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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: Neuropharmacology. 2021 Sep 20;199:108800. doi: 10.1016/j.neuropharm.2021.108800

Pharmacokinetics and Pharmacodynamics of the Synthetic Cannabinoid, 5F-MDMB-PICA, in Male Rats

Alex J Krotulski 1,3, Nancy Garibay 2, Donna Walther 2, Sara E Walton 1,3, Amanda LA Mohr 1, Barry K Logan 1,3,4, Michael H Baumann 2
PMCID: PMC8511335  NIHMSID: NIHMS1743204  PMID: 34547333

Abstract

5F-MDMB-PICA is a popular synthetic cannabinoid associated with analytically confirmed intoxications. In vitro studies show 5F-MDMB-PICA is a potent cannabinoid-1 receptor (CB1) agonist, but little information is available about in vivo pharmacokinetics and pharmacodynamics. To this end, the present study had three aims: 1) to develop a validated method for detection of 5F-MDMB-PICA and its metabolites in rat plasma, 2) to utilize the method for investigating pharmacokinetics of 5F-MDMB-PICA in rats, and 3) to relate 5F-MDMB-PICA pharmacokinetics to pharmacodynamic effects. 5F-MDMB-PICA and its metabolites were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) and method validation followed forensic standards. Male Sprague-Dawley rats bearing surgically implanted jugular catheters and subcutaneous (s.c.) temperature transponders received 5F-MDMB-PICA (50, 100, or 200 μg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, 240, and 480 min post-injection, and plasma was assayed using LC-MS/MS. At each blood draw, body temperature, and catalepsy scores were recorded. Maximum plasma concentrations (Cmax) of 5F-MDMB-PICA rose linearly with increasing dose (1.72-6.20 ng/mL), and plasma half-life (t1/2) ranged from 400-1000 min. 5F-MDMB-PICA-3,3-dimethylbutanoic acid and 5OH-MDMB-PICA were the only metabolites detected, and plasma concentrations were much lower than the parent drug. 5F-MDMB-PICA induced robust hypothermia and catalepsy-like symptoms that were significantly correlated with concentrations of 5F-MDMB-PICA. Radioligand binding in rat brain membranes revealed 5F-MDMB-PICA displays high affinity for CB1 (IC50=2 nM) while metabolites do not. In summary, 5F-MDMB-PICA is a potent CB1 agonist in rats whose pharmacodynamic effects are related to circulating concentrations of the parent drug and not its metabolites.

Keywords: 5F-MDMB-PICA, Body Temperature, Cannabinoid-1 Receptor, Catalepsy, Forensic Toxicology, Metabolites, Method Validation, Quantitation, Synthetic Cannabinoid

1. Introduction

The constant influx of new drugs of abuse, especially the vast number of novel psychoactive substances (NPS), presents challenges for the research efforts of forensic chemists, pharmacologists, and toxicologists (Madras, 2017; Peacock et al., 2019). As new substances appear on non-medical (i.e., recreational) drug markets, limited information is available about their molecular mechanisms of action, pharmacological effects, and toxic potential, which can complicate interpretation of results from human casework. The uncertainties about NPS are compounded by their rapid rate of emergence and disappearance from drug markets, often times so quickly that in-depth pharmacology and toxicology studies cannot be completed (Chung et al., 2014; Halter et al., 2020; Hudson and Ramsey, 2011; Krotulski et al., 2020). As researchers struggle to keep pace with the evolving array of NPS, new methods for quickly characterizing in vitro pharmacology are being developed, and efforts to study the pharmacokinetics and pharmacodynamics of NPS in animal models are needed to increase the body of knowledge about these substances.

Synthetic cannabinoids represent a large and diverse class of NPS, and their involvement in forensic and clinical casework continues to raise concerns. As described in the literature, synthetic cannabinoids are associated with numerous adverse effects, including confusion, agitation, sedation, vomiting, psychosis, seizures, tachycardia, and cardiotoxicity, sometimes leading to death (Gerostamoulos et al., 2015; Hvozdovich et al., 2020; Labay et al., 2016; Seely et al., 2012). As generations of new cannabinoids evolve, scientists have noted a trend for increased binding affinity and functional efficacy at cannabinoid-1 receptors (CB1), which represents a dangerous combination of effects for recreational drug users (Antonides et al., 2019; Banister et al., 2016, 2015; Pinson et al., 2020). In 2016, a new synthetic cannabinoid, 5F-MDMB-PICA (also known as methyl 2-[[1-(5-fluoropentyl)indole-3-carbonyl]amino]-3,3-dimethyl-butanoate, 5F-MDMB-2201, or MDMB-2201), emerged among the drug supply in Europe (Mogler et al., 2018; Risseeuw et al., 2017). At the time, only one study was available regarding this new substance, which identified the drug as a potent CB1 agonist (Banister et al., 2016). Two years later, 5F-MDMB-PICA emerged in the United States of America (USA), and this compound became the predominant synthetic cannabinoid detected in confiscated drug products and human casework during much of 2018 and 2019 (NPS Discovery, 2019a). A number of studies carried out in transfected cell systems have confirmed that 5F-MDMB-PICA is a potent CB1 agonist (Noble et al., 2019; Sachdev et al., 2019), but few investigations have examined the in vivo effects of the drug (Musa et al., 2020).

Research aimed at determining the pharmacokinetics of NPS provides key information about how organisms respond to drug administration. Forensic casework can provide data about the concentrations of cannabinoids in discrete samples from human users, but little information about drug pharmacokinetics can be gleaned from single samples. Preclinical data from laboratory animals offer a more complete picture of the biological effects of synthetic cannabinoids, however, these reports often come long after the substance has faded from the market (Bilel et al., 2019; Carlier et al., 2018; Kevin et al., 2017; Pinson et al., 2020; Toennes et al., 2017). Given these considerations, we sought to characterize the pharmacokinetics and pharmacodynamics of 5F-MDMB-PICA, which remains a popular synthetic cannabinoid in the USA (“NPS Discovery,” 2019b). First, we developed a validated analytical method to quantitate 5F-MDMB-PICA and its major metabolites in rat plasma. Mogler et al. (Mogler et al., 2018) and Truver et al. (Truver et al., 2020) previously identified 12 and 22 metabolites of 5F-MDMB-PICA in vitro, respectively. Using this information, paired with data and knowledge about synthetic cannabinoid metabolites found in vivo and their prevalence, the list was narrowed down to five suspected major metabolites of 5F-MDMB-PICA (see Figure 1 for chemical structures), and we had these metabolites custom-synthesized for our study [www.caymanchem.com]. We then used the analytical method to examine the pharmacokinetics of 5F-MDMB-PICA and its metabolites in male rats receiving subcutaneous (s.c.) administration of the drug. Rats in our study were fitted with surgically implanted jugular catheters and s.c. temperature transponders, which allowed for simultaneous determination of plasma analyte concentrations and pharmacodynamic endpoints in the same subjects (Carlier et al., 2018). We hypothesized that 5F-MDMB-PICA administration would induce hypothermia and catalepsy in rats related to circulating concentrations of the parent drug or active metabolite(s).

Figure 1.

Figure 1.

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Metabolism of 5F-MDMB-PICA as determined in vitro.

2. Materials and Methods

2.1. Chemical and reagents

5F-MDMB-PICA, 5F-MDMB-PICA 3,3-dimethylbutanoic acid, 5OH-MDMB-PICA, 6’OH-5F-MDMB-PICA, 2COOH-MDMB-PICA, 4OH-5F-MDMB-PICA 3,3-dimethylbutanoic acid, 5F-MDMB-PICA-d5, and 5F-MDMB-PICA 3,3-dimethylbutanoic acid-d5 were custom synthesized and purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All materials and reagents used for liquid chromatography and mass spectrometry analyses were of LC/MS grade purity. Water, hexane, ethyl acetate, methyl tert-butyl ether (MTBE), methanol, and other necessary solvents were purchased from Honeywell (Charlotte, NC, USA). Formic acid ampoules (1 mL) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Phosphoric acid was purchased from BDH VWR (Radnor, PA, USA). Tris pH 7.4, disodium EDTA, MgCl2 × 6 H2O, and bovine serum albumin (BSA, heat shock fraction, protease free, fatty acid free), and sodium metabisulfite were obtained from Sigma-Aldrich, Millipore (St. Louis, MO, USA).

For the drug administration studies, 5F-MDMB-PICA (Cayman Chemical) was dissolved in a solution of DMSO, Tween-80, and saline (5%:5%:90%, v:v:v), which served as the vehicle for injection. DMSO and Tween-80 were obtained from Sigma-Aldrich, Millipore, whereas sterile 0.9% saline was purchased from Hospira (Lake Forest, IL, USA). Briefly, each milligram (mg) of powdered drug was initially dissolved in 50 μL of DMSO using sonication, followed by the addition of 50 μL of Tween-80 with vortexing, and a final addition of 900 μL of 0.9% sterile saline to yield a stock solution of 1 mg/mL 5F-MDMB-PICA in vehicle. Serial dilutions of the 1 mg/mL stock were prepared in vehicle to yield 200, 100, and 50 μg/mL solutions for injection. Doses were administered as μg/kg body weight.

2.2. Analytical methods

Rat plasma samples were prepared alongside calibrators and controls for quantitative analysis. Eight non-zero calibrators ranging from 1 ng/mL to 200 ng/mL were prepared from standard sub-stocks containing 5F-MDMB-PICA and all metabolites purchased. An additional calibrator at 0.5 ng/mL for 5F-MDMB-PICA only was also included. Control samples were prepared at 3, 40, and 150 ng/mL. The matrix volume was 200 μL. Internal standard (20 μL, 0.1 ng/μL) was added to all calibrators, controls, and rat plasma samples.

Samples were prepared using a liquid-liquid extraction (LLE) protocol (Krotulski et al., 2020). One mL of 5% phosphoric acid in water was added and samples were vortexed. Three mL of extraction solvent was then added consisting of hexane, ethyl acetate, and MTBE (80:10:10, v:v:v). Samples were capped and rotated for 15 minutes followed by centrifugation at 4,600 rpm for 10 minutes. The supernatant was removed via freezing the aqueous layer and pouring over to a new test tube. The supernatant was dried to completion under air using a TurboVap at 40°C for 30 minutes. Samples were reconstituted in 200 μL of initial LC conditions and transferred to autosampler vials for analysis.

Instrumental analysis was performed using a Waters Xevo TQ-S micro tandem mass spectrometry coupled with a Waters Acquity I-class ultra-performance liquid chromatograph (Milford, MA, USA). Chromatographic separation was achieved under gradient elution (Supplementary Table 1). Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in methanol. The analytical column was an Agilent InfinityLab Poroshell 120 EC-C18 (3.0 × 100 mm, 2.7 μm). The flow rate was 0.4 mL/min, whereas injection volume was 5 μL. The column temperature was 30 °C. Positive electrospray ionization (ESI+) was used. Multiple reaction monitoring (MRM) was used for mass filtration and detection. Specific MRM parameters and retention times (RT) are listed in Supplementary Table 2.

2.3. Method validation

The analytical method was validated in reference to the AAFS Standards Board (ASB) Standard Practices for Method Validation in Forensic Toxicology (AAFS Standards Board, 2019). Performance characteristics evaluated included: calibration model, precision, accuracy (bias), limits of detection and quantitation, carryover, interferences, matrix effects, and processed sample stability.

The calibration model was assessed using eight or nine non-zero calibration points over five days (metabolites vs. 5F-MDMB-PICA, respectively). The origin was excluded during data analysis. Curve model and weighting were selected based on fitness of data. The coefficient of determination (R2) was required to exceed 0.98 and back calculation of standards was required to not exceed 20%. Precision and accuracy were assessed using low, mid, and high control concentrations over five days. Acceptable precision was less than 20% and acceptable accuracy was within 20% of target concentration. Limits of detection and quantitation were administratively set and analyzed in replicates of six over three days. The limit of detection (LOD) for all compounds was set to 0.1 ng/mL, and the signal was required to exceed a signal-to-noise (S/N) ratio of 3. The limit of quantitation (LOQ) for 5F-MDMB-PICA was assessed at 0.5 ng/mL and for the metabolites at 1 ng/mL. The signal was required to exceed a S/N ratio of 10, and the quantitative value was required to meet accuracy and precision criteria.

Carryover (n=5) was evaluated each day by analyzing two blank injections after a 500 ng/mL sample. The method was assessed for interferences from matrix, analyte, internal standard, and other substances, including therapeutic, abused, and emerging drugs. More than 250 toxicologically relevant and commonly encountered substances were included in this assessment. For carryover and all interference types, the presence of a chromatographic peak was monitored and expected not to exceed 10% area of the LOQ. Matrix effects (n=5), recovery (n=5), and process efficiency (n=5) were assessed by comparison of unextracted standards, processed samples fortified before extraction, and processed samples fortified after extraction (all at 50 ng/mL). Ideally, analytical methods should have minimal matrix effects (<±20%), recover the majority of drug (>80%), and have good process efficiency (>80%); however, if these values were less than anticipated, but had no impact on the performance of the method for its intended use, the parameters were determined to be acceptable. Processed sample stability (n=3 days) was evaluated by re-analyzing previously prepared controls (n=3) against newly prepared calibrators; results were expected to pass accuracy and precision criteria. Unprocessed sample stability was not included as part of this study but has been reported by Krotulski et al. (Krotulski et al., 2021); 5F-MDMB-PICA was found to be stable under frozen conditions (−20 °C) for at least 35 days. During this study, the rat plasma samples were stored at −80 °C for their lifetime prior to analysis.

2.4. Animals and surgery

Male Sprague-Dawley rats (300-400 g) purchased from Envigo (Frederick, MD, USA) were double-housed under conditions of controlled temperature (22 ± 2 °C) and humidity (45% ± 5%), with ad libitum access to food and water. Lights were on between 7:00 a.m. and 7:00 p.m. The Institutional Animal Care and Use Committee of the National Institute on Drug Abuse (NIDA), Intramural Research Program (IRP), approved the animal experiments, and all procedures were carried out in accordance with the National Institutes of Health, Guide for the Care and Use of Laboratory Animals. The approved NIDA IRP Animal Study Proposal was 19-MTMD-5. Vivarium facilities were fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Experiments were designed to minimize the number of animals included in the study.

Rats were anesthetized with intraperitoneal (i.p.) ketamine and xylazine (75 and 5 mg/kg) (Covetrus, Dublin, OH, USA), and venous catheters were surgically implanted into the right jugular vein. Catheters were constructed of Silastic medical grade tubing (Dow Corning, Midland, MI, USA) linked to vinyl tubing (SCI, Lake Havasu City, AZ, USA) using a 23 g stainless steel connector. In brief, the proximal Silastic end of the catheter was advanced to the atrium, while the distal vinyl end was exteriorized on the nape of the neck and plugged with a metal stylet. Immediately after catheter implantation, while rats were still under anesthesia, temperature transponders (model IPTT-300; Bio Medic Data Systems, Seaford, DE, USA) were surgically implanted to allow for noninvasive measurement of body temperature (Elmore and Baumann, 2018). The temperature transponder emits radio frequency signals received by a compatible handheld reader system (DAS-7006/7r; Bio Medic Data Systems). The transponders are cylindrical in shape (14×2 mm) and were implanted s.c. along the midline of the back, posterior to the shoulder blades, via a prepackaged sterile guide needle delivery system. Rats were single housed postoperatively and given at least one week to recover from surgery.

2.5. Animal experiments

Rats were brought into the laboratory in their home cages on the day of an experiment and allowed 1 h to acclimate to the surroundings. Polyethylene extension tubes were attached to 1 mL tuberculin syringes and filled with sterile saline before being connected to the vinyl end of the catheters. Blood sampling, performed by an investigator remote from the animal, was facilitated by threading the extension tubes outside the cage. Catheters were flushed with 0.3 mL of 48 IU/mL heparin saline (Thomas Scientific, Swedesboro, NJ, USA) to facilitate blood withdrawal. To prepare the 5F-MDMB-PICA for injection, a 200 μL aliquot of 1 mg/mL 5F-MDMB-PICA was serially diluted in its DMSO:Tween-80:saline vehicle to yield concentrations of 50, 100, and 200 μg/mL. Groups of rats received s.c. injections of either vehicle (control) or 50, 100, or 200 μg/kg 5F-MDMB-PICA on the lower back between the hips. Doses were administered as μg/kg body weight. Rats were randomly assigned to each dose group. Blood samples (400 μL) were withdrawn via catheters immediately before and at 15, 30, 60, 120, 240, and 480 min after injection.

Samples were collected into 1 mL tuberculin syringes and transferred to 1.5 mL plastic tubes containing 5 μL of 1000 IU/mL heparin (Thomas Scientific) as an anticoagulant and 5 μL of 250 mM sodium metabisulfite as a preservative. Blood was centrifuged at 1000 g for 10 min at 4 °C. Plasma was decanted into cryovials and stored at −80 °C until analysis. To maintain volume and osmotic homeostasis, rats received an equal volume of saline solution via the intravenous catheter after each blood withdrawal.

Body temperature and catalepsy scores were determined prior to each blood withdrawal. Catalepsy was operationally defined by the presence of three specific symptoms: 1] immobility, 2] flattened body posture, and 3] splayed limbs, as described below. The rats that received 5F-MDMB-PICA in our experiments displayed varying degrees of catalepsy-like rigid immobility. We chose to gauge catalepsy by observing the rats, rather than using the more traditional bar test, because we did not want to physically handle the rats or disturb their implanted catheters during the serial blood sampling. Rat behaviors were observed by an experienced rater for 1 min just prior to the measurement of body temperature via the handheld reader system. The behavioral rater was blind to treatment conditions. On each test day, one investigator prepared 5F-MDMB-PICA solutions and administered the drug to rats, whereas another investigator performed the behavioral scoring without knowing the dose being administered to the animal. During the 1 min observation period, catalepsy behaviors were scored based on the presence of 1] immobility (i.e., frozen in place), 2] flattened body posture (i.e., belly pressed to the cage floor), and 3] splayed limbs (i.e., forelimbs and/or hindlimbs extended laterally away from the body) (Elmore and Baumann, 2018). At each time point, each symptom was scored as either 1=absent or 2=present. For each animal, catalepsy scores at each time point were summed, yielding a minimum score of 3 and a maximum possible score of 6.

2.6. Binding experiments

Rat cerebella were obtained from BioIVT (Westbury, NY, USA), and membranes were prepared by homogenizing tissues with a motor driven pestle at 1500 rpm in 20 volumes (w/v) of ice-cold Tris buffer (100 mM Tris, 1.0 mM EDTA, pH adjusted to 9.0). The tissue homogenate was centrifuged at 900 x g for 10 min at 4˚C. The supernatant was saved, and the pellet resuspended with ice-cold Tris buffer pH 9.0. The tissue homogenate was centrifuged at 900 x g for 10 min at 4˚C. Supernatants were combined and centrifuged at 11,500 x g for 25 min at 4˚C. The pellets were resuspended in cold tissue buffer (50 mM Tris pH 7.4, 1 mM EDTA, 3 mM MgCl2 × 6 H2O) to a concentration of 10 mg/mL wet tissue weight. Assays were conducted in 0.5 mL of tissue buffer plus 3 mg/mL Bovine Serum Albumin (heat shock fraction, protease free, fatty acid free) at 30°C for 60 min. Binding assays were initiated by adding 1 mg of tissue homogenate to each tube containing 0.7 nM [3H]SR141716A (specific activity 42 Ci/mmol) (PerkinElmer Health Sciences Inc., Boston, MA, USA) and the test compound. Dose response curves were generated by testing 8 different drug concentrations in triplicate. Nonspecific binding was determined by using 0.01 mM SR141716A (Cayman Chemical). The binding reaction was stopped by adding 1 mL ice-cold wash buffer (0.9% NaCl, 2 mg/mL BSA) then rapid filtration through Whatman GF/B filters (Whatman Inc. Clifton, NJ, USA) presoaked in ice-cold wash buffer. The filters were washed with ice-cold wash buffer (3 × 4 mL) and the bound radioactivity measured by liquid scintillation spectrometry.

2.7. Data analysis and statistics

Pharmacokinetic and pharmacodynamic data were statistically evaluated using GraphPad Prism version 8.02 (GraphPad Software, San Diego, CA, USA). Time-concentration profiles for 5F-MDMB-PICA and its metabolites were evaluated using two-way analysis of variance (dose x time) followed by Dunnett’s multiple comparisons test to compare dose effects at each time point. Time course data were further analyzed using Kinetica software (Thermo Fisher, Philadelphia, PA, USA) to calculate pharmacokinetic constants including maximal concentration (Cmax), time of maximal concentration (Tmax), area-under-the-curve at 8 h (AUC8h), elimination constant (Ke) and plasma half-life (t1/2). Pharmacokinetic constants for each analyte were compared by one-way analysis of variance (dose) followed by Tukey’s tests to determine differences between dose groups. Time course data for body temperature and catalepsy scores were evaluated using two-way analysis of variance (dose x time) followed by Dunnett’s multiple comparisons test. The relationships between plasma concentrations of analytes and body temperature or catalepsy score were assessed using a Pearson’s correlation analysis. Specifically, for each subject, the mean value for 5F-MDMB-PICA concentration across all time points was plotted with respect to the mean temperature or catalepsy score across all times points. For the radioligand binding studies, IC50 values were calculated by nonlinear regression analysis using GraphPad Prism. p < 0.05 was used as the minimum threshold for statistical significance for all comparisons.

3. Results

3.1. Method validation

The described analytical method was successfully validated and acceptable for its intended use in quantitation of 5F-MDMB-PICA, 5F-MDMB-PICA 3,3-dimethylbutanoic acid, 5OH-MDMB-PICA, 2COOH-MDMB-PICA, and 4OH-5F-MDMB-PICA 3,3-dimethylbutanoic acid. Chromatographic separation is shown in Figure 2. 6’OH-5F-MDMB-PICA failed criteria for a quantitative method and was assessed for qualitative purposes only.

Figure 2.

Figure 2.

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Chromatographic separation achieved and relative abundance for 5F-MDMB-PICA and its metabolites at 1 ng/mL (the metabolite LOQ). Peaks present in the chromatogram correspond to [R to L] a) 5F-MDMB-PICA, b) 5F-MDMB-PICA 3,3-Dimethylbutanoic Acid, c) 5OH-MDMB-PICA, d) 2COOH-MDMB-PICA, and e) 4OH-5F-MDMB-PICA 3,3-Dimethylbutanoic Acid.

Quantitation parameters selected and accompanying results are shown in Supplementary Table 3. After assessment of data using varying setpoints, a quadratic calibration model and 1/x weighting were selected for all analytes. The coefficient of determination (R2) for all analytes was greater than 0.999 and all calibrators were within 20% of target. The y-intercept for all analytes was less than 0.34 and deemed acceptable. Measured concentration and S/N ratios for all LOQ samples were acceptable; S/N ratios for all LOD samples were acceptable.

Validation results for accuracy, precision, recovery, matrix effect, process efficient, and processed stability are shown in Supplementary Table 4. All accuracy calculations were within 20% of the target concentration. All precision calculations were less than 20%. Overall, the method was highly accurate and precise. Analyte recovery ranged from 33.0% to 84.5%, however, no impact on performance or quantitative abilities was observed, as well as minimal variability (<7% CV). Lower recovery may be expected due to the varying chemistries of parent vs. metabolites and the static pH of the LLE. The method was not impacted by gross matrix effects; slight ion suppression was observed which also was not variable (<9% CV). Processed sample stability showed all analytes were stable for at least six days (except for 5OH-MDMB-PICA which was stable for 5 days). No interferences from matrix, drug, internal standard, or commonly encountered substances were observed (i.e., no identifiable chromatographic peak). The method was free from carryover (i.e., no identifiable chromatographic peak).

3.2. Pharmacokinetics

5F-MDMB-PICA was detected in all plasma samples collected after drug administration, whereas no drug was detected in the samples from vehicle control rats. Figure 3A depicts the time-concentration profiles for 5F-MDMB-PICA in plasma, whereas Table 1 shows pharmacokinetic constants calculated from the time-course data. Plasma concentrations of 5F-MDMB-PICA were significantly affected by dose (F[2,105]=232.7, p<0.0001) and time (F[6,105]=52.39, p<0.0001), with a significant dose x time interaction (F[12,105]=7.166, p<0.0001). In general, concentrations of 5F-MDMB-PICA rose linearly with the dose administered, and the drug was eliminated slowly. Dunnett’s post hoc tests revealed that concentrations of 5F-MDMB-PICA after the 100 and 200 μg/kg doses were significantly greater than those after the 50 μg/kg dose for all time points.

Figure 3.

Figure 3.

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Time-concentration profiles for plasma 5F-MDMB-PICA (Panel A) and its metabolite 5F-MDMB-PICA 3,3-dimethylbutanoic acid (Panel B) in rats. Male Sprague Dawley rats received s.c. injection of 5F-MDMB-PICA (50, 100, or 200 μg/kg), and serial blood samples (0.4 mL) were withdrawn immediately before and at 15, 30, 60, 120, 240, and 480 min post-injection. Plasma was assayed for 5F-MDMB-PICA and its metabolites as described in Materials and Methods. Data are ng/mL concentrations expressed as mean ± SEM for N=6 rats per group. Vertical lines through the symbols represent SEM; when no vertical line is visible, the SEM is within the symbol. Solid symbols indicate significant effects compared to the 50 μg/kg dose group at a given time point (Dunnett’s p<0.05)

Table 1.

Pharmacokinetic constants for plasma 5F-MDMB-PICA after systemic administration of the drug to male rats

Dose (μg/kg, s.c.) Cmax (ng/mL) Tmax (min) AUC8h (min * ng/mL) Ke (1/min) t1/2 (min)
50 μg/kg 1.72±0.15 42.5±7.48 516.7±31.7 0.0023±0.0004 413.5±96.3
100 μg/kg 3.08±0.19 45.0±6.12 1021.8±40.0* 0.0018±0.0005 621.0±184.8
200 μg/kg 6.20±1.06* 170.0±69.48 2263.5±133.8*# 0.0011±0.0004 1028.3±435.2
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Data mean ±SEM for N=6 rats per group

n.d. = not determined

*

p<0.01 compared to 50 μg/kg dose

#

p<0.01 compared to 50 and 100 μg/kg doses

As expected, the dose of 5F-MDMB-PICA significantly influenced Cmax (F[2,15]=6.289, p<0.01) and AUC (F[2,15]=118.2, p<0.0001) (see Table 1). Tukey’s post hoc tests showed that Cmax after 200 μg/kg 5F-MDMB-PICA was greater than Cmax after 50 μg/kg but not after 100 μg/kg. AUC after 200 μg/kg 5F-MDMB-PICA was greater than corresponding values for the 100 and 50 μg/kg doses, whereas AUC after 100 μg/kg 5F-MDMB-PICA was greater than that after 50 μg/kg. Tmax for 5F-MDMB-PICA occurred around 45 min for the 50 and 100 μg/kg doses, and was somewhat delayed for the higher dose, though Tmax was not significantly affected by dose (F[2,15]=3.24, p<0.0677). The t1/2 for 5F-MDMB-PICA increased incrementally with increasing dose (range 413.46 – 1028.35 min), but this variable was not significantly affected by dose (F[2,15=1.238, p<0.3179), perhaps due to high variance in the data at the highest dose administered.

During this study, only two of five 5F-MDMB-PICA metabolites were detected: 5F-MDMB-PICA 3,3-dimethylbutanoic acid and 5OH-MDMB-PICA. 5F-MDMB-PICA 3,3-dimethylbutanoic acid was qualitatively identified in all samples positive for 5F-MDMB-PICA, however, quantitative confirmation (>1 ng/mL) was only possible in plasma samples after the highest dose of 5F-MDMB-PICA administered. Because the butanoic acid metabolite was only quantitated for one dose, two-factor ANOVA evaluation was not performed for this analyte. Figure 3B shows the time-concentration profile for 5F-MDMB-PICA 3,3-dimethylbutanoic acid in plasma after the 200 μg/kg dose of 5F-MDMB-PICA. Cmax for 5F-MDMB-PICA 3,3-dimethylbutanoic acid after 200 μg/kg 5F-MDMB-PICA was 1.72±0.12 ng/mL and Tmax occurred 200.20±23.09 min post injection. 5OH-MDMB-PICA was qualitatively confirmed in all plasma samples where 5F-MDMB-PICA was positively identified, but this metabolite did not reach sufficient concentrations for quantitation.

3.3. Pharmacodynamic effects

Figures 4A and 4B show the time-course effects for body temperature and catalepsy score after s.c. administration of 5F-MDMB-PICA. Temperature was measured noninvasively using a handheld reader. Temperature responses to 5F-MDMB-PICA were significantly affected by dose (F[3,133]=277.0, p<0.0001) and time (F[6,133]=79.31, p<0.0001), with a dose x time interaction (F[18,133]=12.67, p<0.0001) (see Fig 3A). 5F-MDMB-PICA induced marked and sustained hypothermia, reaching 4-5 °C below normal. Dunnett’s post hoc tests showed that the 50 μg/kg dose of 5F-MDMB-PICA decreased temperature with respect to vehicle control for 240 min post injection, whereas the 100 and 200 μg/kg doses induced hypothermia that lasted for the entire experimental session. In general, larger temperature drops were observed at higher doses, which also showed delayed recovery times. Maximum temperature drops were observed between 120 min and 240 min post injection.

Figure 4.

Figure 4.

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Time course effects of 5F-MDMB-PICA on body temperature (Panel A) and catalepsy scores (Panel B). Male Sprague Dawley rats received s.c. injection of 5F-MDMB-PICA (50, 100, or 200 μg/kg) or its DMSO:Tween-80:saline (1:1:18) vehicle, and pharmacodynamic measures were determined immediately before blood sample withdrawal at 0 (pre-injection),15, 30, 60, 120, 240, and 480 min post-injection. Body temperature was measured non-invasively using a handheld reader, whereas catalepsy score was determined using a numerical scale ranging from 3 (no evidence of catalepsy) to 6 (maximal cataleptic response). Data are expressed mean±SEM for N=6 rats per group. Vertical lines through the symbols represent SEM; when no vertical line is visible, the SEM is within the symbol. Solid symbols indicate significant effects compared to the vehicle control group at a given time point (Dunnett’s p<0.05)

Catalepsy was determined by rating the presence or absence of immobility, flattened body posture, and splayed limbs. The catalepsy score induced by 5F-MDMB-PICA was significantly influenced by dose (F[3,133]=62.48, p<0.0001) and time (F[6,133]=19.84, p<0.0001), with a dose x time interaction (F[18.133]=2.877, p<0.0003) (See Fig 3B). Dunnett’s post hoc tests revealed that the 50 μg/kg dose induced modest cataleptic effects that resolved by 240 min, whereas the higher doses had greater and more sustained effects. At the 200 μg/kg dose of 5F-MDMB-PICA, catalepsy was still evident at 480 min post injection. Maximum catalepsy score was observed between 15 min and 60 min post injection and slowly resolved thereafter.

3.4. Correlation analysis

Because we collected pharmacokinetic and pharmacodynamic data from the same individual rats, we were able to examine relationships among the variables measured using Pearson’s correlation analysis. Correlations between mean circulating concentrations of 5F-MDMB-PICA and mean temperature measures for individual rats are shown in Figure 5A. Body temperature was negatively correlated with 5F-MDMB-PICA plasma concentrations (r = −0.842, p<0.001). Correlations between mean circulating concentrations of 5F-MDMB-PICA and mean catalepsy scores are shown in Figure 5B. Catalepsy score was positively correlated with 5F-MDMB-PICA concentrations (r = 0.778, p<0.0001).

Figure 5.

Figure 5.

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Correlations between plasma 5F-MDMB-PICA concentrations and body temperature (Panel A) or catalepsy score (Panel B). Mean plasma concentration of 5F-MDMB-PICA, mean body temperature and mean catalepsy score over the 8 h experimental session were determined for each rat (N=18), and values were subjected to Pearson’s correlation analyses. 5F-MDMB-PICA was negatively correlated with body temperature (p<0.001) and positively correlated with catalepsy score (p<0.001).

3.5. Binding affinity

Figure 6 depicts the results of radioligand binding assays to measure the affinity of 5F-MDMB-PICA and its metabolites at CB1 receptors in rat cerebellar membranes. As expected, 5F-MDMB-PICA displayed high-affinity binding to CB1 receptors labeled with [3H]SR141716A (IC50=2.00±0.24 nM). Of the metabolites, 5OH-MDMB-PICA displayed 50-fold lower affinity than the parent compound (IC50=105.5±11.4 nM), followed by even weaker affinity for 5F-MDMB-PICA 3,3-dimethylbutanoic acid (IC50=3,336±889 nM), and nearly no binding for 2COOH-MDMB-PICA (IC50=10,511±2,674 nM). It is noteworthy that the IC50 of the plant-based cannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC), was 59.77±7.07 nM. The binding results confirm an important role for the 5-fluoropentyl chain in the CB1 affinity of 5F-MDMB-PICA.

Figure 6.

Figure 6.

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Radioligand binding of 5F-MDMB-PICA and its metabolites at cannabinoid-1 receptors (CB1) labeled with the CB1 antagonist [3H]SR141716A in rat cerebellar membranes. Dose-response relationships were evaluated by incubating 8 concentrations of 5F-MDMB-PICA or its metabolites with 0.7 nM [3H]SR141716A. IC50 values for inhibition of binding were calculated using nonlinear regression analyses and given in the text. Binding data are expressed as mean±SD for N=3 separate experiments performed in triplicate. Vertical lines through the symbols represent SD; when no vertical line is visible, the SD is within the symbol.

4. Discussion

5F-MDMB-PICA was the most commonly encountered synthetic cannabinoid compound in the USA during 2019 and 2020, and the drug remains popular today (Drug Enforcement Administration, 2020; NPS Discovery, 2019a). 5F-MDMB-PICA was first reported in the scientific literature by Banister et al. (2016) who showed the drug is a potent and efficacious agonist at CB1 (EC50=0.45 nM) and cannabinoid-2 receptors (CB2) (EC50=7.5 nM) in transfected cells in vitro (Banister et al., 2016). In 2016, 5F-MDMB-PICA began appearing in human toxicology samples and confiscated street drug materials in Europe (Mogler et al., 2018; Risseeuw et al., 2017). The emergence of 5F-MDMB-PICA in the USA was delayed until 2018, possibly due to market dominance of indazole-containing cannabinoids like 5F-ADB and FUB-AMB (Drug Enforcement Administration, 2018; Krotulski et al., 2018). Despite its popularity and widespread use, the in vivo effects of 5F-MDMB-PICA in animal models have received little attention (Musa et al., 2020). Here, we report the first characterization 5F-MDMB-PICA pharmacokinetics and pharmacodynamics in rats. Our study reveals three main findings: 1) 5F-MDMB-PICA displays dose-proportional plasma pharmacokinetics, and two of its metabolites are present in plasma at low concentrations, namely 5F-MDMB-PICA 3,3-dimethylbutanoic acid and 5OH-MDMB-PICA; 2) 5F-MDMB-PICA induces dose-related hypothermia and catalepsy-like effects that correlate with circulating concentrations of the parent compound; 3) 5F-MDMB-PICA exhibits high-affinity binding to CB1 in rat brain membranes (IC50=2.00±0.24 nM), while its metabolites are much weaker in this regard.

Our pharmacokinetic findings with 5F-MDMB-PICA demonstrate that Cmax and AUC values increase in a dose-proportional fashion, indicative of simple linear kinetics, at least for the doses tested. On the other hand, a large portion of the AUC for the highest dose tested was unknown (i.e., beyond the 8 h time point), so there is the possibility of non-linear accumulation of 5F-MDMB-PICA after doses greater than 100 μg/kg. The plasma concentrations of 5F-MDMB-PICA observed during our study are somewhat lower than those reported for other synthetic cannabinoids in rats, even when accounting for differences in dose or route of administration (Bilel et al., 2019; Carlier et al., 2018; Kevin et al., 2017; Toennes et al., 2017). For example, following the 100 μg/kg dose of 5F-MDMB-PICA, we observed a Cmax of 3.08±0.19 ng/mL. In the investigation of Carlier et al. (Carlier et al., 2018), an identical dose of s.c. AM2201 led to a Cmax of 8.2±1.7 ng/mL in rat plasma, more than twice that of 5F-MDMB-PICA shown here. In one of the few studies examining pharmacokinetics of synthetic cannabinoids in humans, Toennes et al. (Toennes et al., 2017) reported peak serum concentrations of JWH-018 ranging from 3.5-9.0 ng/mL after a 2 mg (~30 μg/kg) smoked dose. The exact reason(s) for the low concentrations of 5F-MDMB-PICA observed in our samples are unclear, but it should be noted that our plasma Cmax values overlap with serum concentrations of the drug found in human casework (range 0.14-7.0 ng/mL) (Kleis et al., 2020), suggesting our rat model has translational value.

During this study, our confirmatory analysis detected only two of the five identified metabolites for 5F-MDMB-PICA in rat plasma samples, and these metabolites were found at much lower concentrations than the parent drug. The low number of 5F-MDMB-PICA metabolites in vivo is consistent with a previous report examining plasma pharmacokinetics of AM2201 in rats, where only three of 13 identified metabolites were observed (Carlier et al., 2018). This phenomenon of fewer in vivo metabolites could be due to a number of factors including route of drug administration, assay sensitivity, or biological matrix collected and tested. It is also a possibility that conjugated metabolites were not detected by our sample analysis workflow. We opted for the s.c. route of administration in the present work, but the i.p. route may have generated more metabolites at higher concentrations because this route facilitates hepatic metabolism. Additional metabolites of 5F-MDMB-PICA may have been present in our plasma samples, but their concentrations were below the level of detection for our assay procedure. The inability to detect low amounts of metabolites could be viewed as a limitation of our study, and a lower quantitative range may have yielded more sensitive results for those additional metabolites – this issue should be considered in future work related to detection of potent synthetic cannabinoids and other NPS in animal models. A final issue to consider is that in vivo pharmacokinetic data from rat plasma are not directly comparable to data from in vitro metabolism studies or human urine sample analysis. In metabolism studies, saturating drug concentrations are incubated with liver microsomes or hepatocytes to induce biotransformation of the parent drug. For instance, Truver et al. (2020) used a final concentration of 10 μM 5F-MDMB-PICA in their incubation medium to identify metabolites formed by human liver microsomes in vitro. Our in vivo data suggest that actual circulating concentrations of 5F-MDMB-PICA after s.c. administration are orders of magnitude lower than 10 μM, in the range of 6.2 ng/mL (~16 nM). Thus, the in vitro conditions used to identify drug metabolites may not always be relevant to the in vivo conditions after systemic drug administration.

The t1/2 of 5F-MDMB-PICA has not been previously reported. Here, we show that 5F-MDMB-PICA displays a t1/2 in the range of 7-16 h following s.c. administration in rats, an elimination rate that is somewhat slower than that of other synthetic cannabinoids. Previous studies in rats demonstrate that t1/2 values for s.c. AM2201 are in the range of 5-6 h (Carlier et al., 2018), whereas t1/2 values for i.p. CUMYL-PICA and 5F-CUMYL-PICA are in the range of 7-12 h (Kevin et al., 2017). The extended half-life of 5F-MDMB-PICA may have clinical implications, since this phenomenon could result in increased toxicity or adverse effects, as the drug remains in the biological system for a longer period. The t1/2 values of 5F-MDMB-PICA and other cannabinoids are likely affected by route of drug administration, and future studies should compare the effects of vaporized versus injected routes on plasma pharmacokinetics. Finally, it should be noted that the t1/2 for 5F-MDMB-PICA tended to increase with increasing dose administered, though this factor did not reach statistical significance, suggesting the possibility of nonlinear kinetics at higher drug doses.

We found that 5F-MDMB-PICA induces robust hypothermia and catalepsy at doses less than 1 mg/kg. These same pharmacodynamic effects have been reported for other synthetic cannabinoids in rodents, including AM-2201 (Carlier et al., 2018), AKB48 (Bilel et al., 2019), CUMYL-PICA (Kevin et al., 2017), HU-210 (Leker Ronen R. et al., 2003), and halogenated JWH-018 analogues (Vigolo et al., 2015). However, the doses required to evoke these effects with 5F-MDMB-PICA are much lower than those previously reported for other substances, confirming the high potency of this drug in rats. It is important to emphasize that we operationally defined catalepsy based on observable symptoms, rather than using the traditional bar test. We developed the behavioral scoring method specifically to avoid handling of the animals or disturbing the intravenous catheters during blood sampling. Musa et al. (2020) recently reported that 10 μg/kg i.p. 5F-MDMB-PICA increases extracellular dopamine in mouse nucleus accumbens, indicating potential addictive effects of the drug at extraordinarily low doses. We found the in vivo efficacy of 5F-MDMB-PICA to be greater than other synthetic cannabinoids. As an example, Bannister et al. (2016) examined the hypothermic effects of MDMB-FUBINACA, a potent indazole-containing synthetic cannabinoid, and showed body temperature reductions of 2-3 °C in rats (Banister et al., 2016). Here, we show robust decreases in body temperature that reach 5 °C below normal (or 32 °C) that are maintained for more than 8 h. The high potency and efficacy for 5F-MDMB-PICA may present risks for human users, since the drug has been associated with serious intoxications, characterized by adverse events and toxicity (Kleis et al., 2020). It seems unlikely that effects of 5F-MDMB-PICA in rats can replicate the complex profile of effects in humans, but clinical data reveal that synthetic cannabinoids can induce serious and sustained hypothermia in some users (Nacca et al., 2018; Seywright et al., 2016). Death resulting from the use of 5F-MDMB-PICA has been observed in our forensic toxicology casework; publication of these data is pending.

When examining the toxicity of an emerging drug, it is important to study the potential for effects resulting from parent drug and its metabolites, as some biotransformation products can have substantial potency or efficacy. Here, we show for the first time in rat brain tissue that 5F-MDMB-PICA displays high affinity for CB1 (IC50=2.00±0.24 nM), and this finding agrees with the high potency of the drug determined in various cell systems transfected with human CB1 receptors (Noble et al., 2019; Sachdev et al., 2019). Studies carried out in cells transfected with human CB1 report EC50 values for 5F-MDMB-PICA which vary widely, ranging from 0.62 to 27.6 nM (Banister et al., 2016; Noble et al., 2019; Sachdev et al., 2019; Truver et al., 2020). Importantly, we show that metabolites of 5F-MDMB-PICA are essentially inactive at CB1, except for 5OH-MDMB-PICA which is about 50-fold weaker than the parent compound (IC50=105.5±11.4 nM), though this affinity value is in the same range as Δ9-THC (IC50=59.77±7.07). Our binding results with 5OH-MDMB-PICA agree with the reduced activity of similar tail group-hydroxylated metabolites for synthetic cannabinoids (Gamage et al., 2019). Overall, our data show that pharmacodynamic effects of ingested 5F-MDMB-PICA are likely due to 5F-MDMB-PICA itself rather than a combination of effects from metabolites.

In summary, we report the first characterization of pharmacokinetics and pharmacodynamics for the popular synthetic cannabinoid 5F-MDMB-PICA in any species. We successfully developed and validated a sensitive quantitative single-injection assay for 5F-MDMB-PICA and four of its primary metabolites. We show that 5F-MDMB-PICA displays dose-proportional plasma pharmacokinetics in rats and has an extended t1/2 that can exceed 16 h. Despite the previous identification of numerous 5F-MDMB-PICA metabolites in vitro and in human urine, only two of the metabolites are present in rat plasma, and metabolite concentrations are much lower than the parent drug. We demonstrate that 5F-MDMB-PICA exhibits high affinity for CB1 receptors in rat brain tissue, which agrees with data from transfected cell systems. Accordingly, the drug elicits profound hypothermia and catalepsy, effects that directly correlate with circulating concentration of 5F-MDMB-PICA. When the popularity of 5F-MDMB-PICA begins to wane, similar analogs, such as 4F-MDMB-BICA, may be increasingly confiscated in drug products (Krotulski et al., 2021). The in vitro potency of 4F-MDMB-BICA is reportedly less than that of 5F-MDMB-PICA (Cannaert et al., 2020), but continued research is needed to assess the risk posed by 4F-MDMB-BICA and the evolving list of new synthetic cannabinoid agents.

Supplementary Material

Supplementary file

Highlights.

  • 5F-MDMB-PICA is a potent CB1 agonist in rats.

  • 5F-MDMB-PICA induces sustained hypothermia and catalepsy in rats (50–200 μg/kg).

  • Plasma concentrations of 5F-MDMB-PICA increase in parallel with dose administered.

  • Only two metabolites were detected in plasma, both at low levels.

  • Pharmacodynamic effects are related to circulating levels of 5F-MDMB-PICA.

7. Acknowledgements

The authors would like to acknowledge Melissa Fogarty of CFSRE for her assistance during this study. We thank Dr. Inger Lise Bogen for her assistance with the pharmacokinetic calculations using Kinetica. Finally, the authors would like to acknowledge Waters Corporation for providing LC-MS/MS instrumentation through a collaborative partnership.

6. Funding

Funding for analytical testing was received from the National Institute of Justice (NIJ) of the U.S. Department of Justice (DOJ) (Award Number 2017-R2-CX-0021). The experiments conducted in Dr. Baumann’s laboratory are generously supported by the Intramural Research Program (IRP) of the National Institute on Drug Abuse (NIDA), National Institutes of Health, grant DA 000523-13. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of DOJ, NIJ or NIH.

Footnotes

5.

Conflicts of Interest

The authors have no conflicts of interest to declare.

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