studies MDMA endocrine-effects, along with modifi cations of astrocytes, increases oxidative stress, cannabinoids have antioxida- microglia reactive markers, CB1R, and SERT expression. tive and antiinfl ammatory properties, supporting the Specifi cally, coadministration of Δ 9 -THC and MDMA im- idea that cannabinoids might also have neuroprotective paired working memory in female rats, and attentional effects against MDMA-neurotoxicity in humans. capabilities in both sexes, more severely than when ad- The literature on the long-term effects of each sub- ministered alone. Moreover, both drugs—either sepa- stance and/or their combination on cognitive functions, rately or in combination—induced increases in reactive memory, mood, and impulsivity is inconsistent. Indeed, microglia cells, suggesting that chronic administration of several reports demonstrated that defi cits in memory, these drugs might account for persistent modifi cations learning, or verbal fl uency, as well as self-reported psy- of microglia reactivity. chopathological problems such as depression, anxiety, As regards the METH + cannabis association, only paranoid ideation, and obsessive-compulsive behavior two groups, including ours, have investigated the abil- are mainly associated with cannabis use, rather than ity of the ECS to prevent METH-induced neurotoxicity with ecstasy ( Gouzoulis-Mayfrank & Daumann, 2006 ). in rats and mice ( Castelli et al., 2014 ; Nader, Rapino, However, other studies revealed a clear relationship Gennequin, & Chavant, 2014 ). Specifi cally, pre- and between neuropsychobiological defi cits and MDMA posttreatment with Δ 9-THC reduced overexpression of use, rather than cannabis use, that is, memory/cognitive nNOS and astrogliosis in the caudate-putamen and pre- defi cits were related to ecstasy, but not to cannabis use. frontal cortex, respectively, in rats exposed to a neuro- Indeed, higher self-rated psychiatric symptoms were toxic regimen of METH. SR reversed the neuroprotective reported in both light and heavy ecstasy polydrug us- effects of Δ 9-THC on METH-induced nNOS expression, ers than in the control group that included cannabis us- while it failed to counteract the effect of Δ 9 -THC on de- ers ( Parrott, 2006 ). Several factors might explain these creasing METH-induced astrogliosis. Our fi ndings indi- confl icting fi ndings, including the relative use of can- cate that Δ 9 -THC prevents METH-induced neurotoxicity nabis and ecstasy, the amount of each drug used (heavy through inhibition of nNOS and astrogliosis via both versus light users), and the psychobiological function CB1 receptor-dependent and -independent mechanisms examined. MDMA and cannabis may also infl uence dif- (Castelli et al., 2014). According to this, it has been re- ferent cognitive aspects, that is, cannabis use has been ported that Δ 9-THC, URB597, and JZL184 (inhibitors of associated with everyday memory problems, while degradation of anandamide and 2-arachidonoyl glycer- MDMA use accentuates prospective memory problems ol, respectively) attenuated the decrease in TH levels in (Parrott, 2006). the striatum, demonstrating that, in mice, stimulation of As regards the potential neuroprotective effect of can- the ECS reduced METH neurotoxicity to dopaminergic nabis on ecstasy users, cannabis + MDMA users were terminals ( Nader et al., 2014 ). found to have fewer psychobiological problems (ie, As concerns cathinone derivatives, no animal stud- lower rates of anger, hostility, depression, and nega- ies are currently available on the concomitant use of tive symptoms) than noncannabis users ( Gouzoulis- cannabis. Mayfrank & Daumann, 2006 ). Moreover, altered brain activation and poor working performance were detected Human Studies in MDMA users, and the concomitant use of cannabis was suggested to improve the MDMA-induced altera- In contrast to basic research, human studies investi- tions ( Parrott, 2006 ). Notably, coadministration of these gating acute and long-term effects of the consumption two drugs did not worsen cognitive impairments in- of cannabis + MDMA are limited and often contradic- duced by either drug (Dumont, van Hasselt, de Kam, & tory. When consumed acutely, cannabis can enhance Van Gerven, 2011 ). MDMA-induced subjective effects and counteract ad- Despite the wide consumption of METH + cannabis, verse MDMA effects, such as fatigue, insomnia, reduced only a few clinical studies have investigated the interac- appetite, irritability, panic attacks, visual hallucinations, tions between these two drugs, and their acute and long- and paranoid delusions that appear when “coming lasting effects. off” MDMA. As previously demonstrated in animals A positron emission tomography study showed that ( Morley et al., 2004 ), cannabis can also acutely attenuate the combined use of these two drugs caused reduced re- body temperature in dance club recreational ecstasy us- gional cerebral glucose metabolism in the frontal, tem- ers ( Parrott et al., 2007 ). poral, and striatal brain areas of METH + marijuana (MJ) Both drugs taken alone may have detrimental ef- users during completion of a continuous performance fects on memory and cognition (Parrott, 2013; Solowij, task, in comparison to individuals that abused METH

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alone, but no differences were observed in continuous methcathinone), often present in combinations of two or performance task accuracy between the two groups more drugs ( Papaseit, Farrè, Schifano, & Torrens, 2014 ). ( Gouzoulis-MayFrank & Daumann, 2006 ). Accordingly, Most worrisome, the recent trend seems to be to mix cognitive functioning in several ability areas (ie, verbal different types of designer drugs like cathinones (stim- fl uency, abstraction/executive functioning, attention/ ulants) or tryptamines (hallucinogens) with synthetic working memory, learning, and delayed recall/ cannabinoids. retention) in METH + MJ were found not to differ signif- icantly from the control group, or METH users without a history of MJ dependence (Gonzalez et al., 2004). Re- MINI-DICTIONARY cently, decreased frontal N-acetylaspartyl glutamate levels, an indicator of intact neuronal integrity, were found Amphetamine-like substances Although there are a variety of in adolescents using METH + MJ, compared to healthy amphetamines, in this chapter, amphetamine-like substances controls and METH groups, supporting the idea that (ALS) stands for amphetamine (AMPH), methamphetamine (METH), 3,4-methylenedioxymethamphetamine (MDMA), and concomitant heavy cannabis and METH use may induce cathinones. The fi rst three compounds are frequently coabused neurotoxicity in the adolescent brain (Sung, Carey, Stein, with cannabis, while cathinones are the most popular new & Ferrett, 2013 ). psychoactive substances (NPS). Further clinical and preclinical studies are needed to Chatinone 2-amino-1-phenylpropanone, a natural compound of clarify if concurrent use of cannabis and MDMA/METH the khat plant. Drug addiction A chronic, relapsing brain disease that causes worsens or improves neurocognitive functions. compulsive drug-seeking and use, despite harmful consequences Finally, a paucity of clinical data are available on the to the drug addict and those around them. effects of cathinone derivatives given alone or in combi- Dopamine A monoamine neurotransmitter. In particular, the nation with cannabis. In humans, cathinones induce pri- mesolimbic dopamine system that projects from the ventral marily stimulant effects such as euphoria, elevated mood, tegmental area to the nucleus accumbens has been implicated in the rewarding effects of drugs of abuse. mental stimulation, intensifi cation of sensory sensations, Ecstasy or MDMA This drug is a phenylethylamine, a increased energy and sociability, empathy connection, molecule that belongs to the group of synthetic derivatives of and decreased inhibition ( Prosser & Nelson, 2012 ). How- methamphetamine. It is an illegal drug, synthesized for the fi rst ever, high doses of cathinones lead to hallucinations, time in 1912, with a prevailing action on the serotonergic system. paranoia, agitation, self-mutilation, psychosis, tachycar- NPS A new psychoactive substance is defi ned as a new narcotic or psychotropic drug, in pure form or in preparation. dia, increased blood pressure, and hemorrhage. Polydrug use A common phenomenon defi ned as the Chronic khat use is associated with impaired inhibito- consumption of more than one drug during a specifi c time period. ry control, and long-term use of cathinone is associated Reward system The brain circuit that controls an individual’s with dysfunctions in the prefrontal cortex, and reduced response to natural rewarding stimuli, such as food, sex, and DA levels in the striatum (Colzato, Ruiz, van den social interaction. Wildenberg, & Bajo, 2011). Even before clubbing (and after recent drug use), mephedrone users performed worse References than nonusers on cognitive tasks and, contrary to con- Baumann , M. H. , Ayestas , M. A. , Jr. , Partilla , J. S. , Sink , J. R. , et al. ( 2012 ) . trols, showed decreased performance in verbal learning The designer methcathinone analogs, mephedrone and methylone, and fl uency ( Herzig, Brooks, & Mohr, 2013 ). Yet, when are substrates for monoamine transporters in brain tissue . Neuro- subjects were analyzed for their use of other drugs or psychopharmacology , 37 , 1192 – 1203 . preexisting factors, it turned out that prior polydrug use Black , D. L. , Cawthon , B. , Robert , T. , Moser , F. , et al. ( 2009 ) . Multiple (cannabis + alcohol in particular, but also AMPH) and drug ingestion by ecstasy abusers in the United States . Journal of Analytical Toxicology , 33 , 143 – 147 . related psychological traits (depression) were likely to Bortolato , M. , Frau , R. , Piras , A. P. , Luesu , W. , et al. ( 2009 ) . Metham- affect cognitive functioning in the sample studied. phetamine induces long-term alterations in reactivity to environ- The presence of some of these substances was de- mental stimuli: correlation with dopaminergic and serotonergic tected through surveys/questionnaires, studies on drug toxicity . Neurotoxicity Research , 15 , 232 – 245 . samples and biological fl uids, and case reports. In one in- Bortolato , M. , Frau , R. , Bini , V. , Luesu , W. , et al. ( 2010 ) . Methamphet- amine neurotoxicity increases brain expression and alters behav- stance, a driver stopped by offi cers of the Massachusetts ioral functions of CB1 cannabinoid receptors . Journal of Psychiatric State Police and subjected to blood sample analysis re- Research , 44 , 944 – 955 . vealed the presence of clonazepam, 7-aminoclonazepam, Capela , J. P. , Carmo , H. , Remião , F. , Bastos , M. L. , et al. ( 2009 ) . Molecu- carboxy-THC, diphenhydramine, and MDMA ( Elian & lar and cellular mechanisms of ecstasy-induced neurotoxicity: an Hackett, 2014). The analysis of fl uid body samples ob- overview . Molecular Neurobiology , 39 , 210 – 271 . Carroll , F. I. , Blough , B. E. , Mascarella , S. W. , Navarro , H. A. , et al. tained from suspected NPS intoxication cases detected ( 2010 ) . Synthesis and biological evaluation of bupropion analogues synthetic cannabinoids of the JHW series and cathinones as potential pharmacotherapies for smoking cessation . Journal of (methylone, butylone, mephedrone, fl ephedrone, and Medicinal Chemistry , 53 , 2204 – 2214 .

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CC0330.indd0330.indd 108108 114/12/164/12/16 2:432:43 PMPM REFERENCES e109

Castelli, M.P., Madeddu, C., Casti, A., Casu, A., et al. (2014) Mohamed , W. M. , Ben Hamida , S. , Cassel , J. C. , de Vasconcelos , A. P. , Δ9-tetrahydrocannabinol prevents methamphetamine-induced et al. ( 2011 ) . MDMA: interactions with other psychoactive drugs . neurotoxicity. PLoS One . 20, 9(5):e98079. Pharmacology Biochemistry and Behavior , 99 , 759 – 774 . Colzato , S. L. , Ruiz , M. J. , van den Wildenberg , W. P. M. , Bajo , M. T. , Morley , K. C. , Li , K. M. , Hunt , G. E. , Mallet , P. E. , et al. ( 2004 ) . et al. ( 2011 ) . Long-term effects of chronic khat use: impaired inhibi- Cannabinoids prevent the acute hyperthermia and partially protect tory control . Frontiers in Psychology , 219 , 1 – 5 . against the 5HT depleting effects of MDMA(“Ecstasy”) in rats . Neu- Daza-Losada , M. , Miñarro , J. , Aguilar , M. A. , Valverde , O. , et al. ( 2011 ) . ropharmacology , 46 , 954 – 965 . Acute blockade of CB1 receptor leads to reinstatement of MDMA- Nader , J. , Rapino , C. , Gennequin , B. , Chavant , F. , et al. ( 2014 ) . induced conditioned place preference . Pharmacology Biochemistry Prior stimulation of the endocannabinoid system prevents and Behavior , 100 , 3 3 – 3 9 . methamphetamine-induced dopaminergic neurotoxicity in the Dumont , G. J. , van Hasselt , J. G. , de Kam , M. , Van Gerven , J. M. , et al. striatum through activation of CB(2) receptors . Neuropharmacology , ( 2011 ) . Acute psychomotor, memory and subjective effects of 5, pii: S0028-3908. MDMA and THC co-administration over time in healthy volun- NSDUH (2010). Results from the 2009 National Survey on Drug use teers . Journal of Psychopharmacology , 25 , 478 – 489 . and Health. Summary of National Findings, volume 1, n2010 Elian , A. A. , & Hackett , J. ( 2014 ) . A polydrug intoxication involving (september 2010). http://www.oas.samhsa.gov/NSDUH methoxetamine in a drugs and driving case . Journal of Forensic Sci- Paillet-Loilier , M. , Cesbron , A. , Le Boisselier , R. , Bourgine , et al. ( 2014) . ences , 59 , 854 – 858 . Emerging drugs of abuse: current perspectives on substituted cathi- ESPAD (2000). The 1999 alcohol and other drug use among students nones . Substance Abuse and Rehabilitation , 5 , 3 7 – 5 2 . in 30 Eruropean countries, Swedish council for information on al- Panenka , W. J. , Procyshyn , R. M. , Lecomte , T. , MacEwan , G. W. , et al. cohol and other drugs (CAN). The Pompidou group of the Council ( 2013 ) . Methamphetamine use: a comprehensive review of molecu- of Europe. lar, preclinical and clinical fi ndings . Drug and Alcohol Dependence , Fattore , L. , Fadda , P. , & Fratta , W. ( 2007 ) . Endocannabinoid regulation 129 , 167 – 179 . of relapse mechanisms . Pharmacological Research , 5 , 418 – 427 . Papaseit , E. , Farrè , M. , Schifano , F. , & Torrens , M. ( 2014 ) . Emerging Fernández-Ruiz , J. , García , C. , Sagredo , O. , Gómez-Ruiz , M. , et al. drugs in Europe . Current Opinion in Psychiatry , 27 , 243 – 250 . ( 2010 ) . The endocannabinoid system as a target for the treatment of Parrott , A. C. ( 2006 ) . MDMA in humans: factors which affect the neu- neuronal damage . Expert Opinion in Therapeutic Targets , 14 , 387 – 404 . ropsychobiological profi les of recreational ecstasy users, the inte- Fishbein , M. , Gov , S. , Assaf , F. , Gafni , M. , et al. ( 2012 ) . Long-term grative role of bioenergetic stress . Journal of Psychopharmacology , 20 , behavioral and biochemical effects of an ultra-low dose of 147 – 163 . Δ 9-tetrahydrocannabinol (THC): neuroprotection and ERK signal- Parrott , A. C. ( 2013 ) . Human psychobiology of MDMA or ‘Ecstasy’: an ing . Experimental Brain Research , 221 , 437 – 448 . overview of 25 years of empirical research . Human Psychopharmacol- Fleckenstein , A. E. , Volz , T. J. , Riddle , E. L. , Gibb , J. W. , et al. ( 2007 ) . ogy, Clinical and Experimental , 28 , 289 – 307 . New insights into the mechanism of action of amphetamines . An- Parrott , A. C. , Milani , R. M. , Gouzoulis-Mayfrank , E. , & Daumann , J. nual Reviews of Pharmacology and Toxicology , 47 , 681 – 698 . ( 2007 ) . Cannabis and Ecstasy/MDMA (3,4-methylenedioxymeth- Gonzalez , R. , Rippeth , J. D. , Carey , C. L. , Heaton , R. K. , et al. ( 2004) . amphetamine): an analysis of their neuropsychobiological inter- Neurocognitive performance of methamphetamine users discor- actions in recreational users . Journal of Neural Transmission , 114 , dant for history of marijuana exposure . Drug and Alcohol Depen- 959 – 968 . dence , 76 , 181 – 190 . Prosser , J. M. , & Nelson , L. S. ( 2012 ) . The toxicology of bath salts: Gouzoulis-Mayfrank , E. , & Daumann , J. ( 2006 ) . The confounding prob- a review of synthetic cathinones . Journal of Medical Toxicology , 8 , lem of polydrug use in recreational ecstasy/MDMA users: a brief 3 3 – 4 2 . overview . Journal of Psychopharmacology , 20 , 188 – 193 . Robledo , P. , Trigo , J. M. , Panayi , F. , de la Torre , R. , et al. ( 2007 ). Be- Green , B. , Kavanagh , D. , & Young , R. ( 2003 ) . Being stoned: a review of havioural and neurochemical effects of combined MDMA and THC self-reported cannabis effects . Drug and Alcohol Reviews , 22 , 453 – 460 . administration in mice . Psychopharmacology , 195 , 255 – 264 . Halpin , L. E. , Collins , S. A. , & Yamamoto , B. K. ( 2014 ) . Neurotoxicity Sala , M. , & Braida , D. ( 2005 ) . Endocannabinoids and 3,4-methylene- of methamphetamine and 3,4-methylenedioxymethamphetamine. dioxymethamphetamine (MDMA) interaction . Pharmacology Bio- Life Sciences , 97 , 3 7 – 4 4 . chemistry and Behavior , 81 , 407 – 416 . Herzig , D. A. , Brooks , R. , & Mohr , C. ( 2013 ) . Inferring about individual SAMHSA . ( 2013 ) . Substance Abuse and Mental Health Services Adminis- drug and schizotypy effects on cognitive functioning in polydrug tration, Results from the 2012 National Survey on Drug Use and Health: using mephedrone users before and after clubbing . Human Psycho- Summary of National Findings, NSDUH Series H-46, HHS Publication pharmacology , 28 , 168 – 182 . No. (SMA) 13-4795 . Rockville, MD : Substance Abuse and Mental Krasnova , I. N. , & Cadet , J. L. ( 2009 ) . Methamphetamine toxicity and Health Services Administration . messengers of death . Brain Research Reviews , 60 , 379 – 407 . Schulz , S. ( 2011 ) . MDMA & cannabis: a mini-review of cognitive, be- Maldonado , R. , Valverde , O. , & Berrendero , F. ( 2006 ) . Involvement of havioral, and neurobiological effects of co-consumption . Current the endocannabinoid system in drug addiction . Trends in Neurosci- Drug Abuse Reviews , 4 , 8 1 – 8 6 . ence , 29 , 225 – 232 . Shen , E. Y., Ali , S. F., & Meyer , J. S. ( 2011 ) . Chronic administration Manzanedo , C. , Rodríguez-Arias , M. , Daza-Losada , M. , Maldonado , C. , of THC prevents the behavioral effects of intermittent adoles- et al. ( 2010 ) . Effect of the CB1 cannabinoid agonist WIN 55212-2 on cent MDMA administration and attenuates MDMA-induced the acquisition and reinstatement of MDMA-induced conditioned hyperthermia and neurotoxicity in rats. Neuropharmacology , 61 , place preference in mice. Behavior and Brain Function , 22 , 6 – 1 9 . 1183 – 1192 . Marshall , J. F. , & O’Dell , S. J. ( 2012 ) . Methamphetamine infl uences on Solowij , N. , Stephens , R. , Roffman , R. A. , & Babor , T. ( 2002 ) . Does brain and behavior: unsafe at any speed? Trends in Neuroscience , 35 , marijuana use cause long-term cognitive defi cits? JAMA , 287 , 536 – 545 . 2653 – 2654 . Marshall , J. F. , Belcher , A. M. , Feinstein , E. M. , & O’Dell , S. J. ( 2007 ) . Strote , J. , Lee , J. E. , & Wechsler , H. ( 2002 ) . Increasing MDMA use among Methamphetamine-induced neural and cognitive changes in ro- college students: results of a national survey . Journal of Adolescent dents . Addiction , 102 , 6 1 – 6 9 . Health , 30 , 6 4 – 7 2 .

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CC0330.indd0330.indd 109109 114/12/164/12/16 2:432:43 PMPM e110 e10. CANNABIS AND THE USE OF AMPHETAMINE-LIKE SUBSTANCES

Sulzer , D. , Sonders , M. S. , Poulsen , N. W. , & Galli , A. ( 2005 ) . Mecha- Touriño , C. , Zimmer , A. , & Valverde , O. ( 2010 ) . THC Prevents MDMA nisms of neurotransmitter release by amphetamines: a review . Prog- neurotoxicity in mice . PLoS One , 5 , 9143 . ress in Neurobiology , 75 , 406 – 433 . United Nations Ofﬁ ce on Drugs and Crime, UNODC (2010). World Sung , Y. H. , Carey , P. D. , Stein , D. J. , Ferrett , H. L. , et al. ( 2013 ) . De- Drug Report. United Nations Publication. creased frontal N-acetylaspartate levels in adolescents concurrently United Nations Ofﬁ ce on Drugs and Crime, UNODC (2014). World using both methamphetamine and marijuana . Behavior and Brain Drug Report. United Nations Publication. Research , 246 , 154 – 161 . Wish , E. D., Fitzelle , D. B., O’Grady , K. E., Hsu , M. H., et al. Touriño , C. , Ledent , C. , Maldonado , R. , & Valverde , O. ( 2008 ) . CB1 can- ( 2006 ) . Evidence for significant polydrug use among ecstasy- nabinoid receptor modulates 3,4-methylenedioxymethamphetamine using college students. Journal of the American College Health , acute responses and reinforcement. Biological Psychiatry , 63 , 1030 – 1038 . 55 , 9 9 – 1 0 4 .

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