Approaches to evaluate the pharmacology of new psychoactive substances

In the last decade, the rapid emergence of a group of compounds known as novel psychoactive substances (NPS) has become a worrying problem due to their ambiguous legal status, their easy availability, their extensive consumption and their severe adverse effects. NPS are compounds designed to mimic existing established recreational drugs. The most commonly clinically encountered NPS are stimulants (such as mephedrone) and cannabinoids (such as “spice”) and our knowledge of their pharmaco-toxicological profile is very limited. The evaluation of hundreds of substances in a short time period is a challenge for public health and drug policies globally. This thesis aims to contribute to the pharmacological evaluation of NPS by developing a targeted metabolomics approach (on neurotransmitters and steroids) applied to brain tissue as well as in plasma and urine as a tool to predict the pharmacological profile of NPS, circumventing limitations of the current evaluation approach. We focused on specific NPS (i.e. cathinones, synthetic cannabinoids) to demonstrate the proof of principle of the methodological approach developed. By using chromatographic techniques coupled to tandem mass spectrometry, the quantification of endogenous and exogenous compounds, presumably altered after drug intake, was achieved in different biological matrices. We studied more in depth the metabolic clearance of mephedrone in humans focusing on its pharmacokinetics and pharmacogenetics aspects regulating its disposition as well as the interactions with its pharmacodynamics in a set of multi-dose randomized double-blind clinical trials. The pharmacological predictions for novel drugs was accomplished by quantifying the neuro-metabolomics fingerprint alterations of NPS compared to those observed after the intake of classical drugs of abuse. In brief, this work has contributed to describe the human disposition of mephedrone and it also highlight the potential of using targeted metabolomics as a tool to predict the pharmacological profile of NPS.  En l'última dècada, la ràpida aparició d'un grup de compostos coneguts com a noves substàncies psicoactives (NPS) s'ha convertit en un problema preocupant a causa del seu estatus ambigu, el seva fàcil accés, el seu consum extens i els seus efectes adversos greus. Les NPS són compostos dissenyats per imitar les drogues d’abús ja establertes. Les NPS més reportades a nivell clínic són els estimulants (com la mefedrona) i els cannabinoides (com ara "spiece") i el nostre coneixement del seu perfil farmaco-toxicològic és molt limitat. L'avaluació de centenars de substàncies en un curt període de temps és un repte per a la salut pública i les polítiques de drogues a nivell mundial. Aquesta tesi pretén contribuir a l'avaluació farmacològica de les NPS desenvolupant un enfocament metabòlic dirigit (en neurotransmissors i esteroides) aplicat al teixit cerebral, al plasma i a l'orina com a eina per predir el perfil farmacològic de les NPS, evitant les limitacions de l’avaluació actual. Ens hem centrat en NPS específiques (com les catinones i els cannabinoides sintètics) per demostrar la prova del principi de l'enfocament metodològic desenvolupat. Mitjançant l'ús de tècniques cromatogràfiques acoblades a l'espectrometria de masses en tàndem, es va aconseguir la quantificació de compostos endògens i exògens, presumptament alterats després de la ingesta de drogues en diferents matrius biològiques. Es va estudiar més a fons l'eliminació metabòlica de la mefedrona en humans, centrant-nos en els seus aspectes farmacocinètics i farmacogenètics que regulaven la seva depuració, així com les interaccions amb la seva farmacodinàmica en un conjunt d'assaigs clínics aleatoritzats de dosis múltiples. Les prediccions farmacològiques de nous fàrmacs es van assolir quantificant les alteracions neuro-metabolòmiques de les NPS en comparació amb les observades després de la ingesta de drogues d’abús clàssiques. En resum, aquest treball ha contribuït a descriure la disposició humana de la mefedrona i també destaca el potencial d'utilitzar metabolòmica dirigida com a eina per predir el perfil farmacològic de NPS

A Comparison of Acute Pharmacological Effects of Methylone and MDMA Administration in Humans and Oral Fluid Concentrations as Biomarkers of Exposure

Methylone is a synthetic cathinone that is usually used as a substitute for conventional psychostimulants, such as MDMA. Chemically, methylone is considered the β-keto analogue of MDMA, with which it presumably shares similar pharmacological effects. To date, the available data about the human pharmacology of methylone in humans are very scarce and are mainly derived from user experiences, published in internet forums or intoxication reports. Thus, an observational-naturalistic study was conducted to evaluate the acute pharmacological effects and determine biomarkers of exposure in oral fluid of methylone after oral self-administration in comparison to MDMA. Methylone induced the prototypical psychostimulant and empathogenic effects commonly associated with MDMA, although they were of lower intensity. Oral fluid concentrations of methylone can be considered a suitable biomarker of acute exposure, and oral fluid has been proven to be a useful biological matrix of detection. Considered the β-keto analogue of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), 3,4-Methylenedioxymethcathinone (methylone) is a synthetic cathinone. Over the years, methylone has been used as a substitute for conventional psychostimulants, such as MDMA. To date, little is known about the human pharmacology of methylone; the only available information has been provided by surveys or published intoxication reports. In the present observational-naturalistic study, we evaluate the acute subjective and physiological effects of methylone after oral self-administration in comparison to MDMA in healthy poly-drug users. Fourteen participants (10 males, 4 females) selected their single oral doses of methylone from 100 to 300 mg (n = 8, mean dose 187.5 mg) or MDMA from 75 to 100 mg (n = 6, mean dose 87.5 mg) based on their experience. Study variables were assessed at 0, 1, 2, and 4 h (h) and included vital signs (non-invasive blood pressure, heart rate, cutaneous temperature) and subjective effects using visual analogue scales (VAS), the 49-item Addiction Research Centre Inventory (ARCI) short form, and the Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) questionnaire. Additionally, oral fluid concentrations of methylone and MDMA were determined. Acute pharmacological effects produced by methylone followed the prototypical psychostimulant and empathogenic profile associated with MDMA, although they were less intense. Methylone concentrations in oral fluid can be considered a useful biomarker to detect acute exposure in oral fluid. Oral fluid concentrations of MDMA and methylone peaked at 2 h and concentrations of MDMA were in the range of those previously described in controlled studies. Our results demonstrate that the potential abuse liability of methylone is similar to that of MDMA in recreational subjects

Acute Pharmacological Effects of Oral and Intranasal Mephedrone : an observational study in humans

Mephedrone (4-methylmethcathinone) is a synthetic cathinone with psychostimulant properties which remains one of the most popular new psychoactive substances (NPS). It is frequently used orally and/or intranasally. To date, no studies have evaluated the acute effects and pharmacokinetics after self-administration of mephedrone orally (ingestion) and intranasally (insufflation) in naturalistic conditions. An observational study was conducted to assess and compare the acute pharmacological effects, as well as the oral fluid (saliva) concentrations of mephedrone self-administered orally and intranasally. Ten healthy experienced drug users (4 females and 6 males) self-administered a single dose of mephedrone, orally (n = 5, 100-200 mg; mean 150 mg) or intranasally (n = 5, 50-100 mg, mean 70 mg). Vital signs (blood pressure, heart rate, and cutaneous temperature) were measured at baseline (0), 1, 2, and 4 h after self-administration. Each participant completed subjective effects questionnaires: A set of Visual Analogue Scales (VAS), the 49-item Addiction Research Centre Inventory (ARCI), and Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) at baseline, 1, 2, and 4 h after self-administration. Oral fluid and urine were collected during 4 h. Both routes of mephedrone self-administration enhanced ratings of euphoria and well-being effects and increased cardiovascular effects in humans. Although it was at times assessed that the oral route produced greater and larger effects than the intranasal one, concentrations of mephedrone in oral fluid and also the total amount of mephedrone and metabolites in urine showed of mephedrone are considerably higher when self-administered intranasally in comparison to orally. Controlled clinical trials are needed to confirm our observational results

Dose-response pharmacological study of mephedrone and its metabolites: pharmacokinetics, serotoninergic effects, and impact of CYP2D6 genetic variation

Mephedrone (MEPH), the most widely consumed synthetic cathinone, has been associated with acute toxicity episodes. The aim of this report was to study its metabolic disposition and the impact of genetic variation of CYP2D6 on MEPH metabolism, in a dose range compatible with its recreational use. A randomized, crossover, phase I clinical trial was performed. Subjects received 50 and 100 mg (n = 3) and 150 and 200 mg (n = 6) of mephedrone and were genetically and phenotypically characterized for the CYP2D6 allelic variation. Our results showed a linear kinetics of mephedrone at the dose range assayed: plasma concentrations, cardiovascular and subjective effects, and blood serotonin concentrations all correlated in a dose-dependent manner. Mephedrone metabolic disposition is mediated by CYP2D6. Mephedrone pharmacology presented a linear dose-dependence within the range of doses tested. The metabolism of mephedrone by CYP2D6 implies that recreational users with no or low CYP2D6 functionality are exposed to unwanted acute toxicity episodes

Quantification of endogenous neurotransmitters and related compounds by liquid chromatography coupled to tandem mass spectrometry

Neurotransmitters are signaling molecules, playing key roles in neuronal communications in the brain. Drug induced changes in neurotransmitters and other brain metabolite concentration may be used to characterize drugs according to their targeted metabolomics profile. Here, we report the development and validation of a straightforward liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 16 endogenous small polar compounds in rat plasma and brain homogenates. The method enables the quantification of the neurotransmitters γ-aminobutyric acid, glutamate, acetylcholine and adenosine, as well as choline, glutamine, acetylcarnitine, carnitine, creatine, creatinine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. After optimizing the sample preparation, chromatographic and spectrometric conditions, the method was successfully validated using the standard addition approach and a hydrophilic interaction chromatography (HILIC) with an amide column. The method was shown to be linear (r > 0.99) as all the compounds were within the ±25% values of intra and inter-day precision and accuracy acceptance. A matrix effect was corrected with the use of 10 isotopically labelled internal standards and the compound stability was evaluated for all compounds. Relevant exaltation of choline (in plasma) and creatinine (in brain) were solved with −20 °C conditions. The applicability of the method was tested by evaluating brain alterations in the concentrations of neurotransmitters and related compounds after the administration of two psychostimulant drugs of abuse (cocaine and methylenedioxypyrovalerone) to rats. A neuro-metabolic fingerprint of each drug was obtained that reflected their pharmacological profile. Altogether, this methodology presents a valuable targeted metabolomics tool for basic and clinical research studies.This work was supported by the European Commission (Drugs Policy Initiatives, Justice Programme 2014–2020, contract n°. HOME/2014/JDRU/AG/DRUG/7082, Predicting Risk of Emerging Drugs with In silico and Clinical Toxicology). JRM is acknowledged for the EU's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant No. 712949 (TECNIOspring PLUS) from the Agency for Business Competitiveness of the Government of Catalonia. The Spanish Health National System is also acknowledged for the contract of OJP (CPII16/00027

Quantification of endogenous neurotransmitters and related compounds by liquid chromatography coupled to tandem mass spectrometry

Neurotransmitters are signaling molecules, playing key roles in neuronal communications in the brain. Drug induced changes in neurotransmitters and other brain metabolite concentration may be used to characterize drugs according to their targeted metabolomics profile. Here, we report the development and validation of a straightforward liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 16 endogenous small polar compounds in rat plasma and brain homogenates. The method enables the quantification of the neurotransmitters γ-aminobutyric acid, glutamate, acetylcholine and adenosine, as well as choline, glutamine, acetylcarnitine, carnitine, creatine, creatinine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. After optimizing the sample preparation, chromatographic and spectrometric conditions, the method was successfully validated using the standard addition approach and a hydrophilic interaction chromatography (HILIC) with an amide column. The method was shown to be linear (r > 0.99) as all the compounds were within the ±25% values of intra and inter-day precision and accuracy acceptance. A matrix effect was corrected with the use of 10 isotopically labelled internal standards and the compound stability was evaluated for all compounds. Relevant exaltation of choline (in plasma) and creatinine (in brain) were solved with −20 °C conditions. The applicability of the method was tested by evaluating brain alterations in the concentrations of neurotransmitters and related compounds after the administration of two psychostimulant drugs of abuse (cocaine and methylenedioxypyrovalerone) to rats. A neuro-metabolic fingerprint of each drug was obtained that reflected their pharmacological profile. Altogether, this methodology presents a valuable targeted metabolomics tool for basic and clinical research studies.This work was supported by the European Commission (Drugs Policy Initiatives, Justice Programme 2014–2020, contract n°. HOME/2014/JDRU/AG/DRUG/7082, Predicting Risk of Emerging Drugs with In silico and Clinical Toxicology). JRM is acknowledged for the EU's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant No. 712949 (TECNIOspring PLUS) from the Agency for Business Competitiveness of the Government of Catalonia. The Spanish Health National System is also acknowledged for the contract of OJP (CPII16/00027

GC-MS quantification method for mephedrone in plasma and urine: application to human pharmacokinetics

Increasing consumption has been observed among young people of new psychoactive substances, including synthetic cathinone derivatives. The most well known of these is mephedrone whose use has been related to acute intoxication and fatality. Several methods able to detect mephedrone have been reported, although to date, none have been applied to human pharmacokinetic studies in a controlled setting. We developed a gas chromatography-mass spectrometry technique for mephedrone quantification in human plasma and urine. Plasma after deproteinization and urine were submitted to a liquid-liquid extraction and derivatization of the extract with MSTFA prior to analysis. Calibration curves covered concentration ranges in plasma between 5 and 300 ng/mL and in urine between 20 and 1,500 ng/mL. The method has been successfully applied to biological samples obtained from a pilot clinical trial intended to evaluate the human pharmacology of mephedrone and its relative bioavailability and pharmacokinetics. Six healthy males were administered 150 mg of mephedrone by the oral route in a randomized, double-blind, cross-over controlled trial. Peak plasma concentration (Cmax = 122.6 ± 32.9 ng/mL) was reached at 1 hour (0.5-2 h) post-drug administration. Mephedrone showed a rapid elimination half-life (t1/2 = 2.2 h) compared to other psychostimulants. Less than 15% of the dose was excreted in urine as a free-form. Mephedrone concentrations displayed a relevant inter-subject variability

Peripheral endocannabinoid concentrations are not associated with verbal memory impairment during MDMA intoxication

BACKGROUND: Preclinical data have suggested involvement of the endocannabinoid (eCB) system in MDMA-induced memory impairment. Clinical research has shown that blockade of the 5-HT2 receptor nulls memory impairment during MDMA intoxication. Interestingly, studies have demonstrated that the eCB and the 5-HT system interact. It was hypothesized that MDMA would cause an increase in eCB concentrations together with a decrease in memory performance, and that combining MDMA with a 5-HT2 receptor blocker ketanserin would lead to a counteraction of the MDMA effects on eCB concentrations and memory. METHODS: Twenty healthy recreational polydrug users entered a double-blind placebo-controlled within-subject study. Participants received a pre-treatment (ketanserin 40 mg, placebo) followed 30 min later by a treatment (MDMA 75 mg, placebo). Verbal memory was tested by means of a 30-word learning test. Endocannabinoid concentrations (anandamide (2-AG); N-arachidonylethanolamine (AEA)) were assessed in blood at baseline, before (90 min post-treatment) and after cognitive tests (150 min post-treatment). RESULTS: Findings showed that MDMA impaired memory 90 min post-treatment in the word learning task. This effect was a replication of previous studies using the same dose of MDMA (75 mg) and the same learning paradigm. Contrary to our hypothesis, MDMA did not affect eCB concentrations, nor did ketanserin block MDMA-induced memory impairment. Ketanserin caused an increase in AEA concentrations, 180 min after administration. CONCLUSION: Current findings suggest that peripherally measured endocannabinoids are not associated with the verbal memory deficit during MDMA intoxication. TRIAL REGISTRATION NUMBER: NTR3691

GC-MS quantification method for mephedrone in plasma and urine: application to human pharmacokinetics

Increasing consumption has been observed among young people of new psychoactive substances, including synthetic cathinone derivatives. The most well known of these is mephedrone whose use has been related to acute intoxication and fatality. Several methods able to detect mephedrone have been reported, although to date, none have been applied to human pharmacokinetic studies in a controlled setting. We developed a gas chromatography-mass spectrometry technique for mephedrone quantification in human plasma and urine. Plasma after deproteinization and urine were submitted to a liquid-liquid extraction and derivatization of the extract with MSTFA prior to analysis. Calibration curves covered concentration ranges in plasma between 5 and 300 ng/mL and in urine between 20 and 1,500 ng/mL. The method has been successfully applied to biological samples obtained from a pilot clinical trial intended to evaluate the human pharmacology of mephedrone and its relative bioavailability and pharmacokinetics. Six healthy males were administered 150 mg of mephedrone by the oral route in a randomized, double-blind, cross-over controlled trial. Peak plasma concentration (Cmax = 122.6 ± 32.9 ng/mL) was reached at 1 hour (0.5-2 h) post-drug administration. Mephedrone showed a rapid elimination half-life (t1/2 = 2.2 h) compared to other psychostimulants. Less than 15% of the dose was excreted in urine as a free-form. Mephedrone concentrations displayed a relevant inter-subject variability

Acute pharmacological effects of oral and intranasal mephedrone: an observational study in humans

Mephedrone (4-methylmethcathinone) is a synthetic cathinone with psychostimulant properties which remains one of the most popular new psychoactive substances (NPS). It is frequently used orally and/or intranasally. To date, no studies have evaluated the acute effects and pharmacokinetics after self-administration of mephedrone orally (ingestion) and intranasally (insufflation) in naturalistic conditions. An observational study was conducted to assess and compare the acute pharmacological effects, as well as the oral fluid (saliva) concentrations of mephedrone self-administered orally and intranasally. Ten healthy experienced drug users (4 females and 6 males) self-administered a single dose of mephedrone, orally (n = 5, 100-200 mg; mean 150 mg) or intranasally (n = 5, 50-100 mg, mean 70 mg). Vital signs (blood pressure, heart rate, and cutaneous temperature) were measured at baseline (0), 1, 2, and 4 h after self-administration. Each participant completed subjective effects questionnaires: A set of Visual Analogue Scales (VAS), the 49-item Addiction Research Centre Inventory (ARCI), and Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) at baseline, 1, 2, and 4 h after self-administration. Oral fluid and urine were collected during 4 h. Both routes of mephedrone self-administration enhanced ratings of euphoria and well-being effects and increased cardiovascular effects in humans. Although it was at times assessed that the oral route produced greater and larger effects than the intranasal one, concentrations of mephedrone in oral fluid and also the total amount of mephedrone and metabolites in urine showed that concentrations of mephedrone are considerably higher when self-administered intranasally in comparison to orally. Controlled clinical trials are needed to confirm our observational results