Thermolytic Degradation of Synthetic Cannabinoids: Chemical Exposures and Pharmacological Consequences.

Synthetic cannabinoids are manufactured clandestinely with little quality control and are distributed as herbal "spice" for smoking or as bulk compound for mixing with a solvent and inhalation via electronic vaporizers. Intoxication with synthetic cannabinoids has been associated with seizure, excited delirium, coma, kidney damage, and other disorders. The chemical alterations produced by heating these structurally novel compounds for consumption are largely unknown. Here, we show that heating synthetic cannabinoids containing tetramethylcyclopropyl-ring substituents produced thermal degradants with pharmacological activity that varied considerably from their parent compounds. Moreover, these degradants were formed under conditions simulating smoking. Some products of combustion retained high affinity at the cannabinoid 1 (CB1) and CB2 receptors, were more efficacious than (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) in stimulating CB1 receptor-mediated guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding, and were potent in producing Δ9-tetrahydrocannabinol-like effects in laboratory animals, whereas other compounds had low affinity and efficacy and were devoid of cannabimimetic activity. Degradants that retained affinity and efficacy also substituted in drug discrimination tests for the prototypical synthetic cannabinoid 1-pentyl-3-(1-naphthoyl)indole (JWH-018), and are likely to produce psychotropic effects in humans. Hence, it is important to take into consideration the actual chemical exposures that occur during use of synthetic cannabinoid formulations to better comprehend the relationships between dose and effect.

Pharmacodynamic Relationships between Duration of Action of JDTic-like Kappa-Opioid Receptor Antagonists and Their Brain and Plasma Pharmacokinetics in Rats.

JDTic is a potent and selective κ-opioid receptor (KOR) antagonist that reverses U50,488-induced diuresis in rats. It partitions into brain with a duration of action lasting for weeks. In a search for KOR antagonists that do not accumulate in the brain, we compared single doses of five methylated JDTic analogs (RTI-97, -194, -212, -240, and -241) for reversal of U50,488 diuresis and pharmacokinetic (PK) properties. All six compounds showed potent and selective KOR antagonism in a [35S]GTPγS binding assay. Plasma half-lives ranged from 24 to 41 h and brain half-lives from 24 to 76 h. JDTic and RTI-194 showed increasing brain to plasma ratios over time, indicating increasing partitioning into brain and a longer duration of action for reversal of diuresis than did RTI-97. RTI-240 did not show significant brain accumulation. RTI-212 showed no substantive difference between brain and plasma levels and was inactive against diuresis. RTI-241, with a lower brain to plasma ratio than JDTic and RTI-194, formed JDTic as a metabolite, which still reduced diuresis after 9 weeks. The fact that the duration of action was correlated with the brain to blood plasma ratios and area under the concentration-time curves suggests that PK properties could help to predict safety and acceptable duration of action for KOR antagonists.

Preparation and Distribution of Cannabis and Cannabis-Derived Dosage Formulations for Investigational and Therapeutic Use in the United States.

Cannabis is classified as a schedule I controlled substance by the US Drug Enforcement Agency, meaning that it has no medicinal value. Production is legally restricted to a single supplier at the University of Mississippi, and distribution to researchers is tightly controlled. However, a majority of the population is estimated to believe that cannabis has legitimate medical or recreational value, numerous states have legalized or decriminalized possession to some degree, and the federal government does not strictly enforce its law and is considering rescheduling. The explosive increase in open sale and use of herbal cannabis and its products has occurred with widely variable and in many cases grossly inadequate quality control at all levels-growing, processing, storage, distribution, and use. This paper discusses elements of the analytical and regulatory system that need to be put in place to ensure standardization for the researcher and to reduce the hazards of contamination, overdosing, and underdosing for the end-user.

Analytical surveillance of emerging drugs of abuse and drug formulations.

Uncontrolled recreational drugs are proliferating in number and variety. Effects of long-term use are unknown, and regulation is problematic, as efforts to control one chemical often lead to several other structural analogs. Advanced analytical instrumentation and methods are continuing to be developed to identify drugs, chemical constituents of products, and drug substances and metabolites in biological fluids. Several mass spectrometry based approaches appear promising, particularly those that involve high resolution chromatographic and mass spectrometric methods that allow unbiased data acquisition and sophisticated data interrogation. Several of these techniques are shown to facilitate both targeted and broad spectrum analyses, the latter of which are often of particular benefit when dealing with misleadingly labeled products or assessing a biological matrix for illicit drugs and metabolites. The development and application of novel analytical approaches such as these will help to assess the nature and degree of exposure and risk and, where necessary, inform forensics and facilitate implementation of specific regulation and control measures.

Analogues of (3R)-7-hydroxy-N-[(1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic). Synthesis and in vitro and in vivo opioid receptor antagonist activity.

The synthesis of compounds 6, 7a,b, 8a,b, 9a,b, and 10a,b where the amino -NH- group of JDTic (3) was replaced with an aromatic horizontal lineCH-, CH(2), O, S, or SO group was accomplished and used to further characterize the SAR of the compound 3 class of kappa opioid receptor antagonists. All of the compounds showed subnanomolar to low nanomolar K(e) values at the kappa opioid receptor. The most potent compound was 7a, where the amino -NH- group of 3 was replaced by a methylene (-CH(2)-) group. This compound had a K(e) = 0.18 nM and was 37- and 248-fold selective for the kappa relative to the mu and delta opioid receptors, respectively. Similar to compound 3, compound 7a antagonized selective kappa agonist U50,488-induced diuresis after sc administration in rats. In contrast to 3, where kappa antagonist activity lasted for three weeks, compound 7a did not show any kappa antagonist activity after one week.

Effectiveness of analogs of the kappa opioid receptor antagonist (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic) to reduce U50,488-induced diuresis and stress-induced cocaine reinstatement in rats.

RATIONALE: The kappa opioid receptor (KOR) antagonist, JDTic, was reported to prevent stress-induced reinstatement of cocaine-maintained responding and to have antidepressant-like effects. OBJECTIVES: Our objectives were to determine whether analogs of JDTic retained KOR antagonist activity and whether an orally effective analog prevented footshock-induced cocaine reinstatement. METHODS: RTI-194 (i.g. 1-30 mg/kg, s.c. 0.3-10 mg/kg, and i.p. 30 mg/kg), RTI-212 (s.c. 0.3-10 mg/kg and i.p. 30 mg/kg), and RTI-230 (i.g. 3-30 mg/kg and i.p. 1-30 mg/kg) were evaluated for their ability to block diuresis induced by 10-mg/kg U50,488 in rats. RTI-194 was additionally evaluated i.g. (3-100 mg/kg) for its ability to prevent footshock-induced reinstatement of responding previously reinforced with 0.5-mg/kg/inf cocaine. RESULTS: RTI-194 significantly (p < 0.05) attenuated U50,488-induced diuresis when given i.g., s.c., and i.p. RTI-194s effectiveness increased 1 week following administration. RTI-212 was ineffective. RTI-230 was ineffective when given i.g., but blocked diuresis at 24 h and 8 days (1, 10, and 30 mg/kg), 15 days (10 and 30 mg/kg), 22 and 29 days (30 mg/kg) following i.p. administration. Footshock reinstated responding in vehicle-but not RTI-194 (30 and 100 mg/kg)-treated rats. CONCLUSIONS: RTI-194 and RTI-230 are effective KOR antagonists, and RTI-194 is now included with JDTic as the only reported compounds capable of antagonizing the KOR following oral administration. The failure of stress to reinstate cocaine seeking in rats treated with RTI-194 is consistent with results reported with JDTic, although it had less efficacy in lowering response levels than JDTic, suggesting a diminished overall effectiveness relative to it.

Positive allosteric modulation of the human cannabinoid (CB) receptor by RTI-371, a selective inhibitor of the dopamine transporter.

BACKGROUND AND PURPOSE: In our search for an indirect dopamine agonist as therapy for cocaine addiction, several selective inhibitors of the dopamine transporter (DAT), which are 3-phenyltropane analogues, were assayed for their effect on locomotor activity in mice. Interestingly, several of the compounds showed a poor correlation between stimulation of locomotion and DAT inhibition. One of the compounds, 3beta-(4-methylphenyl)-2beta-[3-(4-chlorophenyl)isoxazol-5-yl]tropane (RTI-371), was shown to cross the blood-brain barrier, by binding studies in vivo, and block cocaine-induced locomotor stimulation. As poor pharmacokinetics could not explain the behavioural effects of RTI-371, this compound was screened through our functional assays for activity at other CNS receptors. Initial screening identified RTI-371 as a positive allosteric modulator of the human CB(1) (hCB(1)) receptor. EXPERIMENTAL APPROACH: The effect of RTI-371 and other DAT-selective inhibitors on CP55940-stimulated calcium mobilization was characterized in a calcium mobilization-based functional assay for the hCB(1) receptor. Selected compounds were also characterized in a similar assay for human mu opioid receptor activation to assess the specificity of their effects. KEY RESULTS: RTI-371 and several other DAT-selective inhibitors with atypical actions on locomotor behaviour increased the efficacy of CP55940 in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: These results suggest that the lack of correlation between the DAT-binding affinity and locomotor stimulation of RTI-371 could be due at least in part to its activity as a positive modulator of the hCB(1) receptor.

Effects of dopamine transporter selective 3-phenyltropane analogs on locomotor activity, drug discrimination, and cocaine self-administration after oral administration.

Several studies suggest that a dopamine transporter uptake inhibitor that has a slower onset and longer duration of action than cocaine in animal behavioral measures and decreases cocaine self-administration would be useful as an indirect dopamine agonist pharmacotherapy to treat cocaine addiction. In the present study, we compared five 3-phenyltropane analogs administered orally in locomotor activity in mice and drug discrimination in rats to gain information concerning relative potency, onset, and duration of action. The compounds were also evaluated for reduction of cocaine self-administration in rats after oral administration. In general, the compounds had a slower onset of action than cocaine and reduced cocaine self-administration. 3beta-(4-Chlorophenyl)-2beta-(3-(4'-methylphenyl)-isoxazol-5-yl)tropane (RTI-336) was the most potent in locomotor activity and drug discrimination; it was less stimulatory than cocaine in the first hour and had the slowest onset and longest duration of action. It also reduced self-administration of two infusion doses of cocaine in the rat.

Monoamine transporter binding, locomotor activity, and drug discrimination properties of 3-(4-substituted-phenyl)tropane-2-carboxylic acid methyl ester isomers.

The monoamine transporter binding properties, gross behavior, and locomotor activity effects in mice and drug discrimination results in cocaine-trained rats of the 2 beta,3beta-, 2 beta,3 alpha-, 2 alpha,3beta-, and 2 alpha,3 alpha-isomers of several 3-(4-substituted-phenyl)tropane carboxylic acid methyl esters were compared (2a-f, 3a-f, 4a-f, and 5b,c). The 2 beta,3beta-isomer showed the highest affinity for the dopamine transporter (DAT), and the 2 beta,3 alpha-isomer showed the next highest affinity. The order of potency for the 2 beta,3beta-isomer is 4'-chloro (2c) = 4'-iodo (2e) > 4'-bromo (2d) = 4'-methyl (2f) > 4'-fluoro (2b) > 4'-hydrogen (2a). In the case of the 2 beta,3 alpha-isomer, the order of affinity was 4'-bromo (3d) > 4'-iodo (3e) = 4'- chloro (3c) > 4'-methyl (3f) > 4'-fluoro (3b) > 4'-hydrogen (3a). The 4'-hydrogen, 4'-fluoro, and 4'-methyl 2 alpha,3beta-isomers, 4a, 4b, and 4f, had the lowest affinity for the DAT. While most of the compounds showed their highest affinity at the DAT, none were selective relative to the other two monoamine transporters. In general, the 2 alpha,3 alpha- and 2 alpha,3beta-isomers were more toxic (death and convulsions) than the 2 beta,3beta- and 2 beta,3 alpha-isomers. With the exception of the 2 alpha,3 alpha-isomers, all compounds produced the locomotor activity stimulation typical of dopaminergic drugs. The ED(50) ranges for the 2 beta,3beta- (2a-f), 2 beta,3 alpha- (3a-f), and 2 alpha,3beta-isomers (4a-f) in the locomotor assay were 0.1-1.2, 6.6-21.8, and 2.4-11.7 mg/kg, respectively. With the exception of the 2 alpha,3 alpha-isomer, all compounds generalized to cocaine. The 2 beta,3beta-isomers were at least 10-fold more potent than cocaine and the other three sets of isomers in this test.

Pharmacological properties of JDTic: a novel kappa-opioid receptor antagonist.

Biological studies were conducted on (3R)-7-Hydroxy-N-[(1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl]-1,2,3,4-tetrahydro-3-isoquinoline-carboxamide (JDTic), the first potent kappa-selective opioid receptor antagonist not derived from an opiate class of compounds. In the mouse tail-flick test, JDTic, administered subcutaneously (s.c.), blocked anticociceptive activity for up to 2 weeks. When JDTic was administered either s.c. or p.o. 24 h before the selective KOP (kappa)-opioid receptor agonist, enadoline, AD(50s) of 4.1 and 27.3, respectively, were obtained. A time-course study of JDTic versus enadoline indicated significant antagonist p.o. activity up to 28 days. In contrast, JDTic, s.c., failed to antagonize the analgesic effects of the selective MOP mu-opioid receptor agonist, sufentanil. In the squirrel monkey shock titration antinociception test, JDTic given intramuscularly (i.m.) shifted the trans-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide (U50,488) dose-effect curve to the right. In the U50,488-induced diuresis rat test, JDTic, s.c., suppressed diuretic activity with a greater potency than that of nor-binaltorphimine (nor-BNI). Thus, JDTic is a potent long- and orally acting selective kappa-opioid antagonist.

Synthesis, monoamine transporter binding properties, and behavioral pharmacology of a series of 3beta-(substituted phenyl)-2beta-(3'-substituted isoxazol-5-yl)tropanes.

Several 3beta-(substituted phenyl)-2beta-(3-substituted isoxazol-5-yl)tropanes (3a-t) were evaluated for their ability to inhibit radioligand binding at the DAT, 5-HTT, and NET as well as in gross observation and locomotor activity in mice and in rats trained to discriminate cocaine. All compounds showed high affinity for the DAT. The IC(50) values ranged from 0.5 to 26 nM. With the exception of 3e and 3f, which have no substituent on the 2beta-(1,2-isoxazole) ring, all compounds were selective for the DAT relative to the 5-HTT and NET. No compound showed death when dosed at 100 mg/kg; however, most compounds did show signs typical of dopamine activity. The ED(50) values for 2beta-(1,2-isoxazoles) that caused locomotor stimulation ranged from 0.2 to 12.8 mg/kg. Most compounds showed slower on-set and longer duration of action relative to cocaine. Surprisingly, 3beta-(4-methylphenyl)-2beta-[3-(4'-chlorophenyl)isoxazol-5-yl]tropane (3p) and 3beta-(4-methylphenyl)-2beta-[3-(4'-methylphenyl)isoxazol-5-yl]tropane (3r) did not produce significant increases in locomotor activity. In the cocaine discrimination test, all analogues showed full or at least 50% generalization with the exception of 3p, which did not show generalization. Importantly, both the locomotor activity and the cocaine discrimination ED(50)values were correlated with the DAT binding but not 5-HTT and NET binding. This provides further support for the dopamine hypothesis of cocaine abuse. High DAT affinity and selectivity, increased locomotor activity with slow onset and long duration of action, and generalization to cocaine shown by the 3beta-(substituted phenyl)-2beta-(3-substituted isoxazol-5-yl)tropanes are properties thought necessary for a pharmacotherapy for treating cocaine abuse.