Description: The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OP-related neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons. Here, using a time-lapse imaging technique, we evaluated the OP-nerve agent diisopropylfluorophosphate (DFP) across a wide range of concentrations (subnanomolar to micromolar) for effects on fast axonal transport of membrane-bound organelles (MBOs) that contain the amyloid precursor protein (APP) tagged with the fluorescent marker Dendra2 (APPDendra2). Both 1 and 24 hours of exposure to DFP and a positive control compound, colchicine, resulted in a decrease in the velocity of anterograde and retrograde movements of MBOs and an increase in the number of stationary MBOs. These effects occurred at picomolar (100 pM) to low nanomolar (0.1 nM) concentrations that were not associated with compromised cell viability or cytoskeletal damage. Moreover, the effects of DFP on axonal transport occurred at concentrations that did not inhibit AChE activity, and they were not blocked by cholinergic receptor antagonists. Given the fundamental importance of axonal transport to neuronal function, these observations may explain some of the long-term neurologic deficits that have been observed in humans who have been exposed to OPs.

Description: Synthetic cannabinoids (SCs) are an emerging class of abused drugs that differ from each other and the phytocannabinoid 9 -tetrahydrocannabinol (THC) in their safety and cannabinoid-1 receptor (CB 1 R) pharmacology. As efficacy represents a critical parameter to understanding drug action, the present study investigated this metric by assessing in vivo and in vitro actions of THC, two well-characterized SCs (WIN55,212-2 and CP55,940), and three abused SCs (JWH-073, CP47,497, and A-834,735-D) in CB 1 (+/+), (+/–), and (–/–) mice. All drugs produced maximal cannabimimetic in vivo effects (catalepsy, hypothermia, antinociception) in CB 1 (+/+) mice, but these actions were essentially eliminated in CB 1 (–/–) mice, indicating a CB 1 R mechanism of action. CB 1 R efficacy was inferred by comparing potencies between CB 1 (+/+) and (+/–) mice [+/+ ED 50 /+/– ED 50 ], the latter of which has a 50% reduction of CB 1 Rs (i.e., decreased receptor reserve). Notably, CB 1 (+/–) mice displayed profound rightward and downward shifts in the antinociception and hypothermia dose-response curves of low-efficacy compared with high-efficacy cannabinoids. In vitro efficacy, quantified using agonist-stimulated [ 35 S]GTP S binding in spinal cord tissue, significantly correlated with the relative efficacies of antinociception ( r = 0.87) and hypothermia ( r = 0.94) in CB 1 (+/–) mice relative to CB 1 (+/+) mice. Conversely, drug potencies for cataleptic effects did not differ between these genotypes and did not correlate with the in vitro efficacy measure. These results suggest that evaluation of antinociception and hypothermia in CB 1 transgenic mice offers a useful in vivo approach to determine CB 1 R selectivity and efficacy of emerging SCs, which shows strong congruence with in vitro efficacy.

Description: Synthetic cannabinoids (SCs) represent an emerging class of abused drugs associated with psychiatric complications and other substantial health risks. These ligands are largely sold over the internet for human consumption, presumably because of their high cannabinoid 1 receptor (CB 1 R) affinity and their potency in eliciting pharmacological effects similar to 9 -tetrahydrocannabinol (THC), as well as circumventing laws illegalizing this plant. Factors potentially contributing to the increased prevalence of SC abuse and related hospitalizations, such as increased CB 1 R efficacy and non-CB 1 R targets, highlight the need for quantitative pharmacological analyses to determine receptor mediation of the pharmacological effects of cannabinoids. Accordingly, the present study used pA 2 and pK B analyses for quantitative determination of CB 1 R mediation in which we utilized the CB 1 R-selective inverse agonist/antagonist rimonabant to elicit rightward shifts in the dose-response curves of five SCs (i.e., A-834,735D; WIN55,212-2; CP55,950; JWH-073; and CP47,497) and THC in producing common cannabimimetic effects (i.e., catalepsy, antinociception, and hypothermia). The results revealed overall similarity of pA 2 and pK B values for these compounds and suggest that CB 1 Rs, and not other pharmacological targets, largely mediated the central pharmacological effects of SCs. More generally, affinity estimation offers a powerful pharmacological approach to assess potential receptor heterogeneity subserving in vivo pharmacological effects of SCs.

Description: Synthetic cannabinoids are a class of novel psychoactive substances that exhibit high affinity at the cannabinoid type-1 (CB 1 ) receptor and produce effects similar to those of -9-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis. Illicit drug manufacturers are continually circumventing laws banning the sale of synthetic cannabinoids by synthesizing novel structures and doing so with little regard for the potential impact on pharmacological and toxicological effects. Synthetic cannabinoids produce a wide range of effects that include cardiotoxicity, seizure activity, and kidney damage, and they can cause death. Six synthetic cannabinoids, recently detected in illicit preparations, MMB-FUBINACA, MDMB-FUBINACA, CUMYL-PICA, 5F-CUMYL-PICA, NNEI, and MN-18 were assessed for: 1) receptor binding affinity at the human CB 1 and human CB 2 receptors, 2) function in [ 35 S]GTP S and cAMP signaling, and 3) THC-like effects in a mouse drug discrimination assay. All six synthetic cannabinoids exhibited high affinity for human cannabinoid receptors type-1 and type-2 and produced greater maximal effects than THC in [ 35 S]GTP S and cAMP signaling. Additionally, all six synthetic cannabinoids substituted for THC in drug discrimination, suggesting they probably possess subjective effects similar to those of cannabis. Notably, MDMB-FUBINACA, a methylated analog of MMB-FUBINACA, had higher affinity for CB 1 than the parent, showing that minor structural modifications being introduced can have a large impact on the pharmacological properties of these drugs. This study demonstrates that novel structures being sold and used illicitly as substitutes for cannabis are retaining high affinity at the CB 1 receptor, exhibiting greater efficacy than THC, and producing THC-like effects in models relevant to subjective effects in humans.

Description: Diversion of synthetic cannabinoids for abuse began in the early 2000s. Despite legislation banning compounds currently on the drug market, illicit manufacturers continue to release new compounds for recreational use. This study examined new synthetic cannabinoids, AB-CHMINACA ( N -[1-amino-3-methyl-oxobutan-2-yl]-1-[cyclohexylmethyl]-1 H -indazole-3-carboxamide), AB-PINACA [ N -(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1 H -indazole-3-carboxamide], and FUBIMINA [(1-(5-fluoropentyl)-1 H -benzo[ d ]imadazol-2-yl)(naphthalen-1-yl)methanone], with the hypothesis that these compounds, like those before them, would be highly susceptible to abuse. Cannabinoids were examined in vitro for binding and activation of CB 1 receptors, and in vivo for pharmacological effects in mice and in 9 -tetrahydrocannabinol ( 9 -THC) discrimination. AB-CHMINACA, AB-PINACA, and FUBIMINA bound to and activated CB 1 and CB 2 receptors, and produced locomotor suppression, antinociception, hypothermia, and catalepsy. Furthermore, these compounds, along with JWH-018 [1-pentyl-3-(1-naphthoyl)indole], CP47,497 [rel-5-(1,1-dimethylheptyl)-2-[(1 R ,3 S )-3-hydroxycyclohexyl]-phenol], and WIN55,212-2 ([(3 R )-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3- de ]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone, monomethanesulfonate), substituted for 9 -THC in 9 -THC discrimination. Rank order of potency correlated with CB 1 receptor-binding affinity, and all three compounds were full agonists in [ 35 S]GTP S binding, as compared with the partial agonist 9 -THC. Indeed, AB-CHMINACA and AB-PINACA exhibited higher efficacy than most known full agonists of the CB 1 receptor. Preliminary analysis of urinary metabolites of the compounds revealed the expected hydroxylation. AB-PINACA and AB-CHMINACA are of potential interest as research tools due to their unique chemical structures and high CB 1 receptor efficacies. Further studies on these chemicals are likely to include research on understanding cannabinoid receptors and other components of the endocannabinoid system that underlie the abuse of synthetic cannabinoids.