European Monitoring Centre for Drugs and Drug Addiction (2016) EMCDDAâEuropol Joint Report on a new psychoactive substance: MDMB-CHMICA. Results p.
Inhibition of human CaV3.2 channels by synthetic cannabinoid MDMB-CHMICA in vitro. 1 Bladen ,
Chris Marina 1 and Mark Connor .
1 Santiago ,
Mitchell
2 Longworth ,
Michael
2 Kassiou ,
Sam
3 Banister ,
Dept of Biomedical Sciences, Macquarie University1, Sydney, NSW, School of Chemistry, University of Sydney2, Sydney, NSW, Dept of Pathology, Stanford University, School of Medicine3, Stanford, CA, USA.
Introduction
Results
Methyl-2-[[1-(cyclohexylmethyl)indole-3-carbonyl]amino]-3,3dimethylbutanoate (MDMB-CHMICA) is a synthetic cannabinoid associated with severe adverse effects including; seizures, arrhythmias and also dozens of fatalities have been attributed to its consumption (EMCDDA, 2016). We recently showed that MDMB-CHMICA has a higher efficacy and 20-fold greater in vitro potency as a CB1 agonist than the psychoactive ingredient of cannabis, ∆9-tetrahydrocannabinol (Banister et al., 2016). The CB1 potency and efficacy may underlie some of the severe hallucinogenic and psychotomimetic effects of this drug, however, the mechanisms responsible for other symptoms such as heart arrhythmias and peripheral toxicity remain to be established.
Fig 2. Using the voltage protocol in Fig 1A, log concentrations of MDMB-CHMICA were applied to HEK cells expressing hCaV3.2 to generate a dose-response curve that yielded an IC50 of 1.5 ± 0.2 µM.
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Aims
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Cannabinoids directly interact with several ion channel classes, and the aim of this study was to explore whether MDMBCHMICA and other closely related synthetic cannabinoids interact with human T-type calcium channels (hCaV3.2), which are known to regulate rhythmicity in both the heart and the brain (Catterall et al., 2008).
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Fig 4A. Standard drug screening at 10 µM of 13 other synthetic cannabinoids structurally related to MDMB-CHMICA, revealed several compounds that blocked > 50% of hCaV3.2 current with AMB-CHMINACA showing the most block. Fig 4B. As in Fig 2, log concentrations of AMB-CHMINACA were applied to HEK cells expressing hCaV3.2 to generate a dose-response curve that yielded an IC50 of 0.74 ± 0.2 µM.(MDMB-CHMICA is included for comparison).
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MDMB-CHMICA MDMB-CHMINACA 5F-AMB-PINACA MDMB-FUBINACA AMB-PICA MDMB PICA 5F-AMB-PICA 5F-MDMB-PICA AMB-FUBICA MDMB-FUBICA AMB-CHMICA MDMB-PINICA AMB-FUBINICA AMB-CHMINACA
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Whole-cell voltage clamp recordings were made from HEK293 cells stably expressing hCaV3.2. Drugs were applied using home-made, gravity-fed perfusion system. All data were acquired using Molecular Devices Hardware and pClamp software. All data points represent n=6 cells or more.
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Fig 3A. When applied at a concentration of 1 µM, MDMB-CHMICA did not significantly affect the half-activation potential of hCaV3.2 (42 ± 1 mV in control, -43.3 ± 1 mV in drug). Fig 3B. Similarly, MDMB-CHMICA (1 µM) did not significantly affect the steady stateinactivation potential of hCaV3.2 (-78.9 ± 1 mV in control, -80.2 ± 1 mV in drug). Fig 3C. Representative hCaV3.2 current traces using standard I-V protocol with -30mV trace highlighted in red.
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Fig 1A. Representative current traces showing block of hCaV3.2 by 10 µM MDMB-CHMICA and subsequent washout. The inset shows the whole cell voltage command protocol used to evoke T-type current (200 ms step from holding potential of -100 mV to -30 mV, every 15 s). Fig 1B. Time course showing rapid onset of current block by 10 µM MDMB-CHMICA followed by rapid washout to almost pre-drug current levels.
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This report is the first to show that like phytocannabinoids (Ross et al., 2008), illicit synthetic cannabinoids potently inhibit human T-type calcium channels. Although the mechanisms underlying synthetic cannabinoid toxicity are not yet established, many of the most severe symptoms, including arrhythmia, seizures and low blood pressure, are associated with altered hCaV3.2 activity.
References:
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Banister SD et al., (2016) ACS Chem Neurosci 7: 1241-1254. Ross HR et al., (2008) J Biol Chem 283: 16124–16134. 60 120 180 240 300 360 420 480 Catterall WA et al., (2008) J. Neurosci 28: 11768 –11777. European Monitoring Centre for Drugs and Drug Addiction (2016) EMCDDA–Europol Joint Report on a new Time (Secs) psychoactive substance: MDMB-CHMICA.
