Pharmacology of novel psychoactive substances

This PhD work consists of an in vitro and in vivo part. In the in vivo part, we investigated the role of dopamine in the acute clinical effects of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) in healthy human subjects. The role of dopamine in the addictive effects of drug of abuse is well established, but whether it contributes to the acute psychotropic effects of MDMA is unclear. In this pharmacological interaction study, we used the dopamine and weak norepinephrine transporter inhibitor bupropion (Stahl et al. 2004) as a pharmacological tool to block the MDMA-induced dopamine release and to study the role of dopamine in the effects of MDMA. We hypothesized that bupropion would decrease the subjective effects of MDMA to the extent that they depend on MDMA-induced release of dopamine. We included 16 healthy human subjects in this double-blind, placebo-controlled, crossover study. Bupropion pretreatment slightly increased MDMA plasma concentration and prolonged but not reduced the subjective effects contrary to our hypothesis. Additionally, bupropion reduced the MDMA-induced elevations in plasma norepinephrine concentrations and the heart rate response to MDMA. These findings support a role for norepinephrine in the MDMA-induced cardiostimulant effects but no role for MDMA-induced transporter-mediated dopamine release in the elevated mood effects after MDMA administration. Possibly, most of the acute psychotropic effects of MDMA are mediated via transporter-mediated release of serotonin and norepinephrine as previously shown (Hysek et al. 2011, Hysek et al. 2012). In the second and main part of this work we characterized the pharmacological profiles of novel psychoactive substances (NPS). Specifically, we studied whether and how potently NPS interacted with the human transporters for norepinephrine, dopamine, and serotonin, stably expressed in human embryonic kidney (HEK293) cells. Additionally, we assessed binding affinity to the serotonin 5-HT1A, 5-HT2A, 5-HT2C-receptors and the activation potency and activation efficacy at 5-HT2A and 5-HT2B receptors. Furthermore, binding to alpha1A/2A-adrenergic, dopamine D1-3, histamine H1 receptors, as well as trace amine-associated receptor 1 (TAAR1) was also assessed. The NPS studied in this project included para-4-halogenated amphetamine derivatives, which were shown to be relatively more serotonergic than their non-4-halogenated counterparts and pyrovaleronering-substituted cathinones, which were highly potent dopamine transporter inhibitors with a high risk for abuse. Para-halogenated drugs (4-fluoroephedrine, 4-fluoroamphetamine, 4-fluoromethamphetamine, 4-fluoromethcathinone, and 4-bromomethcathinone) also released monoamines, similar to MDMA, whereas pyrovalerones were found to be pure uptake inhibitors. Most benzofurans were similar to MDMA but slightly more serotoninergic than MDMA and additionally activated the serotonin 5-HT2B receptor. The last big group of NPS studied in this project, were novel hallucinogens, which predominantly interacted with the 5-HT2A receptor. This serotonin receptor subtype mediates the hallucinogenic and hallucinogenic-like visual effects of classic serotonergic hallucinogens (Vollenweider et al. 1998, Nichols 2004, Halberstadt et al. 2013, Halberstadt et al. 2014, Halberstadt 2015). Compounds tested in this project included the benzodifuran 8-Bromo-2,3,6,7-benzo-dihydro-difuran-ethylamine (2C-B-FLY), 2C-drugs with their highly potent N-(2-methoxy)benzyl (NBOMe)-derivatives, and lysergic acid diethylamide (LSD). Interestingly, NBOMe derivatives displayed higher affinities at the 5-HT2A receptor than LSD, together with a high selectivity for 5-HT2A over the 5-HT1A receptor, contrary to LSD. NBOMes were partial 5-HT2A receptor agonists, similar to LSD. These novel drugs likely carry a high hallucinogenic potential when used recreationally by humans and the high binding to α1A-receptor (Ki < 1µM) may result in additional vasocontrictive and cardiovascular stimulant effects. Taken together, this PhD contributed to the understanding of the role of dopamine in the effects of MDMA, an important recreational substances. Additionally, we characterized the in vitro pharmacology of many novel designer drugs, which will be helpful in the prediction of the clinical toxicological effects of these newly used recreational drugs

Monoamine transporter and receptor interaction profiles of novel psychoactive substances: para-halogenated amphetamines and pyrovalerone cathinones

The pharmacology of novel psychoactive substances is mostly unknown. We evaluated the transporter and receptor interaction profiles of a series of para-(4)-substituted amphetamines and pyrovalerone cathinones. We tested the potency of these compounds to inhibit the norepinephrine (NE), dopamine (DA), and serotonin (5-HT) transporters (NET, DAT, and SERT, respectively) using human embryonic kidney 293 cells that express the respective human transporters. We also tested the substance-induced efflux of NE, DA, and 5-HT from monoamine-loaded cells, binding affinities to monoamine receptors, and 5-HT2B receptor activation. Para-(4)-substituted amphetamines, including 4-methylmethcathinone (mephedrone), 4-ethylmethcathinone, 4-fluoroamphetamine, 4-fluoromethamphetamine, 4-fluoromethcatinone (flephedrone), and 4-bromomethcathinone, were relatively more serotonergic (lower DAT:SERT ratio) compared with their analogs amphetamine, methamphetamine, and methcathinone. The 4-methyl, 4-ethyl, and 4-bromo groups resulted in enhanced serotonergic properties compared with the 4-fluoro group. The para-substituted amphetamines released NE and DA. 4-Fluoramphetamine, 4-flouromethamphetamine, 4-methylmethcathinone, and 4-ethylmethcathinone also released 5-HT similarly to 3,4-methylenedioxymethamphetamine. The pyrovalerone cathinones 3,4-methylenedioxypyrovalerone, pyrovalerone, α-pyrrolidinovalerophenone, 3,4-methylenedioxy-α-pyrrolidinopropiophenone, and 3,4-methylenedioxy-α-pyrrolidinobutiophenone potently inhibited the NET and DAT but not the SERT. Naphyrone was the only pyrovalerone that also inhibited the SERT. The pyrovalerone cathinones did not release monoamines. Most of the para-substituted amphetamines exhibited affinity for the 5-HT2A receptor but no relevant activation of the 5-HT2B receptor. All the cathinones exhibited reduced trace amine-associated receptor 1 binding compared with the non-β-keto-amphetamines. In conclusion, para-substituted amphetamines exhibited enhanced direct and indirect serotonergic agonist properties and are likely associated with more MDMA-like effects. The pharmacological profile of the pyrovalerone cathinones predicts pronounced stimulant effects and high abuse liability

Comparing attitudes towards compulsory interventions in severe and persistent mental illness among psychiatrists in India and Switzerland

BACKGROUND: Psychiatrists face a major ethical challenge when deciding whether to make use of coercive measures in the treatment process of patients suffering from severe and persistent mental illness (SPMI). As India and Switzerland show major cultural, political and financial differences, it is hypothesized that attitudes towards coercive measures among Indian and Swiss psychiatrists will vary too. Exploring differences in attitudes between cultures strengthens the critical reflection on one's own stances and in consequence, on our way of action. Especially when it comes to situations involving power imbalances between patients and health practitioners, self-reflection is essential to prevent ethically inappropriate behavior. METHODS: An online survey on aspects of care for patients with SPMI was sent to 3'056 members of the Indian Psychiatric Society between April and June 2020 and to 1'311 members of the Swiss Society for Psychiatry and Psychotherapy between February and March 2016. The respondents' answers were compared. This article deals with the questionnaire's items on autonomous decision making and the implementation of coercive measures in clinical practice. More precisely, participating psychiatrists were asked to rate the importance of patient's autonomy in general and their willingness to apply coercive measures regarding two specific case vignettes depicting a patient with schizophrenia and one with depression. The statistical analysis, namely descriptive data analysis and calculation of arithmetic means, Shapiro Wilks tests and Mann-Whitney U tests, was carried out using IBM SPSS Statistics version 27. RESULTS: Answers were received from 206 psychiatrists in India and 457 psychiatrists in Switzerland. Indian participants tended to value autonomous decision making as slightly less important than Swiss participants (62.2% vs. 91%, p =.01). Regarding a case of severe and persistent depression, psychiatrists in the Indian group were on average more in favor of acting against the wishes of the patient (55% vs. 34.1%, p <.0001) as well as of accepting a temporary decrease in quality of life due to coercion (40% vs. 23%, p =.008). Answers concerning a case of schizophrenia revealed that Indian participants were more in favor of acting against the patient's wishes than Swiss participants (39% vs. 37%, p =.007), whereas the comparison whether to accept a temporary decrease in quality of life regarding this case showed no significant difference (p =.328). CONCLUSIONS: The significant difference in attitudes towards coercive measures among Indian compared to Swiss psychiatrists found in this study might arise from a predominantly more collectivist society in India compared to Switzerland. Moreover, differences in financial resources, the organization of the health care system, and the historical background might have an influence. Continuous and critical reflection on one's own views and behavior is essential, especially if ethical principles and individual rights could be violated through a power imbalance, as in the case of coercive measures

Pharmacological profile of novel psychoactive benzofurans

Benzofurans are newly used psychoactive substances, but their pharmacology is unknown. The aim of the present study was to pharmacologically characterize benzofurans in vitro.; We assessed the effects of the benzofurans 5-APB, 5-APDB, 6-APB, 6-APDB, 4-APB, 7-APB, 5-EAPB and 5-MAPDB and benzodifuran 2C-B-FLY on the human noradrenaline (NA), dopamine and 5-HT uptake transporters using HEK 293 cells that express the respective transporters. We also investigated the release of NA, dopamine and 5-HT from monoamine-preloaded cells, monoamine receptor-binding affinity and 5-HT2A and 5-HT2B receptor activation.; All of the benzofurans inhibited NA and 5-HT uptake more than dopamine uptake, similar to methylenedioxymethamphetamine (MDMA) and unlike methamphetamine. All of the benzofurans also released monoamines and interacted with trace amine-associated receptor 1 (TA1 receptor), similar to classic amphetamines. Most benzofurans were partial 5-HT2A receptor agonists similar to MDMA, but also 5-HT2B receptor agonists, unlike MDMA and methamphetamine. The benzodifuran 2C-B-FLY very potently interacted with 5-HT2 receptors and also bound to TA1 receptors.; Despite very similar structures, differences were found in the pharmacological profiles of different benzofurans and compared with their amphetamine analogues. Benzofurans acted as indirect monoamine agonists that interact with transporters similarly to MDMA. The benzofurans also interacted with 5-HT receptors. This pharmacological profile probably results in MDMA-like entactogenic psychoactive properties. However, benzofurans induce 5-HT2B receptor activation associated with heart valve fibrosis. The pharmacology of 2C-B-FLY indicates predominant hallucinogenic properties and a risk for vasoconstriction

Receptor interaction profiles of novel psychoactive tryptamines compared with classic hallucinogens

The present study investigated interactions between the novel psychoactive tryptamines DiPT, 4-OH-DiPT, 4-OH-MET, 5-MeO-AMT, and 5-MeO-MiPT at monoamine receptors and transporters compared with the classic hallucinogens lysergic acid diethylamide (LSD), psilocin, N,N-dimethyltryptamine (DMT), and mescaline. We investigated binding affinities at human monoamine receptors and determined functional serotonin (5-hydroxytryptamine [5-HT]) 5-HT2A and 5-HT2B receptor activation. Binding at and the inhibition of human monoamine uptake transporters and transporter-mediated monoamine release were also determined. All of the novel tryptamines interacted with 5-HT2A receptors and were partial or full 5-HT2A agonists. Binding affinity to the 5-HT2A receptor was lower for all of the tryptamines, including psilocin and DMT, compared with LSD and correlated with the reported psychoactive doses in humans. Several tryptamines, including psilocin, DMT, DiPT, 4-OH-DiPT, and 4-OH-MET, interacted with the serotonin transporter and partially the norepinephrine transporter, similar to 3,4-methylenedioxymethamphetamine but in contrast to LSD and mescaline. LSD but not the tryptamines interacted with adrenergic and dopaminergic receptors. In conclusion, the receptor interaction profiles of the tryptamines predict hallucinogenic effects that are similar to classic serotonergic hallucinogens but also MDMA-like psychoactive properties

Pharmacological profiles of aminoindanes, piperazines, and pipradrol derivatives

Aminoindanes, piperazines, and pipradrol derivatives are novel psychoactive substances found in "Ecstasy" tablets as replacements for 3,4-methylenedioxymethamphetamine (MDMA) or substances sold as "ivory wave." The pharmacology of these MDMA- and methylphenidate-like substances is poorly known. We characterized the pharmacology of the aminoindanes 5,6-methylenedioxy-2-aminoindane (MDAI), 5-iodoaminoindane (5-IAI), and 2-aminoindane (2-AI), the piperazines meta-chlorophenylpiperazine (m-CPP), trifluoromethylphenylpiperazine (TFMPP), and 1-benzylpiperazine (BZP), and the pipradrol derivatives desoxypipradrol (2-diphenylmethylpiperidine [2-DPMP]), diphenylprolinol (diphenyl-2-pyrrolidinemethanol [D2PM]), and methylphenidate. We investigated norepinephrine (NE), dopamine (DA), and serotonin (5-hydroxytryptamine [5-HT]) uptake inhibition using human embryonic kidney 293 (HEK 293) cells that express the respective human monoamine transporters (NET, DAT, and SERT). We also evaluated the drug-induced efflux of NE, DA, and 5-HT from monoamine-preloaded cells and the binding affinity to monoamine transporters and receptors, including trace amine-associated receptor 1 (TAAR1). 5-IAI and MDAI preferentially inhibited the SERT and NET and released 5-HT. 2-AI interacted with the NET. BZP blocked the NET and released DA. m-CPP and TFMPP interacted with the SERT and serotonergic receptors. The pipradrol derivatives were potent and selective catecholamine transporter blockers without substrate releasing properties. BZP, D2PM, and 2-DPMP lacked serotonergic activity and TAAR1 binding, in contrast to the aminoindanes and phenylpiperazines. In summary, all of the substances were monoamine transporter inhibitors, but marked differences were found in their DAT vs. SERT inhibition profiles, release properties, and receptor interactions. The pharmacological profiles of D2PM and 2-DPMP likely predict a high abuse liability

Receptor interaction profiles of novel N-2-methoxybenzyl (NBOMe) derivatives of 2,5-dimethoxy-substituted phenethylamines (2C drugs)

N-2-methoxybenzyl-phenethylamines (NBOMe drugs) are newly used psychoactive substances with poorly defined pharmacological properties. The aim of the present study was to characterize the receptor binding profiles of a series of NBOMe drugs compared with their 2,5-dimethoxy-phenethylamine analogs (2C drugs) and lysergic acid diethylamide (LSD) in vitro.; We investigated the binding affinities of 2C drugs (2C-B, 2C-C, 2C-D, 2C-E, 2C-H, 2C-I, 2C-N, 2C-P, 2C-T-2, 2C-T-4, 2C-T-7, and mescaline), their NBOMe analogs, and LSD at monoamine receptors and determined functional 5-hydroxytryptamine-2A (5-HT2A) and 5-HT2B receptor activation. Binding at and the inhibition of monoamine uptake transporters were also determined. Human cells that were transfected with the respective human receptors or transporters were used (with the exception of trace amine-associated receptor-1 [TAAR1], in which rat/mouse receptors were used).; All of the compounds potently interacted with serotonergic 5-HT2A, 5-HT2B, 5-HT2C receptors and rat TAAR1 (most Ki and EC50: &lt;1 μM). The N-2-methoxybenzyl substitution of 2C drugs increased the binding affinity at serotonergic 5-HT2A, 5-HT2C, adrenergic α1, dopaminergic D1-3, and histaminergic H1 receptors and monoamine transporters but reduced binding to 5-HT1A receptors and TAAR1. As a result, NBOMe drugs were very potent 5-HT2A receptor agonists (EC50: 0.04-0.5 μM) with high 5-HT2A/5-HT1A selectivity and affinity for adrenergic α1 receptors (Ki: 0.3-0.9 μM) and TAAR1 (Ki: 0.06-2.2 μM), similar to LSD, but not dopaminergic D1-3 receptors (most Ki:&gt;1 μM), unlike LSD.; The binding profile of NBOMe drugs predicts strong hallucinogenic effects, similar to LSD, but possibly more stimulant properties because of α1 receptor interactions

Effects of GaAs laser and stretching on muscle contusion in rats

Laser e alongamento são usados para tratar lesões musculares. Este estudo objetivou avaliar os efeitos do laser GaAs e alongamento na morfologia do músculo Tibial anterior (TA) após contusão. Trinta e seis ratos (349±23 g) foram divididos em seis grupos (n=6): grupo controle (GC); grupo lesão e laser (GLL); grupo lesão e alongamento (GLA); grupo lesão, laser e alongamento (GLLA) e grupo alongamento (GA). Foi realizada lesão no TA por meio de um aparato de contusão. O tratamento com laser GAAS foi usado com dose de 4,5 J/cm2 durante 32 s, iniciando 48 h após lesão, por 7 dias. Alongamento passivo manual consistiu de 10 repetições de 1 minuto de duração, iniciando no 8º dia, 3 vezes por semana, durante 3 semanas. Após 4 semanas, os ratos foram eutanasiados para retirada do TA para análise de: peso e comprimento musculares, área de secção transversa das fibras musculares (ASTFM), número de sarcômeros em série (NSS), comprimento dos sarcômeros e porcentagem de tecido conjuntivo. A comparação entre os grupos deu-se por meio da ANOVA e post hoc Tukey, com nível de significância ≤ 0,05. O número de sarcômeros em série do GLLS foi maior que o GLS. O comprimento dos sarcômeros no GLA foi superior ao GLL, GLLA e GA. No GA houve aumento do NSS comparado com o GC, enquanto a porcentagem de tecido conjuntivo do GA diminuiu em comparação com o GLLA. Assim, a sarcomerogênese dos músculos lesionados foi aumentada pelo uso do laser, alongamento e pela associação destes. O alongamento foi suficiente para aumentar o NSS em músculos intactos.Láser y estiramiento son utilizados para sanar lesiones musculares. Este estudio tuvo como objetivo evaluar los efectos del láser GaAs y del estiramiento en la morfología del músculo tibial anterior (TA) después de contusión. Treinta y seis ratones (349±23 g) fueron divididos en seis grupos (n=6): grupo control (GC); grupo lesión y láser (GLL); grupo lesión y estiramiento (GLE); grupo lesión, laser y estiramiento (GLLA) y grupo estiramiento (GA). Se realizó lesión en el TA mediante un aparato de contusión. Se utilizó el tratamiento con láser GaAs con dosis de 4,5 J/cm2 durante 32 s, iniciado 48 h después de la lesión, durante 7 días. El estiramiento pasivo manual consistió de 10 repeticiones de 1 minuto de duración, iniciado en el 8º día, 3 veces por semana, durante 3 semanas. Después de 4 semanas, los ratones sufrieron eutanasia para la retirada del TA para análisis de: peso y longitud musculares, área de sección trasversa de las fibras musculares (ASTFM), número de sarcómeros en serie (NSS), longitud de los sarcómeros y porcentaje de tejido conjuntivo. La comparación entre los grupos ocurrió mediante la Anova y post hoc Tukey, con nivel de significancia de ≤0,05. El número de sarcómeros en serie del GLLS fue mayor que el GLS. La longitud de los sarcómeros en el GLA fue superior al GLL, GLLA y GA. En el GA hubo aumento del NSS en comparación con el GC, mientras el porcentaje del tejido conjuntivo del GA disminuyó en comparación al del GLLA. Así, la sarcomerogénesis de los músculos lesionados fue aumentada por el uso del láser, estiramiento y por su asociación. El estiramiento fue suficiente para aumentar el NSS en músculos intactos.Laser and stretching are used to treat skeletal muscle injuries. This study aimed to evaluate the effects of GaAs laser and stretching in the morphology of the tibialis anterior (TA) muscle after contusion. Thirty-six male rats (349±23g) were divided into six groups (n=6): control group (CG); lesion group (LG); lesion and laser group (LLG); lesion and stretching group (LSG); lesion, laser and stretching group (LLSG); and stretching group (SG). TA was wounded by a contusion apparatus. We used GaAs laser 4.5 J/cm2 dose for 32 s each, beginning 48 h after lesion, for 7 days, once a day. Manual passive stretching was applied by 10 repetitions for 1 minute, initiating on the 8th day, once a day, 3 times a week, during 3 weeks. After 4 weeks, rats were euthanized and we analyzed: muscle weight and length, cross sectional area of muscle fibers (CSAMF), serial sarcomere number (SSN), sarcomere length, and percentage of connective tissue. Comparisons among groups were made by ANOVA and post hoc Tukey tests, with the significance level set at ≤ 0.05. The serial sarcomere number of LLSG was higher than LSG. The sarcomere length of LSG was superior to LLG, LLSG, and SG. SG increased SSN compared to CG, while the percentage of connective tissue of SG decreased in comparison to LLSG. Thus, the sarcomerogenesis of injured muscles was enhanced by laser therapy, stretching, and association of both. The stretching protocol was enough to increase SSN of intact muscles

Protocol for a Wnt reporter assay to measure its activity in human neural stem cells derived from induced pluripotent stem cells

The canonical Wnt signaling is an essential pathway that regulates cellular proliferation, maturation, and differentiation during neurodevelopment and maintenance of adult tissue homeostasis. This pathway has been implicated with the pathophysiology of neuropsychiatric disorders and was associated with cognitive processes, such as learning and memory. However, the molecular investigation of the Wnt signaling in functional human neural cell lines might be challenging since brain biopsies are not possible and animal models may not represent the polygenic profile of some neurological and neurodevelopmental disorders. In this context, using induced pluripotent stem cells (iPSCs) has become a powerful tool to model disorders that affect the Central Nervous System (CNS) in vitro, by maintaining patients' genetic backgrounds. In this method paper, we report the development of a virus-free Wnt reporter assay in neural stem cells (NSCs) derived from human iPSCs from two healthy individuals, by using a vector containing a reporter gene (luc2P) under the control of a TCF/LEF (T-cell factor/lymphoid enhancer factor) responsive element. Dose-response curve analysis from this luciferase-based method might be useful when testing the activity of the Wnt signaling pathway after agonists (e.g. Wnt3a) or antagonists (e.g. DKK1) administration, comparing activity between cases and controls in distinct disorders. Using such a reporter assay method may help to elucidate whether neurological or neurodevelopmental mental disorders show alterations in this pathway, and testing whether targeted treatment may reverse these. Therefore, our established assay aims to help researchers on the functional and molecular investigation of the Wnt pathway in patient-specific cell types comprising several neuropsychiatric disorders

Generation of induced pluripotent stem cells from two ADHD patients and two healthy controls

Attention-deficit hyperactivity disorder is a neurodevelopmental disorder which prevalence has been increasing in the past decades, affecting more than 5% of children, adolescents worldwide. Regarding etiology, polygenic, environmental factors contribute to the occurrence of ADHD even though molecular mechanisms are not known. Understanding the pathophysiology in patient-specific cells is crucial for the discovery of potential predictive markers, the establishment of new therapeutic targets. In this study, we generated further lines from ADHD patients, healthy controls using Sendai virus transduction, which may help on the study of ADHD at the molecular, cellular levels