Affinity and Efficacy Studies of Tetrahydrocannabinolic Acid A at Cannabinoid Receptor Types One and Two

Abstract

Introduction:Cannabis biosynthesizes Δ⁹-tetrahydrocannabinolic acid (THCA-A), which decarboxylates into Δ⁹-tetrahydrocannabinol (THC). There is growing interest in the therapeutic use of THCA-A, but its clinical application may be hampered by instability. THCA-A lacks cannabimimetic effects; we hypothesize that it has little binding affinity at cannabinoid receptor 1 (CB₁). Materials and Methods: Purity of certified reference standards were tested with high performance liquid chromatography (HPLC). Binding affinity of THCA-A and THC at human (h) CB₁ and hCB₂ was measured in competition binding assays, using transfected HEK cells and [³H]CP55,940. Efficacy at hCB₁ and hCB₂ was measured in a cyclic adenosine monophosphase (cAMP) assay, using a Bioluminescence Resonance Energy Transfer (BRET) biosensor. Results: The THCA-A reagent contained 2% THC. THCA-A displayed small but measurable binding at both hCB₁ and hCB₂, equating to approximate K_i values of 3.1μM and 12.5μM, respectively. THC showed 62-fold greater affinity at hCB₁ and 125-fold greater affinity at hCB₂. In efficacy tests, THCA-A (10μM) slightly inhibited forskolin-stimulated cAMP at hCB₁, suggestive of weak agonist activity, and no measurable efficacy at hCB₂. Discussion: The presence of THC in our THCA-A certified standard agrees with decarboxylation kinetics (literature reviewed herein), which indicate contamination with THC is nearly unavoidable. THCA-A binding at 10μM approximated THC binding at 200nM. We therefore suspect some of our THCA-A binding curve was artifact-from its inevitable decarboxylation into THC-and the binding affinity of THCA-A is even weaker than our estimated values. We conclude that THCA-A has little affinity or efficacy at CB₁ or CB₂.

Keywords: Cannabis; THCA; cannabinoid receptors; pharmacodynamics; pharmacology; phytocannabinoids.

FIG. 1.

Chemical structures of THC (left), THCA-A (center), and THCA-B (right). THC, Δ-tetrahydrocannabinol; THCA-A, Δ-tetrahydrocannabinolic acid A; THCA-B, 4-COOH-THC.

FIG. 2.

Analytical RP-HPLC of THCA-A sample (lower panel) and THC standard (upper panel); absorbance detected at 210 nm. HPLC, high performance liquid chromatography.

FIG. 3.

Binding affinity of THCA-A and THC illustrated in competition binding curves against [³H]CP55,940. CB₁ on the left (A) and CB₂ on the right (B). Data are representative data from a single experiment and data points represent mean±SEM for triplicate data points. CB₁, cannabinoid receptor subtype one; CB₂, cannabinoid receptor subtype two; SEM, standard error of the mean.

FIG. 4.

Efficacy of THCA-A and THC in cAMP assays, CB₁ on the left, CB₂ on the right. (A–D) Show representative images of the biosensor traces of single experiments carried out in duplicate. (A, B) Show that THC (10 μM) inhibits cAMP at both CB₁ (left) and CB₂ (right). (C, D) Show the same assay carried out with THCA-A. THCA-A can be seen to inhibit forskolin mediated cAMP alone and to partially antagonize the ability of CP55,940 to inhibit cAMP at CB₁, but has no effect under equivalent conditions at CB₂. The lower panels (E, F) are summary data for the area under the curve analyses for all replicate assays combined (n=5 for CB₁ or n=6 for CB₂). *p=0.0065 by paired t-test, n=5. ^#p=0.015 t-test compared to 100%. cAMP, cyclic adenosine monophosphase.