Psychoactive substances present in "legal highs" acquired in "smartshops" or via internet

Ana Margarida Carvalho Araújo Master Degree in Forensic Sciences University of Porto September 2013 Supervisors Doutora Paula Guedes de Pinho Professor Doutor Félix Carvalho Professora Doutora Maria de Lourdes Bastos Psychoactive substances present in “legal highs” acquired in “smartshops” or via Internet Chemical characterization and in vitro cytotoxicity studies of synthetic cathinones II Experimental work carried out in REQUIMTE Laboratory, Department of Toxicology, Faculty of Pharmacy, University of Porto III ACKNOLEDGMENTS Agradecimentos A vida é feita de sonhos e desafios, uns mais fáceis de concretizar, outros nem tanto. Contudo, com dedicação e apoio tudo é possível! Este projeto resume-se a mais um desafio que eu impus a mim mesma na minha ainda curta carreira científica. Posto isto, não poderia deixar passar em branco o apoio, direto ou indireto, de todas as pessoas que tornaram este desafio possível. Assim, gostaria de agradecer...  À comissão coordenadora do Mestrado em Ciências Forenses, em especial à Professora Doutora Teresa Magalhães pela criação e orientação do mesmo e pela oportunidade de frequentar um Curso que tanto almejava, permitindo-me realizar o trabalho numa área que me dá tanto gosto.  À Doutora Paula Guedes de Pinho, orientadora deste trabalho não apenas pelos ensinamentos, mas sobretudo pela confiança, incentivo, conselhos, paciência e amizade. Tudo isso me ajudou a tornar-me numa pessoa mais descontraída e segura de mim. Agradecer ainda por me aceitar e acompanhar nesta jornada, mesmo que ao início não fosse a minha área de conforto. Espero ter estado à altura!  Ao Professor Doutor Félix Carvalho pela coorientação deste trabalho. Pela inspiração e confiança que me transmite. Pelos elogios que me deram vontade em ir mais além, em ser melhor...não sei se correspondi às expectativas, mas juro que tentei.  À Professora Doutora Maria de Lourdes Bastos em primeiro lugar por me ter dado a oportunidade de poder integrar esta maravilhosa equipa e ter depositado essa confiança em mim. Espero também não a ter decepcionado. Obrigada ainda por ter coorientado este trabalho e pelas carinhosas palavras de incentivo.  À Professora Doutora Márcia Carvalho pelo incansável apoio e ajuda no isolamento de hepatócitos e ensaios afins. Obrigada por todo o tempo disponibilizado, dedicação e ensinamentos...pela boa disposição e serenidade.  À Doutora Helena Gaspar pela colaboração neste trabalho, nomeadamente na realização de todas as análises de RMN. Obrigada pela disponibilidade e prontidão em esclarecer as minhas dúvidas. IV  À Doutora Ana Cardoso Matias um obrigada igualmente pela colaboração.  A todos os meus colegas e restantes colaboradores do Departamento de Toxicologia da Faculdade de Farmácia da Universidade do Porto por tornarem a integração mais fácil. Obrigada pelos rebuçadinhos que adoçaram os meus dias mais longos, pelos bolos de aniversário e almoços em “família”, pelas cantorias desafinadas de alguns e outros momentos afins...A todos agradeço a forma amiga como me receberam.  Um obrigada especial à Márcia sempre pronta a dar-me uma mãozinha quando precisei, à Maria João pelos ensinamentos e apoio incondicional na última etapa do trabalho e à Diana pela ajuda estatística final.  À pequena Mariana por todas as horas de brincadeira e gargalhadas contagiantes que me ajudaram a fazer uma pausa do trabalho e abstrair das preocupações.  Ao Rui por me ajudar a pôr em prática as minhas ideias criativas, apesar de adorar ser sempre do contra e deixar-me uma pilha de nervos por deixar tudo para a última da hora.  À restante “família emprestada” por me aturar em todos os momentos.  À minha família, em especial à minha mãe e às minha irmãs...espero que se orgulhem de mim! Obrigada por me terem dado ânimo nos momentos em que desanimei, por me terem ensinado a nunca parar, a lutar sempre pelo que quero, por todos os valores que me transmitiram...por serem da maneira que são...especiais!  Por último mas o elemento mais importante de toda esta jornada e outras tantas que já passei: OBRIGADA PEDRO! Obrigada pela grande/enorme/gigante paciência, por todo o amor e apoio e por nunca teres duvidado das minhas capacidades. Obrigada por acreditares sempre em mim, e por me ajudares a ser sempre melhor! Por tudo isto e muitas outras coisas... Obrigada a todos! V CONGRESS PRESENTATIONS The results described in this dissertation were presented in a scientific meeting (49th Congress of the European Societies of Toxicology; Interlaken; Switzerland; 1 – 4 September 2013) as a poster communication (image below). The list of the submitted abstract is presented:  Araújo, Ana Margarida; Bastos, Maria de Lourdes; Carvalho, Márcia; Carvalho, Félix; Gaspar, Helena; Cardoso Matias, Ana; Guedes de Pinho, Paula. (2013) Identification of psychoactive substances in “legal highs” marketed in Portugal. Toxicology Letters, Abstracts. 221S: S59-S256. VI VII ABSTRACT The world’s culture on recreational drugs has recently changed and the consumption of new psychoactive substances, known as "legal highs", is continuously growing, especially among young people. These drugs are typically sold via Internet or "smartshops" as legal alternatives to controlled substances, being announced as “bath salts” and “plant feeders” and claiming to be not intended for human consumption. The astonishing speed at which these compounds appear makes detection, identification and the associated health risks and potential harm difficult. Information about the actual content of these products is still very scarce, raising concern of consumer safety. Although the illusion of being substances more pure and safe for consumption, the number of fatalities has increased, with clinical patterns comparable to those reported for better-studied stimulant drugs like amphetamines. The purpose of this study is to chemically characterize commercial products marketed as “plant feeders” in Portuguese “smartshops”, using highly powerful methodologies, such as GC-MS (Gas Chromatography coupled to Mass Spectrometry) and NMR (Nuclear Magnetic Resonance) analysis. It is intended to further evaluate the hepatotoxic effects of two commercial “legal high” products and individual cathinone derivatives, using HepaRG cells and primary rat hepatocytes cultures as in vitro models. NMR spectroscopy, the most useful analytical technique for determining the structure of molecules, in combination with the highly sensitive analytical methodology GC-MS were applied to unveil the real composition of these products. The main active compounds identified in methanolic solutions of the marked products are synthetic cathinones, namely methedrone, methylone, buphedrone, flephedrone, pentedrone, 4-methylethylcathinone (4-MEC), ethcathinone, methylenedioxypyrovalerone (MDPV), but also aminoalkyl benzofurans derivatives, cocaine analogs (dimethocaine) and caffeine. The content differs between the products; some containing only one active compound, while others have a greater variety. Qualitative and quantitative variability was found between identical products sold in different “smartshops”. In cytotoxicity studies, cells were exposed to the test drugs (0.05 – 5mM), at 37°C for 48 hours. MDMA was used as a reference for comparison effects. Results show that for all agents tested there is an increase of cell death dependent of chemical concentration. In both cell models, methylone proved to be the least hepatotoxic individual agent and MDPV the most potent. The commercial “legal highs” studied ("Bloom" and "Blow") induced a severe hepatotoxicity, with EC50 values significantly lower or equal to MDMA, respectively. Mathematical models of independent action (IA) and concentration addition (CA) were applied for modeling the expected effects of “legal highs” mixtures; the exposure of primary hepatocytes VIII to “Bloom” shows an obvious synergism, while “Blow” reveals an additive effects. Primary rat hepatocytes showed to be more sensitive for hepatotoxicity evaluation since the HepaRG are metabolically less competent. In synopsis, these results show a miscellany of psychoactive compounds present in “legal high” products marketed in Portugal, with hepatotoxic effects extremely pronounced. In addition, potential harmful interactions among synthetic cathinones are expected when these drugs are taken concomitantly. These data contributes to unveil the health hazards likely to arise from exposure to these emerging psychoactive drugs and may influence behavioral changes in consumers worldwide. Key words: Legal highs – Synthetic cathinones – GC-MS – NMR – Hepatotoxicity – Primary rat hepatocytes – HepaRG cell line IX RESUMO A cultura do mundo das drogas recreativas mudou recentemente e o consumo de novas substâncias psicoativas, conhecidas como "drogas legais", tem vindo a crescer continuamente, especialmente entre os jovens. Estas novas drogas são normalmente vendidas através da Internet ou "smartshops" como alternativas legais para substâncias controladas, sendo anunciadas como “sais de banho” e “fertilizantes para plantas”, afirmando não serem destinadas ao consumo humano. A velocidade estonteante a que estes compostos aparecem torna a sua detecção, identificação e avaliação dos riscos de saúde associados e dano potencial difíceis. Informações sobre o conteúdo real destes produtos são ainda muito escassas, conduzindo a um aumento da preocupação com a segurança dos consumidores. Embora exista a ilusão de serem substâncias mais puras e seguras para o consumo, o número de mortes aumentou, com padrões clínicos comparáveis aos relatados para drogas estimulantes bem estudadas, como é o caso das anfetaminas. Um dos objectivos deste estudo passa por caracterizar quimicamente produtos comercializados como “fertilizantes para plantas” à venda em “smartshops” portuguesas, utilizando metodologias altamente poderosas, tais como a análise por GC-MS (cromatografia gasosa acoplada à espectrometria de massa) e RMN (espectroscopia por ressonância magnética nuclear). O trabalho destina-se ainda a avaliar os efeitos hepatotóxicos de dois produtos comerciais adquiridos em “smartshops” e de derivados de catinonas vendidos como agentes individuais através da Internet, usando células HepaRG e cultura primária de hepatócitos de rato como modelos in vitro. A espectroscopia por RMN, uma técnica analítica útil na determinação da estrutura de moléculas, em combinação com uma metodologia analítica altamente sensível, o GC-MS, foram aplicadas para desvendar a composição real destes produtos. Os principais compostos ativos identificados em soluções metanólicas foram catinonas sintéticas, nomeadamente a metedrona, metilona, bufedrona, flefedrona, pentedrona, 4-metiletilcatinona (4-MEC), etcatinona, metilenodioxipirovalerona (MDPV), mas também derivados dos aminoalquil benzofuranos, análogos da cocaína (dimetocaína) e cafeína. O conteúdo entre os produtos mostrou-se variável; alguns contendo apenas um composto ativo, enquanto outros têm uma maior variedade. Foi encontrada variabilidade qualitativa e quantitativa entre produtos idênticos vendidos em “smartshops” diferentes. Nos estudos de citotoxicidade, as células foram expostas a drogas teste (0.05 – 5mM) a 37˚C, durante um período de incubação de 48h. A MDMA foi usada como composto de referência para comparação de efeitos de hepatotoxicidade. Os resultados mostram que para todos os agentes testados existe um aumento da morte celular dependente da concentração química. Nos XVI XVII Figures Index XVIII XIX Figure 1: “Legal highs” sold in Portuguese “smartshops” (Magic mushroom and Euphoria)…......5 Figure 2: New psychoactive substances notified to EMCDDA in 2005-2012……………………………..8 Figure 3: Percentage of young Europeans who consumed, any time during life, new licit drugs…………………………………………………………………………………………………………………………………….9 Figure 4: Market availability of new psychoactive products………….…………………………………………9 Figure 5: Khat (Catha edulis) plant……………………………………………………………………………………….17 Figure 6: (A) Khat chewing; (B) Fresh khat leaves, ready for sale and chewing………………………18 Figure 7: Chemical structure of amphetamine, cathine and cathinone…………………………………...19 Figure 8: Timeline associated with cathinone derivatives……………………………………………………..26 Figure 9: Chemical structures of some elements of the class of synthetic cathinones, divided into four major groups: (A) “Classic” cathinones; (B) Pyrrolidinopropiophenone derivatives; (C) "3,4-methylenedioxy" cathinones; and (D) "Mixed" cathinones……………………………………………...28 Figure 10: Structural relationship between some cathinone derivatives and their amphetamine analogs………………………………………………………………………………………………………………………………..29 Figure 11: Main metabolic pathways of ß-keto derivatives with 3,4-methylenedioxy ring in rats and humans…………………………………………………………………………………………………………………………32 Figure 12: Proposed scheme for the metabolism of mephedrone in rats and humans…………….33 Figure 13: Proposed scheme for the metabolism of MDPV in humans……………………………………34 Figure 14: Mechanism of action of MDMA at the level of the serotonergic neurons………………...36 Figure 15: Summary of procedures involved in “legal highs” characterization by GC-MS………..56 XX Figure 16: Generic derivatization mechanism with the TFAA reagent to obtain the O-TFA and NTFA derivatives……………………………………………………………………………………………………………………57 Figure 17: Schematic representation of the procedure used in the chemical characterization of “legal highs”, referring to the four crucial steps of the whole analysis…….………………………………58 Figure 18: Template of one of the thousands of websites (Sensearomatic.com) from which any person can purchase several “legal highs”, for consumption or other purposes, without any restriction and at a very affordable price. To circumvent all the legal aspects, there is clear information that the products are “not for human consumption” and that do not have any responsibility if the products is illegal in the country to which the intended delivery……………..63 Figure 19: EI mass spectra of methylone, 4-MEC, pentedrone, isopentedrone and MDPV, respectively (direct injection)………………………………………………………………………………………………64 Figure 20: Appearance of some commercial products…………………………………………………………..66 Figure 21: Full scan chromatographic profile of methanolic extracts (Sample 11 – “Bliss”) injected directly (red) into the GC-MS and injected after derivatization with TFAA (green), indicating the potential identification of compounds based on the analysis of mass spectra and new fragmentation patterns of molecules after derivatization with TFAA. The signal strength after derivatization is significantly higher, showing greater sensitivity of the method. 1 – Methedrone; 2 – Methedrone N-TFA……………………………………………………………………………………..70 Figure 22: EI mass spectra corresponding to compounds of the sample 4 (“Blast”). A – Flephedrone EI mass spectrum (direct injection); B – EI mass spectrum of flephedrone N-TFA; C – Caffeine EI mass spectrum (direct Injection); D – Caffeine EI mass spectrum after derivatization………………………………………………………………………………………………………………………71 Figure 23: Different substances found in compositional analysis of the various "legal highs”. Yellow compounds are representatives of the synthetic cathinones class (66.7%), the orange are aminoalkyl benzofurans (7.6%), the red is a representative of amphetamine class (3%), while blue is of the tryptamine class (1.5%). The only cocaine derivative (3%) detected is highlighted in rose while caffeine (18.2%) is in beige………………………………………………………………………………74 XXI Figure 24: Full scan chromatographic profile of methanolic extracts (Sample 1, 2 and 3 – “Bloom”) injected directly into the GC-MS, indicating the potential identification of compounds based on mass spectrum analysis and revealing the qualitative and quantitative variability between products with the same commercial name. 1 – Isopentedrone; 2 – Pentedrone; 3 – 4MEC; 4 – Methylone; 5 – Dimethocaine; 6 – Ethcathinone; 7 – Methedrone; 8 – Caffeine…………76 Figure 25: Analysis of “Rush” and “Kick” samples acquired in 3 different Portuguese “smartshops”: (A) Full scan chromatographic profile of methanolic solutions (1mg/mL) injected directly into the GC-MS, in parallel with the standard compounds. (B) 1H-NMR in MeOD (400.13 MHz) of the samples that allowed to assign the structure of the molecules present in the “plant feeders”……………………………………………………………………………………………………………………………….78 Figure 26: Full scan chromatographic profile of derivatized solution (Sample 14 – “Charlie” in the form of powder and Sample 23 – “M” in the form of tablet) injected after derivatization with TFAA. The powder signal strength is significantly higher, may be an indicator of higher purity of product. The potential compounds identification is indicated based on the analysis of mass spectra and new fragmentation patterns of molecules. 1 – Ethcathinone N-TFA; 2 – Buphedrone N-TFA; 3 – Fluoroamphetamine N-TFA…………………………………………………………………………………81 Figure 27: Semi-quantification of some substances (4-MEC, Pentedrone, Methylone and MDPV) present in “legal high” samples randomly selected and the quantitative relation between the same compound for different solutions. The figure A expresses the amount of 4-MEC, Pentedrone and Methylone (mg) in each gram of “Bloom” (1). Graph B expresses the amount of 4-MEC and MDPV (mg) in each gram of “Blow” (16), while the graph C expresses the amount of Pentedrone (mg) in each gram of “Kick” (19). Finally, the graph D expresses the amount of Methylone (mg) in each tablet of “Bliss” (25). The results are expressed through the mean ± SD, n=3 (* P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 for solution B or solution C vs. solution A). The groupings shown in figure A and B represent comparisons between the solutions as a whole……………………………………………………………………..……………………..…………………………………….84 Figure 28: Schematic representation of the hepatic portal vein cannulation and perfusion system…………………………………………………………………………………………………………………………………93 Figure 29: (A) Microscopic appearance of freshly isolated rat hepatocytes in a total magnification of 100x. Figures B1 and B2 shows microscopic appearance of HepaRG cells to 8090% of confluence in a magnification of 100x and 200x, respectively. Figures C shows the C XXII HepaRG cells after 18 days of culture in the presence of differentiation medium with DMSO in a magnification of 100x (B1) and 200x (B2). H –hepatocyte-like cells; BC – bili canaliculus……..102 Figure 30: Full scan chromatographic profile of “Bloom” stock solution with methylone at 50 mM and methylone standard also at 50 mM, injected directly into the GC-MS, showing equal areas and therefore equal concentrations…………………………………………………………………………...104 Figure 31: Regression models for the cytotoxicity effects of individual cathinones, using isolated rat hepatocytes as a model at 37 °C. The grey line shows the EC50 and ECmax for each response curve and the dashed lines show the 95% confidence interval belt of the fit. Data were normalized to negative (untreated) and positive (1% Triton X-100) controls. Data were obtained from five independent experiments run in triplicate………………………………………..……106 Figure 32: Predicted and observed effects of “Bloom” and “Blow” mixtures and their individual agents in MTT assay. Circles represent individual data points of the “Bloom” and “Blow” mixtures. The dashed lines show the prediction according to concentration addition (CA) and independent action (IA). The grey lines show the EC50 and ECmax for each response curves. Experimental data derive from five independent experiments run in triplicate…………………….107 Figure 33: Concentration-response curves obtained with the MTT reduction cytotoxicity assay for “legal highs” mixtures, in primary rat hepatocytes after 48h incubation at 37 °C and its comparison with pure MDMA in same conditions. The grey lines show the EC50 and ECmax for each response curve and the dashed lines show the 95% CI belt of the fit. Data were normalized to negative (untreated) and positive (1% Triton X-100) controls and were obtained from five independent experiments run in triplicate.………...……………………………………………………………….109 Figure 34: Concentration-response curves obtained with the MTT reduction cytotoxicity assay for “legal highs” mixtures and all individual agents used in study, in HepaRG cells after 48h incubation at 37 °C. Data were normalized to negative (untreated) and positive (1% Triton X100) controls and were obtained from three independent experiments run in triplicate……………………………………………………………………………………………………………………………110 XXIII Tables Index XXIV XXV Table 1: Chromatographic techniques used in qualitative analysis of several "legal highs”……..11 Table 2: Adverse physical effects of khat……………………………………………………………………………...20 Table 3: Effects of synthetic cathinones and comparison test drugs on transporter-mediated inhibition of uptake and stimulation of release in rat brain synaptosomes……………………………..37 Table 4: Effects associated with the consumption of synthetic cathinones reported by consumers and medical entities……………………………………………………………………………………………39 Table 5: Results of external analysis of cathinone derivatives standards ………………………………65 Table 6: Main features of "legal highs" (powders) acquired for analysis………………………………...67 Table 7: Main features of "legal highs" (tablets) acquired for analysis……………………...……………68 Table 8: Active ingredients detected in 27 “legal highs” acquired in three distinct “smartshops” after analysis by GC-MS and NMR…………………………………………………………………………………………72 Table 9: Retention time (minutes), base peak (m/z) and other characteristic ions (m/z) of active ingredients detected directly by GC-MS and after derivatization with TFAA………………….73 Table 10: Calibration curves for 4-MEC, Pentedrone, Methylone and MDPV…………………………..82 Table 11: Intra-day precision of the method for the quantification of 4-MEC, Pentedrone, Methylone and MDPV in different “legal highs” products (n=3)……………………………………………...83 Table 12: Parameters derived from nonlinear fits of single agents and “legal highs” mixtures concentration-response data to the asymmetric logit function, in the MTT reduction assay for primary hepatocytes…...……………………………………………………………………………………………………..108 Table 13: Parameters derived from nonlinear fits of single agents and “legal highs” mixtures concentration-response data to the asymmetric logit function, in the MTT reduction assay, in HepaRG cells……………………………………………………………………………………………………………………...111 XXXII NRG - Naphyrone PFPA – Pentafluoropropionic anhydride pFPP – para-Fluorophenylpiperazine R2 – Squared correlation coefficient rpm – Revolutions per minute SD – Standard deviation S.E.M. – Standard error of mean SERT – Serotonin transporter SW – SWGDRUG library SWGDRUG – Scientific working group for the analysis of seized drugs TFAA – Trifluotoacetic anhydride TFMPP – 1-[3-(trifluoromethyl)phenyl]piperazine TH – Tryptophan hydroxylase UK – United Kingdom U/mL – Enzyme unit per milliliter UN – United Nations USA – Unites States of America v/v – Volume concentration, Volume/Volume VMAT2 – Monoaminic vesicular carrier General Introduction 1 3 Chapter 1 – The “Legal Highs” 4 5 In recent years, the world's drug culture has changed substantially and, nowadays, includes a group of products known as "legal highs", "legal drugs" or "designer drugs", referring to a range of natural or artificial psychotropic substances that are marketed as “incense”, “plant feeders” and “bath salts”, although this is not their legitimate practice. These products are purchased to be used as recreational drugs that mimic the effects of illegal drugs of abuse, as cocaine and 3,4-methylenedioxymethamphetamine (MDMA or "Ecstasy"), acting as their legal alternatives, being easily purchased at Internet or through specialized shops – called "smartshops" or "headshops" [1-3]. The packages are very attractive with suggestive names – "Bliss", "Bloom", "Blast", "Kick" – as a marketing strategy to attract consumers (Figure 1). All packages are labeled as "not for human consumption" in order to circumvent the laws that govern the sale of psychoactive substances [4-5]. Figure 1 – “Legal highs” sold in Portuguese “smartshops” (Magic mushroom and Euphoria) 1.1 The problem of “legal highs” These new recreational substances are very similar to the controlled drugs in their chemical structure and/or pharmacodynamics [6]. The term "legal highs" is defined as psychoactive substances whose production, sale and possession, are not covered by the law, not being prohibited [7]. This seems to be the ideal situation from the point of view of producers, sellers and consumers of these new drugs; however, there are a number of problems associated, as will be discussed in this thesis. 6 Despite some legislative initiatives on these products, there are evidences suggesting that little has changed. Consequently, banned substances are still being sold under a new guise. It is not known how many and which of these products contain substances known as legal or illegal substances [5, 8-10]. In addition, new products are also produced through the modification of the chemical structure of substances that have been enacted [11-13]. The emergence of these new substances is extremely challenging, not only due to the large number of different compounds that are theoretically possible to exist, but also due to the number of those that are actually manufactured, distributed and consumed [14]. The new alternative products are immediately promoted, being the Internet an effective medium for its dissemination, once it responds quickly to legal changes. Thus, the online market is an important tool to identify the new trends of drugs of abuse [15]. In view of this panorama, as the law is always a step behind, manufacturers and consumers continue to explore legislative gaps to constantly create new "legal drugs", continuing the “cat and mouse game”. Another problem is that these drugs are sold in packages highly variable and inconsistent concerning its content. This has been verified for products with the same brand name and identical packages. The variability found refers to significant qualitative as quantitative differences. This inconsistency increases the risk of toxicity and injury associated with its use. In accordance, the information provided to consumers on the composition, dosage, duration and even the effects is generally limited or imprecise [6, 8-9, 16]. Despite all the problems associated with these new psychoactive substances, its popularity keeps growing due to the illusory idea of security that was installed on a misinformed public. In addition, other reasons are associated with their incessant demand, where users suggest a decline in purity of illegal drugs, replacing them by these new synthetic drugs. These new substances produce similar effects to illicit drugs and are easily purchased, but the biggest advantage lies on the absence of criminal sanctions [8, 9, 11, 13]. In contrast to what happens with traditional drugs of abuse, there is little or no information available about these new substances, both in terms of their chemical composition and toxicity or long-term effects associated with its use, representing a serious challenge for public health authorities [3, 9, 12]. Based in medico-legal information, excessive use of drugs of abuse is a social danger respecting self-harm and crimes committed under the influence of such substances. The easy access to new recreational products lead to great social burden, as exemplified by the disruption of personality, mental disorders and deterioration of social relations, leading users to a social marginalization [17]. 7 The fact that most of these new drugs are legal in many countries, lead many consumers to believe they are harmless substances. Unfortunately, the consumption of these unknown substances is very risky and many of them appear associated with adverse physical effects and fatalities [18-21]. To increase the problem, it is common for consumers to create "designer mixtures” in order to enhance its effects, which consequently become unpredictable, making it difficult to evaluate the effects of specific substances [17]. Hence, it is necessary and urgent for health care providers to be familiar with the effects of these new psychoactive substances, as well as it is important that the forensic toxicological research community develops strategies for fast and effective recognition of the consumption of new "legal highs" [22]. For correct application of the law and for benefit of the general community, it is vital that a forensic laboratory is able to correctly identify the compounds present in these new drugs and determine if the sample is a controlled substance under the legal system of the country concerned. The process of detection and identification of new drugs is not easy, because many substances exhibit identical chemical, chromatographic and spectral properties, since the structural modifications of a controlled substance may include only the addition of a small chemical group or the formation of isomeric structures. Furthermore, the absence of standards and reference spectra in available bibliotheca data will increase the complexity of identification, being hardly achieved through routine analytical protocols. Thus, sophisticated methods of analysis are needed to test these unknown products, often through complex and expensive processes [6, 11]. 1.2 The new drugs evolution The market of new drugs is characterized by the extreme speed of producers’ reaction to the imposition of control measures and monitoring, providing alternatives to banned products. According to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), between 2005 and 2012, more than 230 new psychoactive substances were notified through the early warning system (EWS). The rhythm at which these drugs appear on the market reached record values in recent years: 24 new substances have emerged in 2009, 41 in 2010, 49 in 2011 and 73 in 2012 (Figure 2). Currently, about two-thirds of notified substances belong to cannabinoids and synthetic cathinones group [23-25]. 8 Figure 2 - New psychoactive substances notified to EMCDDA in 2005-2012 [adapted from 25] Until 2008, the new psychoactive substances that have emerged in the European Union (EU) drug market belong to a restricted number of chemical classes, being the phenethylamines and tryptamines the most accounted for [23]. However, the market has undergone drastic changes in recent years. The increasing number of substances that has been reported corroborates this fact. In the last years, a new chemical class – the synthetic cathinones – with stimulant properties emerged in Europe's drug market. These substances are structurally related to cathinone, the main natural psychoactive substance of khat plant [26]. In 2010, 15 of the 41 novel derivatives detected belonged to synthetic cathinones group, being now the second largest family of drugs monitored by EWS [23]. The newly identified substances list also includes a diverse group of plant-derived and synthetic substances, including indanes, benzodifuranyls and synthetic derivatives of cocaine, ketamine and phencyclidine [6]. 1.3 Prevalence In Europe, studies on the prevalence of use of licit alternatives are scarce and, often, are accompanied by methodological limitations such as lack of representative samples [24, 27]. Year of notification Number of substances 9 During 2008, Polish researchers conducted a study in 1250 students with 18-19 years and concluded that 3.5% of these young people had consumed this type of substances at least once in their lifetime. In 2010, a follow-up study found an increase to 11.4%. When asked about the consumption of licit drugs over the last 12 months, in 2008, 2.6% responded affirmatively and this number increased to 7.2% in 2010. This fact can be explained by the exponential increase of selling points in Poland, rising from 40 “smartshops” in 2008 to 1500 in 2010 [23]. In 2011, a survey was carried out at European countries, including more than 12000 young people (15-24 years), about their attitudes to the new generation of drugs. The analysis of results estimated that 5% of the young surveyed have used "legal highs" (Figure 3). The highest percentages came from Ireland (16%), followed by Latvia, Poland and United Kingdom (10% each). Italy, Finland and Greece were the countries with the lowest values [28]. Figure 3 - Percentage of young Europeans who consumed, any time during life, new licit drugs [adapted from 28] In the same inquiry, it was asked to young people how they purchased these products. The vast majority of respondents (54%) indicated that friends offered them and 36% mentioned that they have been obtained at parties. About a third of participants (33%) admitted having purchased the substances in specialized shops and only 7% via the Internet (Figure 4) [28]. Figure 4 – Market availability of new psychoactive products. [Adapted from 28] Other Such substances were bought over the Internet Such substances were bought in a specialised shop Such substances were offered during a party Such substances were offered by a friend 7 7 33 36 54 No, I never used such substances 95% Yes, I have used such substaces 5% 16 17 2.1 History Khat (Figure 5) (Family: Celastraceae; Specie: Catha edulis) is a slow growing plant, native to Eastern Africa and Arabian Peninsula and has other names and spellings such as kat, chat, quat, catha, tschat, miraa, African salad, African tea, Abyssinian tea, kuses-salahin, and tohai [48]. The origin of the plant is controversial. Many believe that its origin is Ethiopian; others claim that it originated in Yemen before spreading to Ethiopia and neighboring countries. However, according to data collected by British explorers, the Ethiopian origin seems most likely [49-50]. Figure 5 – Khat (Catha edulis) plant Abu Rayhan Al-Biurni, a Persian scientist, carried out the first description of this plant during the 11th century. The oldest scientific report about this specimen dates back to the 18th century, when the botanist Peter Forskal identified the plant in Yemen, naming it Catha edulis [51-53]. The consumption of plants, and especially of khat, to take advantage of its stimulant properties is not a new practice. According to a famous legend, the first human that experienced khat was a Yemeni herder. He observed the effects that leaves caused in his goats, deciding to experience these same effects. To this end, the herder chewed the leaves, which is currently the exclusive method for the consumption of khat [54]. Chewing leaves to take pleasure of its stimulant effects become a deeply entrenched social and cultural tradition, practiced by the inhabitants of these geographical areas (Figure 6A), where there are no laws restricting its cultivation and consumption. It is estimated that about 5 to 10 million people do so daily [53]. This habit involves picking tender leaves of khat, beginning to masticate thoroughly one at time while users engage in discussions and social interactions. During the sessions, the leaves are chewed slowly over several hours and the juice is swallowed while the residue is putted into 18 one side of the mouth [48]. During the last two decades, this practice has gained global prominence as a result of migration, although khat use in western countries such as the United Kingdom, Canada and United States has recently become restricted and is now classified as a controlled substance of abuse [48]. Khat has won a recognized economic value comparable to other crops such as tea, coffee and cocoa [53]. Figure 6 – (A) Khat chewing; (B) Fresh khat leaves, ready for sale and chewing Traditionally, khat is used as a socializing drug although it is also sought by farm workers to reduce physical fatigue and by drivers and students for improving attention [54-55]. 2.2 Active constituents of Khat The alkaloids present in khat have been investigated for years, but it was only in 1930 that norpseudoephedrine or cathine [S, S (+) phenylpropanolamine] was identified in the leaves. Initially the stimulating power of khat was explained by the presence of this substance, until it was shown that its concentration in the leaves and respective potency were too low to justify the effects caused [49, 56]. Subsequently, other studies have indicated the presence of another alkaloid in the plant, with a more potent stimulant power – cathinone [S (-) alpha - aminopropiophenone], a betaketo amphetamine analog (Figure 7), known as "natural amphetamine", since the effects are similar to those produced by other known psychostimulants as amphetamines and their congeners. This substance was found at high concentrations particularly in young leaves [49, 5657]. B A 19 Figure 7 – Chemical structure of amphetamine, cathine and cathinone Other researchers found that cathinone is a precursor that in khat leaves is enzymatically converted into cathine and norephedrine, the latter to a lesser extent. This transformation is fast in the adult leaves and slow in younger leaves. This can be explained presumably because in younger leaves the enzymatic development is still incomplete [49-50]. Thus, users of khat will choose, preferentially, to consume young leaves, where the amount of cathinone is high, thus obtaining the desired stimulating effects with less adverse systemic effects. Cathinone degradation by sunlight or heat was also checked, and to delay the process, the leaves of khat when harvested for sale are usually wrapped in banana leaves (Figure 6B) to retain the moisture and preserve freshness [58]. Many other chemical components were identified in khat plant including tannins, flavonoids, sterols, carotenes, terpenoids, essential oils, amino acids, proteins, vitamins and minerals [50, 58-59]. 2.3 Effects produced by Khat consumption 2.3.1 Physical sequelae Cathinone is structurally and functionally similar to amphetamine (Figure 7). It causes the release of catecholamines originating Central Nervous System (CNS) stimulation in low doses, associated with an alertness, euphoria and sense of well-being. Cathinone has also peripheral sympathomimetic effects, such as increased respiration, body temperature, blood pressure and heart rate. Some cases of anorexia, hyperactivity and loquacity have been reported after khat consumption. Another common effect is insomnia, a condition that users try to overcome with sedatives or alcohol in order to neutralize the stimulating effect caused by khat [52, 56-57]. CH3 NH2 CH3 NH2 OH CH3 NH2 O Amphetamine Cathine or Norpseudoephedrine Cathinone 20 The adverse physical effects in humans resulting from high doses and chronic consumption of khat have been well studied and can be briefly described according to the physiological systems involved (Table 2). 2.3.2 Dependence, tolerance and withdrawal According to some authors, the habit of chewing khat can cause a moderate, but permanent, psychological dependence. After continued use, mild withdrawal symptoms have been reported, including lethargy, mild depression, recurring nightmares and tremors [49]. In the literature there is little information regarding the dependency on khat and regular consumers do not report difficulties to stop consumption. However a cessation of its use results in significant improvement of sleep and appetite [56, 62]. Table 2: Adverse physical effects of khat [51-53, 59-61] Central nervous system Dizziness, impaired concentration, memory deficit, insomnia, headaches, migraines, conjunctival congestion, impaired motor coordination, tremors, stereotyped behaviors Respiratory system Bronchitis, tachypnea, dyspnea, tuberculosis Tachycardia, arrhythmias, palpitations, hypertension, vasoconstriction, ischemia, myocardial infarction, chest pain, pulmonary edema, cerebral hemorrhage Cardiovascular system Gastrointestinal system Dry mouth, polydipsia, tooth decay, brownish teeth, oral cancer, periodontal disease, oral keratotic lesions, chronic gastritis, gastric ulcers, esophagitis, constipation, hemorrhoids, paralytic ileus, duodenal ulcer, anorexia, weight loss, increased risk of upper gastrointestinal malignancy Hepatobiliary system Cirrhosis Genitourinary system Spermatorrhea, change in libido, impotence, urinary retention, renal toxicity, abnormal sperm Obstetric effects Low birth weight, stillbirth, impaired lactation Metabolic and endocrine effects Hyperthermia, sweating, hyperglycaemia Ocular effects Mydriasis, blurred vision Psychiatric effects Lethargy, irritability, depression and psychotic reactions 21 Tolerance is difficult to assess since the tradition defines a maximum amount (100-200 g) of khat to be consumed daily [50]. However, a certain degree of tolerance seems to be associated with an increase in blood pressure, heart rate, respiratory rate and body temperature [63]. 2.3.3 Treatment of intoxications Few reports on this topic have been enumerated. Giannini and collaborators (1992) reported two cases of addiction to khat treated with bromocriptine, following the same protocol as developed for the treatment of cocaine addiction [62]. 2.3.4 Socio-economic effects Apart the negative health impact in the communities where khat is consumed regularly, impact on socio-economic conditions have also been reported. While the consumption of khat can be indirectly associated with absenteeism and unemployment due to the hours dedicated to daily ritual, on the other hand there are those who defend that a moderate intake of khat improves performance due to its stimulant effects, decreasing fatigue. Therefore, the work is extended over hours, due to an increase in the motivation of employees, thereby increasing the productivity and the economy. Another problem arises to the fact that these people spend a large part of the salary to feed the addiction to khat consumption, which leads the idea of possible dependency. Against this background, consumers seek incessantly income to pay for khat and may resort to inappropriate behavior such as criminal behavior and prostitution. However, apart to the losses associated, economic benefits for these producer countries can also be found in the sale and export of khat [57]. 22 23 Chapter 3 – Synthetic Cathinones 24 25 3.1 History Prior the confirmation of the cathine as one of the active ingredients of khat, synthesis procedures were carried out, discovering the first two synthetic cathinones: methcathinone (1928) and mephedrone (1929) [64]. The evident structural relationship of these substances with the amphetamine has led to a strong interest in these compounds, initially for therapeutic purposes. In the 1930s the methcathinone (also known as ephedrone) has been developed as an antidepressant in the Soviet Union, but was never marketed for this purpose due to its strong addictive potential [20]; the amfepramone (or diethylcathinone) was marketed as an appetite suppressant in the late 1950s [65]; from 1970 the pyrovalerone was used for a few years in the treatment of chronic fatigue and lethargy, which was also abandoned due to dependency issues [66]; bupropion was introduced on the market in 1985 as an antidepressant and later used in pharmacotherapy of smoking cessation, being the only cathinone derivative currently used for therapeutic purposes in the United States and Europe [67-68]. For several decades methcathinone was consumed as a recreational drug without any kind of control, confirming its widespread abuse in the former Soviet Union, Russia and Western Europe, sprawling to the USA in the 1990s [20]. In 1994, the U.S. Government recommended the inclusion of methcathinone as a Schedule I controlled substance in the UN Convention on Psychotropic Substances [64]. However, a growing number of new derivatives of cathinones have emerged and its therapeutic potential has continued to be evaluated. In 1996, methylone was patented as antiparkinsonian agent and its similarity to amphetamine, particularly with MDMA was described [69]. Over the next decade, the scenario of cathinone derivatives began to change, appearing now as "legal highs" in some countries. It was the case of methylone, which in 2004 emerged in the Japanese and European market under the name of "Explosion", being one of the first products to be sold in “smartshops” and on the Internet [70]. In turn, mephedrone appeared in Israel in the early 2000s, locally nicknamed "Hagigat", though its consumption was banned in 2008 by the Israeli Government due to the large number of hospitalizations associated with their use [71]. However, other countries have shown interest in this product, starting to be marketed in Europe since 2007 [38]. Growing concerns about the safety of mephedrone and related derivatives have led to a series of restrictions in countries in which it was predominantly consumed, as it was the case of the United Kingdom that in 2010 has introduced these derivatives on a collective group of class B substances, under the Misuse of Drugs Act 1971, having a specific legislation to deal with such substances [38]. After mephedrone, other derivatives appeared on the market, such as flephedrone (4fluoromethcathinone) and two structural isomers (2 - and 3 - fluoromethcathinone) [43], as well 32 Figure 11 – Main metabolic pathways of ß-keto derivatives with 3,4-methylenedioxy ring in rats and humans [adapted from 69] β – Ketone reduction N – Dealkyl metabolites β – OH – metabolites N - Dealkylation O - methylation 4’-OH-3’-MeO metabolites 3’-OH-4’-MeO metabolites Conjugates O O R1 N H R2 O O O R1 NH2 O OH OH R1 N O H R2 R1 N H R2 O O OH CH3 R1 N H R2 O OH O CH3 R1 N HR2 OH O O Regarding cathinones structurally similar to mephedrone, metabolic pathways differ slightly. Based on the identification of metabolites of mephedrone in the urine of rats and humans, the following metabolic pathways have been postulated (Figure 12): N-Dealkylation to the primary amine (metabolites nr. 2 and 3), reduction of the keto moiety to the respective alcohol (metabolites nr. 3 and 4) and oxidation of the tolyl moiety to the corresponding alcohol (metabolites 5 and 6) [80]. The metabolite represented by the number 4 has only been detected in human urine samples. Figure 12 – Proposed scheme for the metabolism of mephedrone in rats and humans [adapted from 80]. Pyrrolidinopropiophenone derivatives and "mixed" cathinones (see topic 3.2. Chemistry), are also extensively metabolized by rats and humans [81-83]. Studies to identify the MDPV metabolites, as well as the cytochrome P450 isoenzymes involved in the process were conducted [82]. Taking into account the urinary metabolites generated, it was concluded that the major metabolic pathways in humans (Figure 13) include demethylenation (metabolite 2, 4, 6 and 7), methylation of the dihydroxy metabolites (metabolite 5, 8 and 9), hydroxylation of the propyl side chain (metabolite 6 and 9), hydroxylation of the 2’-position of the pyrrolidinyl ring 1 2 3 4 5 6 CH3 NH CH3 CH3 O CH3 NH2 CH3 O CH3 NH2 CH3 OH NH2 CH3 O OH NH CH3 O OH CH3 NH CH3 OH OH O CH3 34 followed by dehydrogenation to the corresponding lactams (metabolite 3, 4 and 8) and finally degradation to primary amines (metabolite 7 and 10). A screening study involving the human hepatic CYPs has found that the CYP2C19, CYP2D6 and CYP1A2 isoenzymes are capable to catalyze the reaction (demethylenation) of formation the initial metabolite, the demethylenylMDPV, in vitro. Figure 13 – Proposed scheme for the metabolism of MDPV in humans [adapted from 82]. The excretion occurs mainly by urinary track, where metabolites with a hydroxyl group are excreted in the form of glucuronide and/or sulfate conjugates and another portion is eliminated without modification [22, 79-81]. 3.5 Mechanism of action Neurotransmitters are contained whitin small storage vesicles in the neuronal cells, until it fuses with the cell membrane and expels biogenic amines (as noradrenaline (NA), dopamine (DA) and serotonin (5-HT)) into the synaptic cleft in order to exert their physiological 9 8 7 6 5 4 2 1 O OH CH3 N O CH3 3 CYP2C19, CYP2D6, CYP1A2 O O CH3 N O OH OH CH3 N O O O CH3 N O O OH OH CH3 N O O OH OH CH3 N O OH O OH CH3 N O OH CH3 O OH CH3 N O CH3 O OH OH CH3 NH2 O O OH CH3 NH2 O CH3 35 effect (Figure 14) [84]. Amphetamine and other related phenethylamines exert its stimulant effects through increasing concentration of biogenic amines in the synaptic cleft. This process arises as a result of two main mechanisms (Figure 14). One of the mechanisms implies that the drug inhibits the reuptake of monoamines by membrane-bound carriers due to competition of drugs to substrate binding sites in the carriers. The second mechanism, suggests that the drug causes the release of neurotransmitters from storage vesicles. The release may be mediated by intracellular pH changes or by inhibition of a monoaminic vesicular carrier, the VMAT2 [1-2, 85]. The vesicular pH is slightly acid when compared with the cytoplasmic pH. This difference creates a concentration gradient by preventing the neurotransmitters to be released into the cell cytoplasm. Drugs such as MDMA and other similar change the permeability and the physiology of intracellular vesicles and carriers. These drugs move to the cell body, merge into storage vesicles and cause a hydrogen ion buffer, increasing the vesicular pH with consequent premature release of neurotransmitters into the cytoplasm before the vesicle merge with the cell membrane [84]. Monoaminic carriers are protein structures integrated into the cell membrane, responsible for the reuptake of neurotransmitters existing in the cleft, moving them back into the cytoplasm. In turn, the VMAT2 carrier is instructed to move back into the vesicles to be used again. Such carriers, when inhibited will block the entry of biogenic amines in the vesicle, staying free inside the nerve ending and subsequently in the synaptic cleft [84]. The stimulant drugs also partially inhibit the activity of the enzyme monoamine oxidase (MAO), which is responsible for the metabolism of monoamine neurotransmitters in excess [15, 84]. A schematic representation is given in Figure 14, taking MDMA as an example. 36 Figure 14 – Mechanism of action of MDMA at the level of the serotonergic neurons. (1) MDMA promotes the release of 5-HT by exchange at the SERT carrier. (2) At higher concentrations, MDMA penetrates into synaptic vesicles and promotes 5-HT release contained therein. Increased 5-HT is also due to inhibition of the metabolic degradation of this neurotransmitter by MAO in the mitochondria (3), inhibition neuronal reuptake system of 5-HT by SERT (4) and stimulation of 5-HT release in the synaptic cleft (5). (6) This 5HT release will stimulate postsynaptic receptors. However 5-HT has mechanisms to avoid the excessive increase of its levels in the synaptic cleft by self-regulation of 5-HT release by presynaptic receptors (7), increased formation of metabolites and autoxidation products of 5-HT in the synaptic cleft (8). The MDMA also promotes the decrease of 5-HT reserves for causing the inhibition of biosynthesis of 5-HT by tryptophan hydroxylase (TH) (9) [adapted from 86]. Given that the information about the mechanism of action of these new synthetic drugs is limited, extrapolations are made taking into account the structural similarity with amphetamine derivatives, whereby identical mechanisms are expected. Scientific evidence supporting this hypothesis, indicate that cathinones, such as amphetamines, interact with the plasma membrane transporters of dopamine (i.e., DAT), noradrenaline (i.e., NAT) and serotonin (i.e., SERT). However, various studies in vitro have shown a mechanistic dichotomy between the most common synthetic cathinones, as well as a variable selectivity for carriers [4, 85, 87-88]. A study found that the methylone and the methcathinone showed a lower activity at VMAT2 level 37 as compared with MDMA and methamphetamine, respectively [85]. In vitro studies with rat brain synaptosomes revealed that the mephedrone and methylone block the uptake of DA, NA and 5HT but also act as stimulants in the release of these neurotransmitters. Table 3 summarizes the results obtained, which confirms that mephedrone, methylone and MDMA act as non-selective substrates, unlike amphetamine, which is selective for NAT and DAT. It was also found that mephedrone and methylone exhibit similar release potency in all transporters despite mephedrone is about twice as potent. In turn, substances such as cocaine and MDPV were inactive as releasers. MDPV showed a different pharmacological profile comparing to other cathinones, revealing a potent selective blocker for catecholamines carriers (DA and NA) [4, 88]. Table 3: Effects of synthetic cathinones and comparison test drugs on transporter-mediated inhibition of uptake and stimulation of release in rat brain synaptosomes [adapted from 4] Mephedrone Methylone MDPV MDMA Amphetamine Cocaine DAT uptake IC50 (nM ± S.E.M.) 762 ± 79 1232 ± 133 4.1 ± 0.5 1009 ± 39 93 ± 17 211 ± 19 NAT uptake IC50 (nM ± S.E.M.) 487 ± 66 1031 ± 162 26 ± 8 450 ± 30 67 ± 16 292 ± 34 SERT uptake IC50 (nM ± S.E.M.) 422 ± 26 1017 ± 59 3349 ± 305 125 ± 11 3418 ± 314 313 ± 17 DAT uptake EC50 (nM ± S.E.M.) [Emax %] 51 ± 5 [102 ± 2] 117 ± 12 [96 ± 1] Inactive 42 ± 2 [100 ± 4] 5.8 ± 0.4 [102 ±1] Inactive NAT uptake EC50 (nM ± S.E.M.) [Emax %] 58 ± 11 [99 ± 4] 140 ± 17 [94 ± 2] Inactive 53 ± 7 [95 ± 2] 6.6 ± 0.7 [92 ± 1] Inactive SERT uptake EC50 (nM ± S.E.M.) [Emax %] 122 ± 10 [101 ± 1] 234 ± 35 [98 ± 2] Inactive 39 ± 5 [103 ± 4] 698 ± 71 [97 ± 2] Inactive Another in vitro study [87] with HEK 293 cells was conducted to determine the potency of different cathinone derivatives in inhibiting the transport of DA, NA and 5-HT, confirming previously published data and adding new evidence. It was verified that in addition to methylone and mephedrone, also ethylone, butylone and naphyrone act as nonselective inhibitors, but like MDMA also induce serotonin release. Moreover, cathinone, methcathinone and flephedrone, as well as amphetamine and methamphetamine, act preferentially as inhibitors of catecholamine uptake and induce the release of dopamine. Also pyrovalerone is a potent and selective inhibitor of dopamine and noradrenaline uptake, without producing the release of monoamines. Thus, synthetic cathinones with a high affinity to a specific carrier will inhibit its actions, resulting in a higher concentration of neurotransmitter in the synaptic cleft. In this way, a greater affinity to noradrenaline transporter will increase the sympathetic effects and cause 38 signs and symptoms similar to those caused by ingestion of cocaine, amphetamine and methamphetamine [71]. By another hand, a greater affinity for serotonin has been associated with delusions, hallucinations and paranoia [84]. Of note the predominant action of all cathinones on the dopamine transporter is probably associated with a considerable risk of addition [4, 84, 8788]. 3.6 Toxicological effects There are not scientific studies in humans to determine the adverse effects of these new compounds. All information is based on descriptions reported by consumers and medical entities. However, the fact that the user believes that he consumed a specific substance does not invalidate that actually has consumed a different one and this fact makes it difficult to assess this type of information. Furthermore, in the case of mixture of compounds it is difficult to attribute the adverse effect to a certain substance. Thus, caution is required when analyzing this information [44]. The clinical effects described are consistent with a sympathomimetic toxicity, denoting a clinical history similar to cocaine, methamphetamine or ecstasy intoxication. The desired effects reported by consumers include increased energy, sociability, euphoria, increased alertness, talkativeness and increased libido [2, 71]. Some synthetic cathinones users also report adverse effects such as sweating, palpitations, nausea, vomiting, headaches, muscle pain, muscle spasms, dizziness and short-term memory problems [2, 76]. Consumers who used the nasal route of administration also reported aggressive behavior and psychosis [76]. However, specific effects of cathinones are sometimes difficult to assess since they are often consumed in combination with other substances such as alcohol, tobacco, MDMA, cannabis and cocaine [2, 26]. These new synthetic substances may affect multiple organ systems, as it happens with natural cathinones (Table 4). 39 Table 4: Effects associated with the consumption of synthetic cathinones reported by consumers and medical entities [2, 5, 27, 38, 44, 71] Cardiovascular Palpitations, chest pain, hypertension, myocarditis, tachycardia, disseminated intravascular coagulation Ear – Nose – Throat (ENT) Dry mouth, nose bleeds, nasal irritation, tinnitus, tongue disorder Abdominal pain, loss of appetite, nausea, vomiting, liver failure, abnormal liver function tests Gastrointestinal Skeletal Muscle Arthralgia, changes in extremities (discoloration, numbness, tingling, cyanosis, muscle tension and cramps), increased creatinine kinase, peripheral vasoconstriction, rhabdomyolysis Neurologic Disorientation, altered mental status, collapse, confusion, bruxism, dizziness, headaches, memory loss, tremors, convulsions, drowsiness, dystonia, hyperreflexia, myoclonus, paresthesia Ophthalmologic Blurred vision, mydriasis, nystagmus, dilated pupils Respiratory Shortness of breath, tachypnea, respiratory distress Psychological Anger, irritability, aggressiveness, anxiety, visual and auditory hallucinations, depression, dysphoria, euphoria, fatigue, increased or decreased concentration, talkativeness, incoherent speech, panic, paranoia, perceptual distortions, agitation, delirium, psychosis, anhedonia, suicidal ideation, self-mutilation Genitourinary Impaired renal function, acute renal failure Renal Anorgasmia, erectile dysfunction, increased libido Others Body odor, dehydration, sweating, fever, insomnia, nightmares, rash, hyponatremia, hyperthermia 40 3.6.1 Hyperthermia Hyperthermia is one of the characteristic effects in MDMA-induced toxicity event cases in which body temperature reaching 43°C has been related [89-91]. Such hyperthermic reactions may contribute to several complications, including rhabdomyolysis, disseminated intravascular coagulation (DIC) and acute renal failure [89-91]. Studies with experimental animals show that the effects of MDMA on thermoregulatory system are strongly conditioned by room temperature. In general, animals exposed to MDMA in low ambient temperatures (below 22°C) had hypothermia, while at high temperatures (above 28°C) MDMA causes severe hyperthermia [92-93]. Since these types of psychostimulants are generally consumed in hot and crowded environments (usually "rave" parties), the ambient temperature is frequently high. This fact, together with the prolonged motor hyperactivity, as well as the state of dehydration may potentiate the hyperthermic effects in the human thermoregulatory system [92]. Hyperthermic effects, as well as its consequent effects, are also reported in cases of consumption of synthetic cathinones (see topic 3.6 Toxicological effects). However, studies in rats after subcutaneous administration of mephedrone shows that at low temperatures (below 20°C) there is a decrease in body temperature, but in case of high ambient temperatures (in the order of 30°C) there were no changes in opposite to what has been established for MDMA exposure. In both environmental temperatures studied, it was verified an increase in locomotor activity, although it is more meaningful for higher temperatures. These results show that the mephedrone exhibits, in vivo, thermoregulatory properties distinct from those that are produced by "Ecstasy" [94]. Thus, it can be deduced that despite the structural similarity between the synthetic cathinones and MDMA, not all information relating to MDMA can be extrapolated to these new synthetic drugs. 3.6.2 Adulterant toxic effects One of the potential factors that contribute to adverse reactions caused by recreational drugs is the concomitant use of other substances that can be intentional or through consumption of adulterants existing in marketed products [95]. Adulterants are very commonly found in illicit drugs and may be present for various reasons. To increase the drug volume and consequently the profit, manufacturers deliberately add many substances pharmacologically inactive, as the case of sucrose. But there are also pharmacologically active adulterants that are added in order to enhance and/or mimic the effect of psychotropic substances. A well known 41 example of an adulterant with such characteristics is caffeine. Adulterants may also be present unintentionally as a result of technical production or storage of products, taking as an example the alkaloids, microorganisms or other biological agents [96]. In the case of "legal highs", as has been already explained, information concerning the composition of commercial products is not always explicit or corresponds to reality. However, compositional analyses carried out on a wide variety of "legal highs" sold all over the world have found the presence of several adulterants, including benzocaine, lidocaine, procaine, glaucine, nicotinamide, caffeine, among others [6, 8, 16, 31]. Certain analysis have shown that caffeine is part of the composition of a wide variety of "legal highs", being the substance most commonly found in these new drugs [6]. There has been found that some of them contain only caffeine as pharmacologically active compound or often associated with other compounds but still at significant percentages (sometimes greater than 87%, corresponding approximately to the consumption of 13L of cola drink). Consequently, the recreational use of these new products can result in severe cases of toxicity [33]. In fact, previous studies have shown that caffeine (10 mg/kg) significantly increases the mortality of rats when coadministered with a high dose of MDMA (15mg/Kg), causing also a high and lasting hyperthermic response, showing that caffeine exacerbates the acute toxicity of MDMA [95]. It was also found that the coadministration of caffeine (10 mg/kg) with MDMA (10 mg/kg) induces a deep tachycardia that is not observed in the case of administration of the substances alone, suggesting the existence of interaction between them [97]. Thus, while that for many people substances as caffeine is taken as a safe substance, its intake simultaneously with these new products leads to a synergy of the stimulant effects, also exacerbating the toxicity produced by them alone, representing a risk to the health of users of this kind of recreational drugs [95]. 3.7 Treatment of intoxications The treatment of intoxications in cases of exposure to synthetic cathinones is mainly supportive, and there is no specific antidote. The available information to guide the treatment is restricted, but given the similarities of effects with amphetamines it seems likely that therapeutic strategies are also similar. Sedation is frequent and the use of large doses of benzodiazepines is required to control the agitation, seizures, hypertension and tachycardia. In the case of persistent hypertension the administration of vasodilators such as sodium nitroprusside or nitroglycerin is recommended. The internal and/or external cooling to treat 48 49 Chemical characterization studies 3 50 51 Chapter 1 - Experimental Part: Chemical Analysis 52 53 1. Material and Methods 1.1 Reagents and Standards All the reagents used were of analytical grade. Methanol (MeOH), chloroform, ethyl acetate, acetonitrile, acetone and n-hexane were obtained from Fisher Chemicals (Loures, Portugal) and diethyl ether was obtained from Pronalab (Lisbon, Portugal). Caffeine and trifluoroacetic anhydride (TFAA), 4-hydroxy-3-metoxybenzylamine hydrochloride (IS) were acquired from Sigma-Aldrich (Saint Louis, Missouri). Deuterated methanol was obtained from Cambridge Isotope Laboratories, Inc.. The reference compounds (methylone, pentedrone, 4-MEC and MDPV) were purchased from Sensearomatic website. All the standards were characterized by mass spectrometry and nuclear magnetic resonance. Methylone, 4-MEC and MDPV were also submitted to external analysis (by Instrumental Analysis Service of C.A.C.T.I., University of Vigo) in order to determine its elemental composition and consequently the molecular weight, as well as its purity. 1.2 “Legal highs” products Twenty seven “legal highs” products were analyzed: 8 samples were purchased in Euphoria Smartshop (Porto), 9 in Magic Mushroom Smartshop (Porto), 9 in Magic Mushroom Smartshop (Lisbon) and 1 was delivered to the laboratory for analysis. Of the 27 samples, 22 were powders and 5 were tablets. All products were acquired prior to the approval of Decree 54/2013[73], prohibiting the marketing of these products. 1.3 Qualitative Screening by GC-MS 1.3.1 Solvent selection Powders and tablets were homogenized in a mortar. Homogenized samples were dissolved in different solvents (chloroform, methanol, ethyl acetate, acetonitrile, acetone, diethyl ether and n-hexane) at a concentration of 1 mg/mL. Two µL of each solution was injected directly in GC-MS. 54 1.3.2 Analysis of commercial products by direct injection and after derivatization 1.3.2.1 Standards and samples preparation Methanolic solutions of the standards and samples were prepared at a concentration of 1 mg/mL. Two µL were injected automatically in GC-MS. Analysis after derivatization was also performed. The derivatization procedure was executed as described by Silva et al. [106] with appropriate adaptations. Standards and samples methanolic extracts were prepared at a concentration of 5 µg/mL and 10 µg/mL, respectively and were evaporated to dryness under nitrogen flow. Fifty µL of ethyl acetate and 50 µL of TFAA were added to the dried residue. Incubation was preformed at 70°C for 30 minutes. After cooling to room temperature the solution was dried under nitrogen flow. The obtained residue was dissolved in 100 µL of ethyl acetate and 2 µL was injected on the GC-MS apparatus. A summary of the procedures is shown in figure 15. 1.3.2.2 GC-MS conditions GC-MS analysis was performed with a Varian CP-3800 gas chromatograph (USA) equipped with a Varian Saturn 4000 Ion Trap mass selective detector (USA) and a Saturn GC-MS workstation software version 6.8. The GC was equipped with a VF-5MS (30 m x 0,25 mm x 0,25 µm) capillary column (Varian). A CombiPAL automatic auto sampler (Varian) was used for all experiments. The carrier gas was helium C-60 (Gasin, Portugal), at a constant flow of 1 mL/min. Injections was performed in split mode, with a ratio of 1/40. The injector port was heated to 250 °C. The initial column temperature of 80 °C was held for 1 min, followed by a temperature ramp of 20 °C/min to 250 °C held for 2 min and 10 °C/min to 300 °C held for 20 min. Mass spectrometric detection was performed using an “ion trap” analyzer. The ion trap detector was set as follows: transfer line, manifold and trap temperatures were 280, 50 and 180 °C, respectively. The emission current was 10 µA and the maximum ionization time was 2500 µsec. Ionization was maintained off during the first 4 minutes to avoid solvent overloading. Total separation run time was 36 minutes. All mass spectra were acquired in electron impact (EI) mode and the mass ranged from 40 to 600 m/z. The injection volume was 2 µL and the analysis was performed in Full Scan mode. For the derivatized compounds analysis, the GC-MS conditions were properly adapted. Injections were performed in splitless mode. The injector port was heated to 250 °C, the oven 55 temperature was adjusted of 100 °C held for 1 min, followed by a temperature ramp of 15 °C/min to 300 °C held for 10 min. The ion trap detector was set as follows: transfer line, manifold and trap temperatures were 280, 50 and 180 °C, respectively. The emission current was 30 µA and the maximum ionization time was 2500 µsec. Ionization was maintained off during the first 4 min to avoid solvent overloading. Total separation run time was 20 minutes. All mass spectra were acquired in electron impact (EI) mode and the mass ranged from 40 to 600 m/z. The injection volume was 2 µL and the analysis was also performed in Full Scan mode. 1.3.3 NMR analysis The samples (approximately 30 to 50 mg) were dissolved in 500 µL of deuterated methanol (methanol-d4). The nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance 400 spectrometer with a frequency of 400 MHz for 1H and 104 MHz for 13C. The structures identification with the respective assignment of the proton and carbon signals was based on the analysis of NMR spectra obtained by 1D (1H, 13C, APT) and 2D (including the COSY, HMBC and HSQC experiments) techniques. The research was conduced by Dr. Helena Gaspar at the Faculty of Sciences, University of Lisbon, Portugal. 56 Analysis of Derivatized Samples Figure 15 – Summary of procedures involved in “legal highs” characterization by GC-MS Extraction A Preparation of methanolic extracts - Standards (5 µg /mL) - Samples (10 µg /mL) Derivatization B Dry under nitrogen flow 50 µL ethyl acetate + 50 µL TFAA 30 min at 70 °C Dry under nitrogen flow Dissolve residue in 100 µL ethyl acetate 2 µL of all extracts were injected in GC-MS GC-MS Analysis C Extraction A Preparation of methanolic extracts - Standards (1 mg/mL) - Samples (1 mg/mL) 2 µL of all extracts were injected in GC-MS GC-MS Analysis B Direct Injection Analysis 57 1.3.5 Identification process The identification of each analyte was done by comparison of retention time and mass spectra of the tested compounds with standards injected under the same chromatographic conditions to ensure correct identification. The EI-GC-MS spectra of unidentified peaks were searched through spectra databases supported by NIST05 (National Institute of Standards and Technology) and SWGDRUG (Scientific Working Group for the Analysis of Seized Drugs). We also made our own mass spectra library. In order to confirm the identity of all compounds detected, derivatization with TFAA was applied. The trifluoroacylation reaction occurs in more electrophilic atoms: the nitrogen atom forming N-TFA and the oxygen atom forming O-TFA (Figure 16) [107]. Figure 16 – Generic derivatization mechanism with the TFAA reagent to obtain the O-TFA and N-TFA derivatives NMR spectroscopy was also applied to confirm the structure of molecules identified by GCMS. Thus, all information was compiled in order to reveal the real composition of the products marketed as “plant feeders” obtained in the three “smartshops”. The process used for the products characterization is summarized in Figure 17. - CF3COOH R – NH2 + R – OH O R OF FF NH R OF FF 64 Figure 19 – EI mass spectra of methylone, 4-MEC, pentedrone, isopentedrone and MDPV, respectively (direct injection) (swgdrug) 3,4-Methylenedioxymethcathinone 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 0 50 100 39 42 51 56 58 63 74 77 91 96 121 130 135 149 162 176 188 207 O O O HN (swgdrug) 4-Methyl-N-ethylcathinone 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 0 50 100 42 44 51 56 65 70 72 77 86 89 91 105 115 119 128 132 146 158 174 190 O NH (swgdrug) Pentedrone 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 0 50 100 30 42 44 51 58 62 65 70 74 77 84 86 91 105 115 119 128 132 148 162 O NH (swgdrug) Isopentedrone 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 0 50 100 42 51 54 59 65 68 71 77 86 91 104 116 118 120 128 132 144 158 162 187 HN O (swgdrug) 3,4-methylenedioxypyrovalerone 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 0 50 100 42 50 55 65 69 74 78 84 91 96 102 110 121 126 131 149 164 174 204 230 273 O NO O Methylone 4-MEC Pentedrone Isopentedrone MDPV 65 All cathinone derivative standards (with exception of pentedrone, which was only available in a small amount) were also subjected to elemental analysis in order to determine if they were hydrochlorides and also to determine their purity. The results of analyses carried out are described in Table 5. Table 5: Results of external analysis of cathinone derivatives standards Compound % N % C % H % Cl Theoretical molecular weight Experimental molecular weight Proposed molecular formula % Purity 4-MEC 6.15 63.28 7.98 15.57 227.75 221.99 C12H17NO.HCl 97.47 Methylone 5.75 54.21 4.97 14.55 243.71 238.77 C11H13NO3.HCl 97.97 MDPV 4.50 61.62 7.13 11.37 311.84 304.40 C16H21NO3.HCl 97.61 The results showed that the samples analyzed were actually hydrochlorides, showing all of them purity higher than 97%. All compositions were confirmed and purities obtained were high so that all standards have been considered for the analysis of chemical characterization of commercial products. 2.2 Characterization of commercial products A total of 27 "legal highs" were purchased in three distinct “smartshops” (Euphoria in Porto and Magic Mushroom in Porto and Lisbon). A survey of all products sold as “plant feeders” or “bath salts” in these shops was conducted, being bought at least one sample of each. Some products were purchased on a large number of units, being a random choice in order to check the possible existence of qualitative and quantitative variability between supposedly identical products, as was previously described in the literature [6, 8-9, 16]. All products were packed in sealable silver foil bags or, occasionally, in transparent plastic bags. The packages featured the commercial name of the product, the alleged composition, the product quantity, and the dosage while “plant feeders”, along with the warnings “not for human consumption” (Figure 20). Some of the products presented its lot number but no other information was provided. 66 Figure 20 – Appearance of some commercial products The majority of powder products (86%) had a white color, however a small part had a yellowish color (Table 6), while the predominant color of the tablets was pink (Table 7). Variation was observed in the color of products with the same commercial name, acquired in the same “smartshop” (e.g. sample 1 and 2) or in different “smartshops” (e.g. sample 1 and 3; 6 and 7; or 16, 17 and 18), which immediately raised the possibility of a different composition. The amount of products purchased ranged between 0.5 and 1 gram in the case of the powders. All tablet products had two units per pack. The weight of each tablet was approximately 500 mg. Considering the amount of 1 g for all products, the prices for the powders varied between 30 and 38 euros, while the price of tablets varied between 14 and 20 euros (Table 6 e 7). This could be related to the product purity, leading us to consider the hypothesis that the powders are more potent than the products sold as tablets. Regarding the composition, the ingredient names printed on the packaging were recorded (Table 6 e 7) and the spelling mistakes were not corrected (e.g. phosphoates which should read phosphates). With the exception of samples 22, 26 and 27 all others have printed “ketones”, which can be interpreted as an indicator of the presence of cathinone derivatives. Samples 26 and 27 (“Bloom+”) have described the presence of aminoalkyl benzofurans, which although not derived from cathinones, are structurally similar to amphetamine analogs. Most powder products also referred the presence of caffeine and glucose. In tablets, the printed composition was more elaborated once beyond the principle active other excipients related to the production of tablets were also present. The fact that some spelling errors have been detected and the composition described being extremely vague it seems that these products do not follow any kind of quality control. 67 Table 6: Main features of "legal highs" (powders) acquired for analysis Sample Number Product Name Smartshop Lot Product Color Chemical composition indicated on the label Price per gram (€/g) 1 Bloom Magic Mushroom (Porto) N/A Yellowish Ketones, Vegetable extracts and Glucose 36 2 Bloom Magic Mushroom (Porto) 2012 45X19P White 94% Ketones, 5% Caffeine and 1% Glucose 36 3 Bloom Magic Mushroom (Lisbon) 2012 45X19P White 94% Ketones, 5% Caffeine and 1% Glucose 36 4 Blast Euphoria (Porto) N/A White 89% Ketones, 10% Caffeine and 1% Glucose 36 5 Blast Magic Mushroom (Lisbon) 2012 46X12P White 100% Ketones 36 6 Rush Magic Mushroom (Porto) N/A Yellowish 89% Ketones, 10% Caffeine and 1% Glucose 30 7 Rush Magic Mushroom (Lisbon) N/A White 89% Ketones, 10% Caffeine and 1% Glucose 30 8 Invader (Crabby) Euphoria (Porto) 2012 47X2P White 100% Ketones 37 9 Invader (Cyclop) Euphoria (Porto) 2012 45X2P White 100% Ketones 37 10 Bliss Magic Mushroom (Porto) 2013 X0619P White 95% Ketones, 5% Caffeine and 1% Glucose 30 11 Bliss Euphoria (Porto) 2012 37014P White 100% Ketones 30 12 Bliss Magic Mushroom (Lisbon) 2012 42X16P White 100% Ketones 30 13 Charlie Magic Mushroom (Porto) 2012 40X17P White 100% Ketones 37 14 Charlie Magic Mushroom (Porto) 2012 35017P White 100% Ketones 37 15 Charlie Magic Mushroom (Lisbon) 2012 35017P White 100% Ketones 37 16 Blow Magic Mushroom (Porto) N/A Yellowish Ketones, Vegetable extracts and Glucose 38 17 Blow Euphoria (Porto) N/A White 94% Ketones, 5% Caffeine and 1% Glucose 38 18 Blow Magic Mushroom (Lisbon) N/A White 94% Ketones, 5% Caffeine and 1% Glucose 38 19 Kick Magic Mushroom (Porto) N/A White Ketones, Vegetable extracts and Glucose 30 20 Kick Euphoria (Porto) 2012 36015P White 94% Ketones, 5% Caffeine and 1% Glucose 30 21 Kick Magic Mushroom (Lisbon) 2012 38015P White 94% Ketones, 5% Caffeine and 1% Glucose 30 22 MMB N/A N/A White N/A N/A 68 Table 7: Main features of "legal highs" (tablets) acquired for analysis Sample Number Product Name Smartshop Lot Product Color Chemical composition indicated on the label Price per two tablets (€/2 tablets) 23 M Magic Mushroom (Porto) N/A Pink Ketones, Dicalcium, Phosphoates, Magnesium, Stearate 16 24 M Magic Mushroom (Lisbon) N/A Pink Ketones, Dicalcium, Phosphoates, Magnesium, Stearate 16 25 Bliss Euphoria (Porto) N/A Pink 160 mg Ketones, 120 mg Lactose, 100 mg Corn starch, 50 mg Calcium stearate, 20 mg Magnesium stearate, 20 mg E124, 6 mg E132, 4 mg E142 14 26 Bloom + Euphoria (Porto) N/A Pink 100 mg Aminoalkyl benzofurans, 120 mg Lactose, 100 mg Corn starch, 50 mg Calcium stearate, 20 mg Magnesium stearate, 20 mg E128, 5 mg E142 20 27 Bloom + Magic Mushroom (Lisbon) N/A Pink 100 mg Aminoalkyl benzofurans, 120 mg Lactose, 100 mg Corn starch, 50 mg Calcium stearate, 20 mg Magnesium stearate, 20 mg E128, 5 mg E142 20 69 2.3 Screening of psychoactive substances in “legal high” products Since the products labeled as “plant feeders” or “bath salts” are supposed to contain ketones (hypothetically cathinone derivatives), we started this work by studying the chemical profiling of different commercial products. Due to difficulties in accessing spectra libraries containing the mass spectra of these new substances, we had to create our own database taking into account the information collected, which contains the molecular structure, molecular weight, mass spectrum profile including base peak and other characteristic fragmentation ions (m/z) of the different compounds. Gas chromatography coupled to mass spectrometry technique was chosen to separate and identify the various compounds present in the "legal highs" samples. Initially, the extraction of compounds was tested with different solvents, being the methanol the solvent chosen for all analyzes. This choice is due to the fact that the cathinone derivatives are very polar molecules due to the presence of the ketone group and thus can be easily extracted by polar solvents such as methanol. Methanolic extracts of all samples were directly analyzed by GC-MS, resulting in different chromatographic profiles. All peaks were taken into account for the products characterization. Using the mass spectrum information of each chromatographic peak (e.g. base peak, molecular ion) we were able to establish relationships with the spectra database. By performing the intersection of information obtained from mass spectra interpretation with mass spectra database (NIST05 and SWGDRUG), and later with NMR data, we were able to identify cathinones in each commercial product. We were able to identify methylone, 4-MEC, isopentedrone, pentedrone and MDPV in commercial products by comparing retention times and mass spectra of each chromatographic peak with the respective standards. Positive matches were obtained for all compounds that initially had been identified through the database created. Thus, these six compounds were unequivocally identified. The other compounds were assigned potential identifications through the analysis of their mass spectra and comparison with compounds of our database. Due to the lack of standards to confirm or reject the other identifications, derivatization with TFAA was performed. Even the substances confirmed by standards were subjected to this process. Derivatization allowed us to obtain more clean chromatograms and get better separations with increased resolution and response (Figure 21). 70 Figure 21 – Full scan chromatographic profile of methanolic extracts (Sample 11 – “Bliss”) injected directly (red) into the GC-MS and injected after derivatization with TFAA (green), indicating the potential identification of compounds based on the analysis of mass spectra and new fragmentation patterns of molecules after derivatization with TFAA. The signal strength after derivatization is significantly higher, showing greater sensitivity of the method. 1 – Methedrone; 2 – Methedrone N-TFA Knowing the TFAA affinity for binding to the active hydrogen’s of amine and hydroxyl groups, it was possible to predict the chemical structures of the formed compounds whose identity was intended to determine. Knowing the molecular weight after derivatization and ions resulting from fragmentation, comparisons with the mass spectrum of the derivatized compounds (Figure 22) and derivatized standards were established in order to confirm the identification of compounds. Of note, there are compounds without binding sites for TFAA, such as caffeine (Figure 22) and MDPV, whereby the molecular structure and consequently the mass spectrum are not altered after derivatization. Sample 11 – “Bliss” (Euphoria Porto) Direct injection of methanolic solution Sample 11 – “Bliss” (Euphoria Porto) Injection of derivatized solution 71 A B C D Figure 22 – EI mass spectra corresponding to compounds of the sample 4 (“Blast”) A – Flephedrone EI mass spectrum (direct injection); B – EI mass spectrum of flephedrone N-TFA; C – Caffeine EI mass spectrum (direct Injection); D – Caffeine EI mass spectrum after derivatization N NN N O OCH3 CH3 CH3 N NN N O OCH3 CH3 CH3 Molecular weight: 194 Molecular weight: 194 Molecular weight: 181 Molecular weight: 277 72 Following the entire procedure, the chemical characterizations of all products purchased in different “smartshops” are summarized in Table 8. In Table 9 are presented the retention time, base peak and other characteristic qualitative ions (m/z) for the compounds identified by GCMS before and after derivatization. In a later stage of the experimental work, we had access to a new mass spectra database (SWGDRUG), which includes these new synthetic substances. Thus, all chromatographic peaks (direct injection) were also screened in this new library. It should also be noted that all products have been analyzed by NMR, thus confirming the molecular structure of each compound being an informative methodology in the distinction of positional isomers. These analyses allowed for example the distinction of 3-MEC and 4-MEC isomers. Table 8: Active ingredients detected in 27 “legal highs” acquired in three distinct “smartshops” after analysis by GC-MS and NMR. (*) “Legal high” sold in tablet form. Product name Smartshop Active substances identified by GC-MS and NMR Bloom Magic Mushroom Porto Isopentedrone, Pentedrone, 4-Methylethcathinone (4-MEC), Methylone, Dimethocaine Magic Mushroom Porto Ethcathinone, Pentedrone, Methedrone, Caffeine Magic Mushroom Lisbon Ethcathinone, Pentedrone, Methedrone, Caffeine Blast Euphoria Porto Flephedrone, Caffeine Magic Mushroom Lisbon Flephedrone, Caffeine Rush Magic Mushroom Porto Buphedrone, Caffeine Magic Mushroom Lisbon Isopentedrone, Pentedrone, Caffeine Crabby Euphoria Porto 3,4-Dimethylmethcathinone (3,4-DMMC) Cyclop Euphoria Porto 3,4-DMMC Bliss Magic Mushroom Porto Isopentedrone, Pentedrone, 3,4-DMMC, Methedrone, Caffeine Euphoria Porto Methedrone Magic Mushroom Lisbon Methedrone Charlie Magic Mushroom Porto Ethcathinone, Buphedrone, Caffeine Magic Mushroom Porto Ethcathinone, Buphedrone Magic Mushroom Lisbon Ethcathinone, Buphedrone Blow Magic Mushroom Porto 3-Methylmethcathinone (3-MEC), 4-MEC, Methylenedioxypyrovalerone (MDPV) Euphoria Porto 3-MEC, 4-MEC, Caffeine, MDPV Magic Mushroom Lisbon 3-MEC, 4-MEC, Caffeine, MDPV Kick Magic Mushroom Porto Isopentedrone, Pentedrone Euphoria Porto Buphedrone, Caffeine Magic Mushroom Lisbon Buphedrone, Caffeine MMB N/A Alpha-methyltryptamine (AMT), Dimethocaine M (*) Magic Mushroom Porto 4-Fluoroamphetamine Magic Mushroom Porto 4-Fluoroamphetamine Bliss (*) Euphoria Porto Methylone Bloom + (*) Euphoria Porto 5-(2-Aminopropyl)benzofuran (5-APB), 6-(2-Aminopropyl)benzofuran (6-APB), 5-(2-Aminopropyl)2,3-dihydrobenzofuran (5-APDB) Magic Mushroom Porto 6-APB, 5-APDB Table 9: Retention time (minutes), base peak (m/z) and other characteristic ions (m/z) of active ingredients detected directly by GC-MS and after derivatization with TFAA. Legend: A – available; N/A – not available; SW – SWGDRUG library; N – NIST05 library; m/z – mass to charge ratio Without TFAA With TFAA Compound Standard Library Retention time (minutes) Base Peak (m/z) Characteristic ions (m/z) Retention time (minutes) Base Peak (m/z) Characteristic ions (m/z) Isopentedrone Pentedrone 3-MEC 4-MEC Methylone Ethcathinone Methedrone Flephedrone Buphedrone 3,4-DMMC MDPV 5-APB 6-APB 5-APDB 4-Fluoroamphetamine AMT Dimethocaine Caffeine A A N/A A A N/A N/A N/A N/A N/A A N/A N/A N/A N/A N/A N/A A SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW/N SW/N SW/N 6,5 6,7 6,8 6,9 8,1 6,1 7,5 5,7 6,3 7,3 10,5 6,6 6,7 7,2 4,3 8,4 11,0 8,8 120 86 72 72 58 72 58 58 72 58 126 44 44 44 44 131 86 194 77, 91, 191 77, 105, 191 91, 119, 191 91, 119, 191 91, 121, 149, 207 44, 77, 105, 177 77, 107, 135, 193 95, 123, 181 77, 105, 177 77, 105, 133, 191 84, 121, 149, 275 77, 131, 175 77, 131, 175 77, 134, 177 83, 109, 153 44, 77, 103, 174 58, 120, 278 55, 67, 82, 109 N/A 7,2 7,5 7,7 9,0 6,7 8,3 6,0 6,9 7,9 11,3 7,4 7,6 8,2 5,0 8,7 11,8 9,4 N/A 140 119 119 149 105 135 123 105 133 126 131 131 160 136 226 86 194 N/A 77, 105, 182, 287 91, 168, 287 91, 168, 287 65, 110, 303 110, 168, 273 176, 182, 289 110, 154, 277 71, 77, 168, 273 105, 174, 182, 287 84, 121, 149, 275 140, 158, 270 140, 158, 270 105, 133, 273 89, 109, 248 140, 154, 366 65, 120, 374 55, 67, 82, 109 A total of 18 different substances were detected (Figure 23), belonging the vast majority to the class of synthetic cathinones. Substances of other classes were also found, namely dimethocaine (cocaine derivative), alpha-methyltryptamine (class of tryptamine), as well as other substances derived from phenethylamines class, namely the derivatives of benzofurans (5-APB, 6-APB and 5-APDB). In total we found 11 distinct cathinone derivatives (Figure 23), the pentedrone and buphedrone being the most frequent (9.1%) while flephedrone and methylone the less common (3%). However, despite the synthetic cathinones correspond to the representative class, the most common substance was caffeine (18.2%) (Figure 23), having the function of promoting the stimulant effects, being an adulterant. 80 harm and toxicity associated with their use. This inconsistency also hampers the assessment of the clinical state of the patient and consequently to provide appropriate treatment. When one substance is replaced by another or by a mixture, or the amount is higher than those labeled, the effects can be significantly different. These differences could have an impact on duration of action, the time required to cause the effects, and to the multiplicity of effects and respective interactions, etc, which could result in severe intoxications. Another matter of concern that should be the subject of further studies passes through the drug-drug interactions between the different components of the mixture and in cases of polydrugs these may worsen the clinical consequences. We can speculate that terrible consequences would come from taking these “legal highs”, but there are not enough evidences to state with certainty that the cause of intoxication and/or death is full responsibility of these new products. In addition, all these inconsistencies reported throughout the study demonstrate unequivocally the lack of control associated with these new substances regarded as “safe”, showing only sellers concern at the monetary impact level, putting aside the health of users. Despite all the problems associated with the new drugs phenomenon, it should be noted that none of the analyses detected compounds present in the list of prohibited substances [72, 74], in Portugal, at the time that these products were purchased. However, there are cases in the literature where substances have been identified in these so-called “legal highs” after their ban. Brandt et al. (2010) [8] obtained 24 products in 18 UK-websites over a period of 6 weeks following the ban of mephedrone and found that over 62% of analyzed products had this banned substance. Other study [31] revealed that five samples, also acquired through a UKwebsite, had controlled substances (benzylpiperazine (BZP) and 1-[3- (trifluoromethyl)phenyl]piperazine (TFMPP)) combined with caffeine. Due to the sudden ban on the sale of certain substances sellers continue selling prohibited products, rebranded as “new legal highs”, in order to reduce the stock. Taking into account this information, users of these “new products” are in possession of illegal substances assuming that they are legal and they are unaware of the consequent criminal and health risks. 81 2.5 Semi-quantitative analysis 2.5.1 Semi-quantitative variability in commercial products By comparing the chromatographic profile of a 10 µg/mL of a “legal high” (powder vs. tablet), it was found that the amount of cathinones in the tablet samples is lower (Figure 26). Comparing the majority peaks of each sample, it is verified that the signal of the tablet samples is about two to four times lower than the powder samples, when prepared in the same concentration and under the same conditions. This finding was observed in all “legal highs” purchased in the form of tablets although the signal strength varies slightly between them. This finding associated with the fact that the tablets are sold at a significantly lower price (about half), gives emphasis to the hypothesis previously raised of the “legal highs” tablets being less powerful/pure in comparison to the powders. Figure 26 – Full scan chromatographic profile of derivatized solution (Sample 14 – “Charlie” in the form of powder and Sample 23 – “M” in the form of tablet) injected after derivatization with TFAA. The powder signal strength is significantly higher, may be an indicator of higher purity of product. The potential identification is indicated based on the analysis of mass spectra and new fragmentation patterns of molecules. 1 – Ethcathinone N-TFA; 2 – Buphedrone N-TFA; 3 – Fluoroamphetamine N-TFA. By comparing the chromatographic profile of samples whose real composition is identical, it was found in some cases an apparent quantitative variability (Figure 24 and 25). However, due to the lack of robust standards, it became impossible to perform a complete and reliable quantitative determination. Still, there are studies in the literature [6] that reveal significant differences in the quantitative composition of the products with real qualitative content identical. The content of Sample 14 – “Charlie“ (Magic Mushroom Porto) Sample 23 – “M” (Magic Mushroom Porto) 82 MDPV detected in some preparations was about 10-15 mg per package and more than 100 mg in other packages. MDPV produces weak psychoactive effects for 3 to 5 mg and severe effects after administration of higher doses (higher than 20 mg), depending our route of exposure [5]. Thus, we can infer that administration of more than 100 mg of MDPV could be a health and even life-threatening. 2.5.2 Linearity study In our study, four samples were randomly selected in order to semi-quantify the substances that compose them. Three powders (Sample 1 – “Bloom”; Sample 16 – “Blow”; Sample 19 – “Kick”) and one tablet (Sample 25 – “Bliss”) were selected. In “Bloom”, it was quantified the 4MEC, pentedrone and methylone. In the sample 16 it was quantified 4-MEC and MDPV while in sample 19 only pentedrone. In turn, in the “Bliss” sample just methylone was analyzed, since it was the only component detected in qualitative analysis. Regarding the quantification process, all samples and standards have been derivatized as it was concluded to be a more sensitive methodology. Table 10 shows the calibration curves equations of standards tested for a given concentration range, as well as the squared correlation coefficient associated. Because the concentration found in the commercial products containing MDPV is lower than those containing other cathinones, we chose a range of lower concentrations (10-2000 ng/mL). All analytes showed high linearity for the concentrations range established, R2 values being higher than 0.99 (Table 10). Table 10: Calibration curves for 4-MEC, Pentedrone, Methylone and MDPV Compound Equation (y=mx+b) Range (ng/mL) R2 4-MEC y=0.0002x+78,442 50 – 10000 0.99781 Pentedrone y=0.0002x-83,787 50 – 10000 0.99909 Methylone y=0.0002x+190,11 50 – 10000 0.99555 MDPV y=0.0005x-3,7372 10 – 2000 0.99219 83 2.5.3 Intra-day precision of the method The precision of the method was determined (Table 11), and the results were satisfactory never exceeding values of 20% for intra-day determinations. Table 11: Intra-day precision of the method for the quantification of 4-MEC, Pentedrone, Methylone and MDPV in different “legal highs” products (n=3) Stock Solution Compound Bloom Blow Kick Bliss Mean (mg) SD CV (%) Mean (mg) SD CV (%) Mean (mg) SD CV (%) Mean (mg) SD CV (%) A 4-MEC 296,9 53,4 10,1 623,1 51,0 8,2 -- -- -- -- -- -- Pentedrone 237,6 15,4 6,5 -- -- -- 705,3 31,2 4,4 -- -- -- Methylone 460,2 50,8 11,0 -- -- -- -- -- -- 133,3 7,9 5,9 MDPV -- -- -- 13,4 1,3 9,4 -- -- -- -- -- -- B 4-MEC 208,3 15,0 7,1 845,6 98,0 11,6 -- -- -- -- -- -- Pentedrone 146,1 1,7 1,2 -- -- -- 536,3 31,6 5,9 -- -- -- Methylone 385,0 33,3 8,6 -- -- -- -- -- -- 254,1 19,3 7,6 MDPV -- -- -- 15,1 2,5 16,7 -- -- -- -- -- -- C 4-MEC 219,0 21,7 9,9 746,9 77,5 10,4 -- -- -- -- -- -- Pentedrone 188,4 2,6 1,4 -- -- -- 579,0 40,5 7,0 -- -- -- Methylone 569,3 19,5 3,4 -- -- -- -- -- -- 215,4 9,8 4,6 MDPV -- -- -- 25,7 3,5 13,6 -- -- -- -- -- -- 2.5.4 Study of homogeneity of commercial products In order to verify the homogeneity of selected commercial samples (“Bloom”, “Blow”, “Kick” and “Bliss”) we took from the same sample three different aliquots (stock solution A, B and C). Figure 27 (A-D) results show the quantitative relation between the same component in the different solutions (for example 4-MEC of a “Bloom” solution A vs. 4-MEC of a “Bloom” solution B vs. 4-MEC of a “Bloom” solution C), as well as the relationship between the different solutions as a whole (e.g. solution A of “Bloom” vs. solution B of “Bloom” vs. solution C of “Bloom”). The results presented correspond to mean ± standard deviation (SD), for n=3. 84 Figure 27 – Semi-quantification of some substances (4-MEC, Pentedrone, Methylone and MDPV) present in “legal high” samples randomly selected and the quantitative relation between the same compound for different solutions. The figure A expresses the amount of 4-MEC, Pentedrone and Methylone (mg) in each gram of “Bloom” (1). Graph B expresses the amount of 4-MEC and MDPV (mg) in each gram of “Blow” (16), while the graph C expresses the amount of Pentedrone (mg) in each gram of “Kick” (19). Finally, the graph D expresses the amount of Methylone (mg) in each tablet of “Bliss” (25). The results are expressed through the mean ± SD, n=3 (* P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 for solution B or solution C vs. solution A). The groupings shown in figure A and B represent comparisons between the solutions as a whole. Solution A Solution B Solution C 0 200 400 600 800 Compound Quantity (mg/g Bloom) 4-MEC Pentedrone Methylone ** * * * ** **** *** ** Solution A Solution B Solution C A B C D Solution A Solution B Solution C 0 500 1000 Compound Quantity (mg/g Blow) 4-MEC MDPV ** ** Solution A Solution B Solution C Solution A Solution B Solution C 0 200 400 600 800 Pentedrone (mg/g Kick) ** * Solution A Solution B Solution C 0 100 200 300 Methylone (mg/g Bliss) *** ** 85 Although the samples, visually, appear homogeneous and the same homogenization procedure was applied, it was verified that there are significant differences (P<0.05) (Figure 27) between the amounts of the compounds in the different solutions for the same product. This leads us to believe that the powders in the mixture of different “legal highs” can have different densities and/or granulometries, interfering in the reproducibility of the quantification values. This can be reflected in different amounts of substances presents in different portions of the products if the consumer does not use the entire product at once. This happens mainly in cases where the consumer is inexperient, being advised by the sellers to consume the product splitted. Thus, if there is a lack of product homogeneity, each time the user consume a portion, will consume different substances in unequal amounts at all times, so it is expected that the intensity of effects can vary, even within the same product. For a correct quantification of active principles and thus circumvent their heterogeneity, the ideal would be to perform a methanolic solution of the entire product and, subsequently, do all analysis from this global solution. 86 87 Cytotoxicity study of cathinones 4 88 89 Chapter 1 - Experimental Part: Cytotoxicity studies 96 The spectrophotometric analysis was run at 550 and 690 nm in a 96-well plate reader (PowerWaveX; Bio-Tek, Winooski, VT, USA). Data were obtained from five independent experiments for primary hepatocytes and three independent experiments for HepaRG cells, with each test plate containing quadruplicates of increasing concentrations of the tested drugs. 1.6 Regression modeling Curves of normalized mortality values (effect) versus log of concentration (mM) were constructed and analyzed using the best-fit approach [113]. In the present study, the logit function was employed: Y = θmin + (θmax – θmin)/(1+exp (-θ1-θ2*log (x))), where θmin and θmax are the minimal and maximal observed effects, respectively; x is the concentration of the test drug; θ1 is the parameter for the location and θ2 is the slope parameter. All of the nonlinear regression models describe sigmoidal concentration-response relationships and the plots were constructed using the GraphPad Prism 6 (version 6.0c) for Mac OS X. MTT data are presented as mean ± 95% confidence interval (CI). The EC50 values were determined for each individual drug and mixture, allowing for comparison between drugs. 1.7 Calculation of predicted mixture effects After characterization of concentration-response curves of the individual agents, the effects of the “legal high” mixtures were predicted assuming additive joint responses. The expected effects were calculated using the concentration addition (CA) and independent action (IA) approaches, as described in Payne et al. [114] and used as a reference for the assessment of combination effects in terms of synergism (if the observed effects are greater than additive predictions), additivity (if the experimental mixture outcomes equal the prediction) and antagonism (if the experimental joint effects fall short of additivity) [115]. Briefly, the concept of CA is based on the assumption that the mixture constituents have similar modes of action, which means that any component can be replaced partially or totally with another without changing the overall mixture effect [116]. This means that each individual component contributes to the global joint effect by acting in proportion to its concentration, even at concentrations producing no effect. The IA alternative approach better describes combination effects of drugs with dissimilar mechanisms of action with each agent interacting at differing sites of action [117]. In this case, the fractional response of one individual component is supposed to be independent from those induced by other components, presuming that 97 mixture components present at zero effect concentrations will not contribute to the overall effect. 98 99 Chapter 2 - Results and Discussion of the cytotoxicity studies 100 101 2.1 Features of primary hepatocytes and HepaRG cells In drug screening assays, the hepatotoxicity is one of the parameters of primary concern, since the liver is an organ responsible for biotransformation and elimination of toxic compounds from the human body, as well as being a primary target for allocation by xenobiotics [114]. The vast arsenal of vital biochemical functions performed by the liver requires an appropriate architecture, therefore the vast majority of all hepatic functions, including xenobiotic biotransformation, is performed by hepatocytes that constitute about 80% of the parenchymal liver cells. Hence, most of the hepatic in vitro models for toxicity studies are based on hepatocytes [115]. Taking this into accout, a wide range of liver-derived in vitro models have been developed and are now available for toxicological studies. Primary rodent hepatocyte cultures and hepatoma-derived cell lines are an important part of this in vitro models [116-120]. Primary cultures of rat hepatocytes (Figure 29 A) are a good alternative to human cells often used in toxicological studies because they present higher metabolic responses than the common human cell lines and the inter-donor variability can be minimized by selecting animals of the same sex, age and with similar feeding regimes [121]. Although primary hepatocytes are clinically relevant, cell lines are frequently used as alternatives. The hepatocyte cell lines present some advantages over freshly isolated hepatocytes, such as higher availability, unlimited life, stable karyotype and the fact that they are easy to handle and grow continuously [116, 118]. One disadvantage associated with hepatoma cells involves the limited extent of its biotransformative activity by some cytochrome P450 enzymes or by their low levels of when compared to a normal adult liver [118]. The HepaRG cell line has the ability to differentiate into hepatocyte-like cells and biliary-like cells, reaching maximum differentiation after 2 weeks of exposure to a 2% DMSO supplemented medium (Figure 29), expressing highly differentiated functions [117, 121-125]. For most metabolizing genes, the expression levels are associated with the presence of DMSO and the expression of CYP isoenzymes generally decreases when DMSO is removed from the medium, whereas carriers and the liver-specific factors remain unchanged [126]. Thus, it is suggested a dual effect of DMSO in cells, affecting not only their differentiation as well as gene expression in differentiated cells. Figure 29 shows HepaRG cells non-differentiated (B) and HepaRG cells after differentiation in culture medium containing 2% DMSO (C). 102 Figure 29 – (A) Microscopic appearance of freshly isolated rat hepatocytes in a total magnification of 100x. Figures B1 and B2 shows microscopic appearance of HepaRG cells to 80-90% of confluence in a magnification of 100x and 200x, respectively. Figures C shows the HepaRG cells after 18 days of culture in the presence of differentiation medium with DMSO in a magnification of 100x (B1) and 200x (B2). H – hepatocyte-like cells; BC – bili canaliculus. B1 B2 A C1 C2 H BC H BC 103 2.2 Yield and viability of cell suspensions of isolated rat hepatocytes More important than the amount of isolated hepatocytes (yield), is their quality, i.e., the percentage of viable cells in suspension. After the isolation process, it is preferable to obtain a small number of cells but intact than getting a large quantity being the vast majority non-viable. Thus, after the rat liver perfusion, mechanical dispersion of cells must be performed carefully to minimize the loss of viability of the final suspension. The variability of cell viability was checked during the assays. The cell suspensions used for the experiments showed viability between 81 and 90.2%. These values are within the ones reported by other authors whose work presented values between 80 to 97% also obtained with Wistar rats [127-128]. The number of cells obtained in each suspension ranged between 124 to 245x106 cells per animal. For good results in terms of yield and viability, the experience factor during the perfusion technique implementation also seems determinant. An efficient and rapid cannulation of the portal vein, to ensure fast and uniform perfusion of all hepatic lobes, is very important in order to obtain favorable results. Parts of the liver that does not change color immediately when perfusion start, represent a poor cannulation, indicating further areas which have not been digested by collagenase. 2.3 Test compounds As previously described, the number of intoxications and deaths associated with synthetic cathinones consumption increased considerably [4, 18-19, 99-100]. The inconsistency of effects and their magnitude is hard to explain. However, one possible explanation for this aspect implicates a pattern of polydrug abuse, often associated with the consumption of “legal highs”, as it was evident through the chemical characterization studies. The present study aimed to evaluate the cytotoxic effects of synthetic cathinones derivatives and real mixtures of this derivatives sold as “plant feeders”. Two “legal highs” were selected for cytotoxity study: “Bloom” and “Blow”. The first was chosen since its major component is the methylone, the direct analogue of “ecstasy”. In turn, the “Blow” was chosen as display in its composition 4-MEC that is structurally similar with one of the most popular synthetic cathinone (mephedrone) [108] and MDPV, which is one of the most potent derivatives due to the presence of a tertiary amine group in its molecular structure [1, 15]. In order to accurately estimate the effects of a mixture, information about the individual responses of each of the constituents is required. Thus, all the major substances present in these products were studied alone in the same conditions as the mixtures. 104 One of the most popular drugs in the world [129] belonging to the amphetamine-class, MDMA, was also considered for the study, since it detains structural similarities with the synthetic cathinones, allowing a comparison of the hepatotoxic potency of the different drugs. For this purpose, stock solutions at a concentration of 50 mM for all products were prepared. Stock solutions of “Bloom” and “Blow” products were prepared in such a way that the concentration of major compound (methylone and 4-MEC, respectively) was fixed at 50 mM and the concentration of the remaining components of the mixture has been determined taking into account this main concentration. Thus, the peak area of the main active ingredient of the commercial product, obtained by gas chromatography, was equal to that obtained for the respective pure compound at desired concentration (50 mM) (Figure 30). Figure 30 - Full scan chromatographic profile of “Bloom” stock solution with methylone at 50 mM and methylone standard also at 50 mM, injected directly into the GC-MS, showing equal areas and therefore equal concentrations. Taking into account this principle, the concentration of methylone, 4-MEC and pentedrone in the “Bloom” product used for cytotoxic study is 50, 33 and 31 mM, respectively. In turn, in the “Blow” sample, the concentration is 50 mM for 4-MEC and 1.8 mM for MDPV. 105 Therefore, the stock solutions were diluted maintaining the ratio between each mixture constituent unchanged. Serial dilutions covered a wide range of concentrations (0.05 – 5mM) for all test substances to describe the complete response range, from 0% cell death to maximum induced cell death, when using the MTT cytotoxicity assay. 2.4 Concentration-response relationship of individual agents in primary cultured hepatocytes In the MTT assay, all tested single agents yielded reproducible effects in a concentrationdependent fashion, resulting in increased cell death with the rising of chemical concentration (decreased percentage of cell viability). The shape of the concentration-response curves for the individual drugs tested were relatively similar, as well as their maximal effects, with the exception of pentedrone which presents values of cell death more pronounced to higher concentrations evidencing a higher slope compared to other substances. More significant differences were observed essentially in EC50 values as shown in figure 31. MDMA has an EC50 value of 0.754 mM. Methylone with an EC50 of 1.18 mM shares a similar potency with 4-MEC (EC50 1.29 mM), however with significantly higher values (P<0.05) than MDMA, being consequently drugs which individually offer lower cytotoxicity. In turn, there are no significant differences between MDPV (EC50 0.742 mM) and pentedrone (EC50 0.647 mM), being more cytotoxic than MDMA, although not significantly. Due to the presence of the pyrrolidinyl ring and the tertiary amine group, MDPV, as previously mentioned, is more lipophilic than other cathinones, showing greater facility to cross barriers [1, 15]. Thus, it was expected that MDPV was the most cytotoxic compound in the MTT assay. Although the EC50 values of pentedrone and MDPV were not significantly different, the first derivative appeared to be more cytotoxic at high concentrations, whereas for lower values, MDPV showed a higher cytotoxicity. Thus, through the MTT assay we can conclude that MDPV is actually one of the most powerful cathinones, showing its maximum hepatotoxicity at concentrations below 0.5 mM, as MDMA. 112 sudden lethal intoxications, as well as facilitate the diagnosis and treatment of non-fatal cases. A better understanding of these combined effects may have a considerable influence on public health, raising awareness of the potential for severe toxicity and therefore stimulating behavioral changes in consumers worldwide. 113 Conclusions 5 114 115 Conclusions of the chemical characterization part:  For the unequivocal identification of active principles of the new drugs, in the absence of standards, the GC-MS and NMR combination proved to be a powerful tool.  Products sold as “plant feeders” although labeled to contain ketones (hypotetical synthetic cathinones), also include substances belonging to other classes. However, the synthetic cathinones derivatives group is more representative. Caffeine is the most commonly compound detected in products.  The diversity in composition among these products is one of the greatest problems connected with the “legal highs” phenomenon. The consumers cannot be sure what they get. As has been shown, products with the same trade name have a different content and the same mixture is sold under different trade names.  Inconsistency in both qualitative and quantitative compositions could lead to serious consequences, because users are unware of active dose, time of duration and even the effects. Conclusions of the cytotoxicity studies:  All cathinones, individually or in mixtures, showed concentration-dependent decrease in cell viability, in both cell models.  Methylone and 4-MEC were less cytotoxic, while MDPV and pentedrone are the most cytotoxic cathinone derivatives, when compared with MDMA.  Results with HepaRG cells showed that they are less sensitive to cathinone effects than primary hepatocytes. 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