Chemistry, Crystal Structure, and In Vitro Receptor Binding of Δ10-THC Isomers.

Introduction: The psychoactive properties of Δ10-THC isomers (trans- and cis-Δ10-THC) are poorly understood. To shed more light on the biological effects of these compounds, we studied in vitro receptor binding of Δ10-THC isomers at cannabinoid CB1 and CB2 receptors. Materials and Methods: We first optimized and simplified catalytic synthesis of trans- and cis-Δ10-THC to allow their safe and cheap large-scale synthesis. In our synthesis, BuLi was replaced with KOtBu, and DMSO/anisole or NEt3/heptane solvent systems were used instead of HMPA/toluene. Single crystal X-ray analysis confirmed the structure of both isomers and the configuration of their chiral centers. Results: In the radioligand replacement assay, both isomers showed strong affinity toward the CB1 receptor, with IC50=29.1 nM for the trans isomer and IC50=294.2 nM for the cis counterpart. However, the IC50 values were significantly higher than that of Δ9-THC (2.1 nM), a naturally occurring psychoactive component of cannabis sativa, suggesting a lower affinity of Δ10-THCs toward this receptor. In function assays, in contrast to Δ9-THC, both isomers failed to show any agonist properties at concentrations up to 10 µM suggesting a lack of THC-like psychoactivity for trans- and cis-Δ10-THC. Conclusions: Our results established Δ10-THC isomers among antagonists of the CB1 receptor as both cis and trans isomers antagonized CP55,490 with IC50=460 nM for trans and IC50=1040 nM for cis. This functional property has not been previously observed for any other THC isomers.

Single-Laboratory Validation of an Immunoaffinity Column Cleanup LC Method for the Analysis of Aflatoxins and Ochratoxin A in Cannabis Plant Material, Resins, Vapes, Isolates, and Edible Products.

BACKGROUND: Potential fungal infection of cannabis plants during drying has raised concerns of resulting mycotoxin contamination in leaves and flowers and subsequent contamination of derived products including cannabis-containing edible products. Validated routine methods are essential to monitor cannabis and cannabis products to ensure consumer safety consistent with long-standing controls for mycotoxins such as aflatoxins and ochratoxin A in foodstuffs. OBJECTIVE: To provide single-laboratory validation data to demonstrate the suitability of a method for determining aflatoxins and ochratoxin A in cannabis plant material, resins, vapes, isolates, and edible products such as chocolate. METHOD: Extraction of solid and liquid matrixes with acetonitrile:water, centrifugation, and then dilution of an aliquot of supernatant with phosphate-buffered saline solution containing Tween 20 surfactant. Cleanup by passing through an immunoaffinity column containing antibodies to both aflatoxins and ochratoxin A and analyzing in a single LC chromatographic run with fluorescence detection. RESULTS: For within-day analysis, recoveries were in the range 77 to 99% with RSDs from 0.7 to 9.6% for aflatoxin B1. Similarly, ochratoxin A recoveries were from 64 to 94% and RSDs from 0.9 to 9.5% for mycotoxin mixtures spiked into cannabis flowers, resins, vapes, isolates, chocolate, gummies, edible oils, and beverages. CONCLUSIONS: A method for the determination of aflatoxins and ochratoxin A was successfully developed and single-laboratory validation data has been presented for cannabis plant material, resins, vapes, isolates, and edible products. HIGHLIGHTS: A multi-mycotoxin immunoaffinity column cleanup with LC-fluorescence has been validated and shown to be suitable for routine control of aflatoxins and ochratoxin A in cannabis flowers and a diverse range of edible cannabis products.

Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.

Elucidating gene function is heavily reliant on the ability to modulate gene expression in biological model systems. Although transient expression systems can provide useful information about the biological outcome resulting from short-term gene overexpression or silencing, methods providing stable integration of desired expression constructs (cDNA or RNA interference) are often preferred for functional studies. To this end, lentiviral vectors offer the ability to deliver long-term and regulated gene expression to mammalian cells, including the expression of gene targeting small hairpin RNAs (shRNAmirs). Unfortunately, constructing vectors containing the desired combination of cDNAs, markers, and shRNAmirs can be cumbersome and time-consuming if using traditional sequence based restriction enzyme and ligation-dependent methods. Here we describe the use of a recombination based Gateway cloning strategy to rapidly and efficiently produce recombinant lentiviral vectors for the expression of one or more cDNAs with or without simultaneous shRNAmir expression. Additionally, we describe a luciferase-based approach to rapidly triage shRNAs for knockdown efficacy and specificity without the need to create stable shRNAmir expressing cells.