The recent federal approval of hemp agriculture and its vast array of non-tetrahydrocannabinol cannabinoids has opened up new and exciting possibilities regarding clinical research using these cannabinoids across a variety of disorders. Most intriguing is the use of cannabidiol (CBD) as an anti-proliferative agent in canine cancer, considering limited study in human in-vitro systems suggests a mixed mechanism for dampening cancer cell proliferation at physiological and supra-physiological concentrations. We have recently examined the cytotoxic effects of CBD in canine cell culture showing strong anti-proliferative properties in the 1-3 μg/ml range and apoptosis within 8 hrs of treatment using modestly higher doses. This anti-proliferative effect translates into approximately 3-10 μM which is close to what can be achieved in the bloodstream based on a recent study examining CBD absorption in dogs. These rapid cytotoxic effects suggest alteration in cell signaling events and/or rapid mitochondrial permeability effects that should be explored due to the potential for CBD to alter AKT and MAP kinase signaling, which are current chemotherapeutic targets (rapamycin, c-kit inhibitors) and may possibly alter chemotherapeutic choices. More importantly, it is entirely possible that the anti-oxidant properties of CBD may hinder some chemotherapeutic effects which can be interrogated in cell culture systems prior to clinical use, in hopes of providing the necessary knowledge regarding appropriate doses, timing of administration and safe use of CBD pharmaceuticals or nutraceuticals in oncology patients.
The anti-inflammatory properties of CBD make hemp oil a promising adjunct therapy in veterinary medicine. A formulation of hemp oil containing CBD and CBDA may have superior antiinflammatory effects than CBD alone. Notably, CBDA inhibits cyclooxygenase-2 (COX-2), attenuating inflammation. However, specific anti-inflammatory effects of a mixed CBD/CBDA hemp oil remain unknown in the dog. This study includes three arms of testing to broadly assess the impact of CBD/CBDA on ex vivo and in vitro inflammatory responses using blood from 6 research colony dogs. First, we will test the effects of CBD, CBDA, and mixed CBD/CBDA (mCBD) on canine neutrophil function ex vivo. PMA-induced reactive oxygen species (ROS) generation, phagocytosis of fluorescent Ig-coated latex beads, and eicosanoid concentrations (PGE2 and LTB4) will be measured with commercially available kits my lab routinely uses. The Boyden chamber method will quantify neutrophil chemotaxis to recombinant canine IL-8. Second, we will evaluate the effect of CBD/CBDA on T cell function. We will measure the proliferative response of mitogen-stimulated T cells producing pro inflammatory (IFNgamma, IL-2, IL-4, IL-10, IL-17) and anti-inflammatory (IL-10) cytokine production ex vivo with optimized canine-specific EliSpot assays. Third, we will measure the effects of CBD, CBDA, and mCBD on canine fibroblast response as resident cells of most tissue types can elaborate eicosanoids and cytokines in response to pathogen components such as LPS from gram-negative bacteria. We will measure the effect of CBD/CBDA on LPS stimulated fibroblast eicosanoid production, cytokine production with a commercially available inflammatory array, and proliferation with the MTT assay in vitro. We hypothesize that the combination of CBD and CBDA will have superior anti-inflammatory effects compared to CBD alone. The proposed study lays the groundwork for future studies evaluating the anti-inflammatory effects of CBD/CBDA in vivo.