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Synthetic : general considerations Nik De Brabantera*, Koen Deventera, Veronique Stoveb, Peter Van Eenooa

aGhent University (UGent), Department of Clinical Biology, Microbiology and Immunology, Doping Control Laboratory, Technologiepark 30, B-9052 Zwijnaarde, Belgium. bDepartment of Laboratory Medicine, Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium.

*Corresponding author: Nik De Brabanter, Ghent University (UGent), Department of Clinical Biology, Microbiology and Immunology, Doping Control Laboratory, Technologiepark 30, B-9052 Zwijnaarde, Belgium, Technologiepark 30, B-9052 Zwijnaarde, Belgium. Email: [email protected]

Received: 14.06.2013 Accepted: 07.08.2013 Published: 30.10.2013

Abstract

Around 2008 were found to be present in; and responsible for the psychoactive effects of herbal mixtures with names like ‘Spice’ or ‘K2’. In response to the increased popularity of these products, (inter)national organizations and governments started banning these cannabimimetics gradually. However, the lack of an uniform and international regulation makes it hard to control this issue. For the different types of synthetic cannabinoids the scientific knowledge in terms of pharmacokinetics and pharmacodynamics is limited. This also means that little is known on the health of users, both on short and long term. In the last years effort has been made to make detection of these products possible in different biological matrices. However, since the number of cannabimimetic compounds on the market appears to grow every month, both scientist and legislators run after a moving target.

Keywords: Synthetic cannabinoids, Spice, legislation, detection methods, .

209 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______INTRODUCTION chemistry professor John W. Huffman, who first synthesized these products in the Starting from 2004, a new 1990s. In a similar way AM (e.g. in AM- generation of psychoactive substances 630) refers to professor Alexandros appeared on the market. These products, Makriyannis from Northeastern University with brand names like ‘Spice’ or ‘K2’, are and HU (e.g. from HU-210) to Hebrew sold as herbal mixtures and are available in University. Also, the pharmaceutical many European countries (1). Packed as industry realized the potential value of ‘natural herbal incense’ or ‘room these products, leading to the synthesis of odorizers’, these products can be traded the CP-family (e.g. CP-47,497) by Pfizer legally in headshops and online stores and the WIN-group (e.g. WIN 55,212-2) (2,3). After smoking these mixtures, users by the former Sterling Winthrop reported -like effects on internet Pharmaceuticals. forums. These effects were first explained In general, it are lipid soluble, non- in 2008 by the detection of synthetic polar molecules, containing 20 to 26 cannabinoids like JWH-018 as active carbon atoms (6). Based upon this ingredient (4) , although not mentioned on chemical structure, synthetic cannabinoids the package. can be divided into different classes (Table Throughout the years, more of 1) (1). these products were identified as additives • Classical cannabinoids: structurally in these packages of herbal material. As a related to THC from Cannabis sativa. response to the rising popularity of these • Non-classical cannabinoids: compounds, several countries started cyclohexylphenols or 3-arylcyclohexanols monitoring and even banning these • Hybrid cannabinoids: structural products (5). combinations of both classical and non- The search for compounds with classical cannabinoids. THC-like properties in the human body, • Aminoalkylindoles; i.e. synthetic agonists o Naphthoylindoles or briefly cannabimimetics, started in the o Phenylacetylindoles pharmaceutical industry. In a way to o Naphthylmethylindoles separate the wanted pain-relieving effects o Benzoylindoles from the unwanted psychotropic effects, • : endocannabinoids and several categories of products were synthetic analogs synthesized and subjected to SAR • Others: diarylpyrazoles, naphthoyl- (structure activity relationship) tests. The pyrroles, etc. academic and/or pharmaceutical origin of It should be noted that, depending these compounds is often reflected in the on the source, the classifications can vary. name of the product. In the best known In the light of an internationally uniformed class of JWH-compounds, these initials approach, referring to the stands for the name of the organic (abovementioned) classification of a

210 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______leading institution as the United Nations recommended. Office on Drugs and Crime (UNODC) is

Classical cannabinoids OH e.g. THC, HU-210, AM906, … OH

O

Non-classical cannabinoids OH e.g. CP-47,497-C8, CP-55,940, HU-308, … OH

Hybrid cannabinoids OH e.g. AM-4030 OH

H

O

OH

Naphthoylindoles e.g. JWH-018, JWH-073, JWH-122, JWH- O 200, … N

Phenylacetylindoles e.g. JWH-250, RCS-8, JWH-203, … O

N

O

Naphthylmethylindoles e.g. JWH-175, JWH-184, JWH-185, …

N

211 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______Benzoylindoles e.g. AM-630, AM-2233, RCS-4, … O

N

O

Eicosanoids O OH e.g. , methanandamide, … NH

Others F e.g. JWH-307, CRA-13, … O

N

Table 1. Classification of synthetic cannabinoids according to the UNODC (1), with some typical examples. The underlined compound is illustrated in the second column.

ABUSE OF SYNTHETIC plants and in many cases the ingredients CANNABINOIDS listed on the package do not cover the content either (8,9). The manufacturers of Packages of ‘Spice’ usually contain these blends make users believe the effects approximately 3g of herbal material and are caused by the mix of plant material are often sold in head shops, gas stations or used. However, research on the botanical via internet shops. The price varies around material showed that most of the plant €10/g, which is considered expensive species do not have psychoactive compared to traditional cannabis (4). It is properties and are therefore only used to promised that the inhalation of the blends dilute the added cannabimimetics (9). of psychoactive plants gives the user a Moreover, the producers try to present similar experience as marijuana, only using their products as natural and safe in order legal alternatives. A survey in the US to circumvent the marijuana policy of showed that ‘Spice’ products were governments. The UNODC concluded that primarily smoked, but also administration producers respond very fast to changes in via vaporization, oral and rectal ingestion legislations by making small modifications were reported (7). to the new products launched (1). Little is known on the exact The success of this ‘legal-highs’ composition and the properties of the used business is reflected in the increasing 212 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______number of web shops selling these produced cheaply, it is clear that these products online. In 2009, the European businesses are very lucrative (10). During Monitoring Centre for Drugs and Drugs the production process, the synthesized addiction (EMCDDA) found 115 online synthetic cannabinoids are distributed over shops offering psychoactive substances in the dried plant material. This is usually Europe, in 48% of these ‘Spice’ products done by homogenization with were offered. In a recent report mention is cannabinoids in the crystalline form or by made of an increase to 314 online shops in spraying the products dissolved in an 2011 and 690 in January 2012 (10). organic solvent. However, recently also the Moreover, an investigation via Google® starting materials are being sold as performed by the Belgian Monitoring ‘research chemicals’ via online shops or Centre for Drugs and Drug Addiction traders (13). (BMCDDA), showed that all new products The rate at which a specific product reported in 2010 were already offered for is spreading, is also noteworthy. In Europe, sale online even before their existence was JWH-018 was first reported by Austria in picked up by the Belgium Early Warning December 2008. Only in the first year after System for Drugs (BEWSD) (11). Also the this, eight more neighboring countries list of products with names like ‘Spice confirmed this finding, followed by ten gold’, ‘Yucatan Fire’ and ‘Lava Red’ more in the next months. Similar continued to rise (8). Furthermore, recent developments were reported for other investigation in Poland demonstrated that compounds like JWH-073 (13 countries), many herbal blends contained more than CP47, 497 (10 countries), JWH-122 (14 one psychoactive ingredient (12). countries), JWH-081 (10 countries) and A survey of the member states of AM-2201 (11 countries) (11). In 2011, the European Union by the EMCDDA already 23 new synthetic cannabimimetics showed that in 2009, ‘Spice’ products were were reported through the European Early identified in 21 out of the 30 countries. At Warning System (EWS), in 2012, another that time no products containing synthetic 30 followed. With a current total of 84 cannabinoids were found in Belgium. In compounds (May 2013), the synthetic 2011 however, Belgian laboratories cannabinoid receptor agonists are, despite reported 11 synthetic cannabinoids to the their recent introduction, already the European Early Warning System (EWS) largest drug family monitored by the and for the first time a complete laboratory EMCDDA (14). capable of producing and packing synthetic An internet search learned that cannabinoids was dismantled in Belgium. mostly young people - especially men - Similar facilities were reported in Ireland aged between 25 and 40 are using Spice- and the Netherlands and are the link like products. The reasons are various; between the producers – mainly located in ranging from previous cannabis users China and India – and the customers in looking for a substitute over people in Europe. Since these substances can be search of legal drugs to experimental users

213 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______seeking sensation (8). In 2008, German is sufficient to make a small change in the authorities found a strong increase in the drug structure to stay one step ahead of the interest for these products after a period of legislator. Therefore, the UK and Ireland biased media attention in which their use started using generic definitions to include as legal cannabis substitutes was products which will appear in the future announced. Once the presence of synthetic (8). Recently, other European countries compounds was demonstrated and some of also adopted this strategy in substitution these products were banned, the opposite for the earlier used approach of individual trend was observed, leaving only the users listing of already identified synthetic looking for a cannabis substitute to avoid cannabinoids (10). positive testing (1). In Belgium the first legislative Even in sport drug testing, the first actions were taken in 2011, by adding the cannabimimetics were reported in the first seven compounds to the list of statistics of the World Anti-Doping prohibited psychotropic substances. In Agency (WADA). However, with only 3 2013 six more cannabimimetics were listed positive cases in 2011, the number remains (Table 2). Unfortunately, to this day, no small compared to the traditional THC generic definitions have come into force. abuse in sport competitions (n = 442 in In the United States, the Synthetic 2011) (15). Drug Abuse Prevention Act of 2012 placed cannabimimetic agents in Schedule I, LEGISLATION making manufacturing, distributing, or possessing these products illegal. Besides Since synthetic cannabinoids are this federal law, several states and even currently not controlled under the UN individual cities have taken additional Drug control conventions, the legal status measures to control ‘Spice’ abuse (16). of these compounds depends on the drug Since, similar to classical cannabis, laws of individual countries (1). the synthetic analogues are predominantly In Europe, the first actions by smoked, it is not inconceivable that passive governments were taken in 2009. The first inhalation of the smoke can result in discovered compounds JWH-018, HU-210 positive testing. Once this was observed and CP47,479 and its homologues were for cannabis (17), threshold concentrations included in national drug laws in Austria, were installed to distinguish active from Germany, France, Luxembourg, Sweden, passive use (18). However, up to now, this Estonia, Poland, Hungary and the United possibility has not been studied for the Kingdom. A recent report on the evolution cannabimimetics currently flooding the of the situation in Poland over the last few market. years, showed that both the compounds In general legislation, both within and the way of trading changed in response and outside Europe, is too diverse and to the successive actions of the Polish therefore not efficient to tackle government (12). As mentioned earlier, it international issues as ‘legal highs’.

214 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______

KB 2011-09-26/16, art. 1,011 KB 2013-03-20/08, art. 1,012 Effective date: 23/10/2011 Effective date: 22/04/2013 JWH-018 AM-694 JWH-073 AM-2233 JWH-250 WIN 48,098 JWH-398 JWH-307 CP-47,497 A-796,260 HU-210 XLR-11 (5F-UR144) JWH-210 Table 2. Synthetic cannabinoids present on the list of prohibited psychotropic substances in Belgium.

PHARMACOKINETICS pharmacological data available for these compounds, human administration in order Contrary to the classical THC for to perform excretion studies is ethically which the pharmacokinetics have been questionable. Therefore, most studies use investigated (19,20), no such data are models to reveal the metabolic pathways in available for the synthetic analogues. the human body. In one of the most As described in several reports on the common approaches human liver experiences of ‘Spice’ users, the effect is microsomes are used to investigate the quickly noticeable after smoking a few metabolisation in vitro (22–25), in the grams of herbal material (2,6,16). These search for a more complete model with observations are supported by a recent higher complexity also in vivo mice study on the quantification of JWH-018 in experiments are performed (26). In some blood after smoking the incense ‘Smoke’ cases, human urine samples are available (21); the maximum concentrations were from caught users (27–29) or conducted found 5 min post-smoking. This shows that self-experiments (30). after inhalation, the absorption via the In general, these compounds are lungs and the distribution over organs like excessively metabolized in the human the brain takes place in a few minutes (1). body. In all metabolic studies on It was found that the measured maximum compounds of the aminoalkylindole family blood concentrations of JWH-018 were described up to now a similar series of already decimated after 3h and the parent modifications were found: single or compound was detectable until 48h after multiple hydroxylations, carboxylation, administration (21). dehydrogenation, dealkylation and Investigation of the metabolisation dihydrodiol formation. From the data of cannabimimetics is not as obtained using in vivo models or positive straightforward as for pharmaceutical urine samples, is was found that these approved agents. Since there are little or no metabolites are mainly excreted as

215 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______glucuronide and/or sulphate conjugates in (34). As shown in Table 3, this is different urine (26). Mainly the monohydroxylated for synthetic cannabinoids. The affinity of (31) and carboxylated (28) metabolites are the most prevalent cannabimimetic found in the highest quantities in urine. compounds is significantly higher, especially towards the CB1 receptor. With PHARMACODYNAMICS this in mind, it can be expected that compounds with lower potency (i.e. lower Although the affinity for the CB1 receptor) than classical sativa and derivates are known for THC will not be used in ‘Spice’-like centuries, it was only in the last twenty products. Nevertheless JWH-015 was years that the interactions in the human recently detected in a herbal blend in body were revealed (6,32). Latvia (1). Today, two cannabinoid receptors When using data on receptor are described in the human body. Both affinities, one should be careful when CB1 and CB2 are G-protein coupled using exact numbers. Indeed, depending on receptors with an important function in the used experimental set-up, variation in intercellular signaling. The CB1 receptor is the values is possible (34). In short, the distributed in the brain and the central receptor affinity (Ki) is determined as the nervous system, mainly expressed ability of the given compound to displace a presynaptically, and decreases the release potent radio labeled cannabinoid (usually of neurotransmitters like dopamine (33). tritiated CP-55,940 or tritiated WIN- Activation of the CB1 receptor is 55,512-2) from their binding sites (35). For responsible for the psychotropic effects a potent cannabinoid, low concentrations assigned to cannabis use. will be sufficient to achieve this. Since this CB2 receptors are located in concentration (IC50) is proportional to the immune cells and interfere in the receptor affinity Ki, the more potent the regulation of the inflammatory process cannabinoid, the lower Ki (Table 3). (19). Therefore in the medical field, research has focused on receptor agonists HEALTH RISKS selective for this CB2 receptor aiming for the therapeutic effects and hereby avoiding Although there are case reports the psychotropic effects induced by the describing the effects experienced interaction with the CB1 receptor. immediately after the use of ‘Spice’ Next to endocannabinoids, plant (2,3,16), little or no information is derived and other exogenous cannabinoids available on the long term effects or the act as agonists of both receptors with consequences of regular use. varying affinity. Classical cannabinoids In 2009, Zimmermann et al. like THC have comparable affinity for reported on a patient who showed both receptors, about 40 nM, without a symptoms of a physical withdrawal major selectivity for a particular receptor syndrome after using ‘Spice Gold’ on a

216 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______Compound Ki - CB1 (nM) Ki - CB2 (nM) HU-210 0.06 (34) 0.52 (34) JWH-122 0.7 (36) 1.2 (36) JWH-073 8.9 (35,37) 27 (35), 38 (37) JWH-018 9 (35) 2.9 (35) CP47,497 9.54 (38) JWH-250 11(39) 33(39) Δ9-THC 39.5 (34) 40 (34) JWH-015 383 (34), 13.8 (34,35) 164(35) Table 3. Receptro affinities for both cannabinoid receptors for some common cannabimimetics. daily basis for about 8 months. While the impossible. It was shown that the content patient initially used only 1g of product of these packages varies significantly and every day, the decreasing effect is often not in accordance with the experienced made him increase the dose up indications on the package. Toxicological to 3g daily. Both physical (sweating, data on the used plant material are not tremor, insomnia, nausea, etc.) as available, and then again, mostly the psychological (depression, desperation, indications on the packages with regards to desire for ‘Spice Gold’) effects were the herbal material are not reliable (9). observed the first days of treatment in Concerning the added synthetic hospital (40). In another paper, psychosis cannabinoids, it was shown that was diagnosed in ten patients after concentrations can vary (5) and that some smoking herbal blends containing synthetic blends may contain two or more active cannabinoids, which lasted months after compounds (42). Although research the final use (41). showed that the used chemicals are of high Recently, compound specific data purity (43), the presence of impurities with related to harm assessment have been unknown toxicity cannot be ruled out. In included in the European Database on New that way, it is not possible to estimate the Drugs (EDND). For two compounds, impact when smoking a few grams of a chronic physical damage after use is given mixture. In general, the observed mentioned. It is related to learning effects are very diverse and highly difficulties and cognitive ailment for HU- dependent on the type of herbal blend or 210 and JWH-018, respectively. Moreover, synthetic cannabinoid(s) used. Most for both compounds physical dependence described psychoactive effects are: (withdrawal symptoms) was reported, alterations in mood (from euphoria to together with psychological dependence anxiety) (5), hallucinations, agitated for JWH-018 and JWH-122 (11). behavior and hyperreflexia (16). Medical Predicting the possible effects of a investigation showed symptoms like particular herbal blend, is almost increased pulse rates (6) and blood 217 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______pressure, flushed skin, dilated pupils and compound, the structure is elucidated by nausea (16). It was reported that the major using high resolution mass spectrometry psychotropic and physical effects (HRMS) or NMR (Nuclear Magnetic disappear after 6h to 8h (5,6). Resonance) technology (52–54). Together with the fact that these When it comes to detection in the cannabimimetics have stronger affinities human body, different approaches can be for the cannabinoid receptors compared to used depending on the type of biological THC, it is not unlikely that overdosing sample available. In serum or whole blood, would lead to life-threatening both the unchanged target compound and intoxications. This is confirmed in case its metabolites are present and can be studies (44) and reflected in the increasing extracted and analyzed by means of liquid statistics of Poison Control Centers in the chromatography (LC) (55,56). The US: in 2011 there were reports of over methods developed up till now mostly 4000 synthetic marijuana exposures in a target the parent compounds, since this period of 8 months, which is an increase of eliminates the need for the time-consuming 52% compared to 2010 (45). search for metabolites and allows the quick update of the method after the release of a DETECTION OF SYNTHETIC new compound in the future (57,58). For CANNABINOIDS ABUSE oral fluid testing, detection of this parent compound is possible, even via direct In the past, several screening injection on the LC system (59). However, procedures – both via immunological and it should be noted that detection in the chromatographic techniques – have been latter matrix is limited to a few hours after developed to screen for the use of products consumption (60). from Cannabis sativa in different matrices If urine is the matrix of choice, (18,46–49). The abuse of the growing knowledge on the metabolisation is group of cannabimimetics, synthesized essential, since no unchanged parent over the last years, however, cannot be compound is found to be excreted. To detected with these existing methods. detect these metabolites, an enzymatic For the identification of spiked hydrolysis is usually performed, followed substances in the herbal material in by an extraction and analysis by means of particular, a more or less standard strategy liquid chromatography coupled to mass is used. The herbal material is extracted spectrometry (LC-MS) (61). For JWH-018 and subsequently analyzed by means of a it was found that monohydroxylated (31) chromatographic technique mostly and carboxy metabolites (28,62) are combined with mass spectrometric excreted in the highest concentrations, detection (50). Next, the outcome is which makes these the metabolites of compared with databases containing the choice to implement in routine screening already known synthetic cannabinoids methods. Similar results are found for (51). If it turns out to be an unknown other indole-based cannabimimetics

218 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______(26,29,63,64). Based upon these findings, market (6). The latter makes it also chromatographic methods are developed difficult to keep screening methods up to and validated to screen for synthetic date, since the existing methods are not cannabinoid metabolites in urine (65,66). able to detect non-target (i.e. currently Also commercial tests for synthetic unknown) compounds. To close this gap, cannabinoids became available, promising an open screening approach whereby the a detection window of 72 h after a single method is capable of detecting a class of use. Peer-reviewed data on detection times cannabinoids in a non-targeted way could - although rare - indicate similar ranges be a solution. (30,67). No information is available on the accumulation in the body for chronic users. PERSPECTIVES - CONCLUSIONS However, in those cases detection in urine would be possible up to 3 weeks (67). Despite the increasing number of Immunochemical-based detection actions taken by governments and other methods have the advantage of being (inter)national institutions, the ‘Spice’ cheaper and faster than the issue is still expanding. The list of chromatographic procedures referred to synthetic cannabinoids detected continues above, but the development and to grow and the statistics on implementation was long in coming. hospitalizations due to the use of these Indeed, developing such immunoassays is herbal blends are following the same trend. a challenging task given the great Although effective interventions of the structural variety between the compounds authorities are necessary to tackle these of the cannabimimetics family. Only problems, strict legislation also has a recently, the first screening method, using downside. The total ban on these products enzyme linked immunosorbent assays takes away the opportunity to investigate (ELISA) for the detection of metabolites of the therapeutic properties. Taking into the naphthoylindole group in urine, has account the successful use of plant derived been described in literature (68). cannabis in medicine, there is demand to Gradually, also commercial kits for high- provide the possibility to do research that throughput screening of synthetic leads towards the medicinal use of these cannabinoids have become available synthetic analogues (71). (69,70). For any positive outcome It is clear that further research in however, a confirmatory analysis by means this field is necessary. When it comes to of the more selective chromatographic pharmacodynamics, so far only the techniques remains essential. properties of the parent compound are It should be noted that correct investigated. However, recent data show identification of these products remains a that also the formed metabolites remain difficult task since the availability of active in the human body by binding to reference material is lagging behind on the both cannabinoid receptors (72). rapid release of new products on the For routine testing, methods should

219 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______be developed to improve detection in more common, there will be a need for different biological matrices. Given the uniform regulations taking also into rapidly growing number of products account the problem of passive inhalation, appearing on the market, an open- as known for THC smoke. screening approach could be a big step forward. When routine screening becomes

220 P Belg Roy Acad Med Vol. 2:209-225 N. De Brabanter et al. ______

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