Neuropathic pain is a pathological condition characterized by spontaneous pain, hyperalgesia and allodynia. Clinical data indicate that approximately 50% of treated individuals were unresponsive to current pharmacotherapies, and in those that receive some benefit, pain relief was typically incomplete (Bonezzi and Demartini, 1999). Of particular therapeutic interest to us was neuropathic pain associated with chemotherapy-induced peripheral neuropathy (CIPN). In this adverse effect produced by many chemotherapeutic regimens, 30–40% of patients experience a progressive, enduring, and sometimes irreversible condition featuring pain, numbness, tingling and sensitivity to cold in the hands and feet (Gutierrez-Gutierrez et al 2010). In many cases, this neuropathic pain associated with CIPN can result in a dose-limiting side effect which, in severe cases, can progress to an irreversible condition (Argyriou et al., 2014). Associated effects on peripheral nerves can lead to oxidative stress and inflammation, sensitization and spontaneous activity of peripheral nerve fibers, and hyperexcitability in the dorsal column of the spinal cord leading to ascending pain pathway sensitization (Peters et al 2007). In addition to neuronal responses, Schwann cells and microglia respond to chemotherapy by stimulating the release of substances that enhance excitability which may contribute to pain hypersensitivity (Koyanagi et al.,2021; Shan et al., 2024).
With neuropathic pain being unresponsive to current therapies, the use of medical cannabis and CBD has achieved an anecdotal level of acceptance by caregivers. To date, the treatment for neuropathic pain by CBD has lacked a demonstration of efficacy in a large, randomized clinical trial. However, a small clinical study of oral CBD has been reported recently to attenuate early symptoms of CIPN in a trial that utilized CBD pretreatment prior to the initiation of chemotherapy (Nielsen et al., 2022). There have been no reports of a reversal of CIPN in any human trial with any drug, including cannabinoids. While verifiable human data has yet to be achieved, significant efficacy has been reported in animal models of CIPN focused on mechanical and cold allodynia in rodents treated with paclitaxel (Ward et al., 2014; King et al., 2017). These studies, which have focused on the ability of CBD to prevent the onset of allodynia associated with paclitaxel treatment, provided supportive evidence in a reproducible model that has driven our interest in this therapeutic area.
Despite the promising efficacy of CBD in preventing CIPN, the pharmacological limitations of this compound have been concerning: low potency, reduced efficacy, liver safety, and oral bioavailability (Brenneman et al., 2018; Foss et al, 2021). With the recognition that optimization of CBD was warranted, the development of CBD analogues was undertaken with the eventual emergence of KLS-13019 (Kinney et al, 2016), a novel compound which has been shown to be effective in both the prevention and reversal of allodynia in a mouse model of CIPN (Foss et al, 2021). Because of the previously demonstrated overlap of preventative efficacy in a mouse model of CIPN, these two cannabinoids (CBD and KLS-13019) were chosen to compare for their effects on reversing inflammation.
While various animal models of CIPN have shown that peripheral and central glial cells exhibit increased pro-inflammatory responses (Lees et al, 2017), our studies have shown that paclitaxel treatment also increases pro-inflammatory responses in DRG neurons, indicating an end point targeting for inflammatory activation of this cell type as well (Brenneman et al., 2022). Because of the suggested importance of inflammation in CIPN (Makker et al., 2017; Fumagalli et al., 2021), the focus of our studies pivoted to selecting relevant molecular targets for inflammation in neurons. As with the reported neuroprotective effects mediated by regulation of mNCX-1 (Brenneman et al., 2019), the strategy for selecting target candidates relevant to inflammation was based on CBD and endocannabinoid pharmacology (Bih et al., 2015; Guerrero-Alba et al, 2019). GPR55 has been described as an endocannabinoid GPCR associated with pain and inflammation (Staton et al., 2008). More recently, a study has reported positive effects of GPR55 receptor antagonism in a rodent model of formalin-induced inflammatory pain (Okine et al., 2020). Thus, with the focus of our mechanistic studies shifting to inflammation, consideration of GPR55 and the inflammasome-3 (Kelley et al., 2019) were addressed experimentally in the dorsal root ganglion (Krames, 2014). Previous studies had indicated that GPR55 played an important role in pain modulation (Schuelert and McDougall, 2011; Staton et al., 2008). Furthermore, earlier data had suggested a proinflammatory role for GPR55 in innate immunity (Chiurchiu et al., 2015). Based on these previous reports and our own observations which demonstrated that paclitaxel elicited increases in GPR55 immunoreactive area in DRG cultures (Brenneman et al., 2022), we decided on GPR55 as the major focus for our neuroinflammation studies relevant to CIPN. In addition, an emerging concept was that chemotherapeutic agents (including paclitaxel) promote inflammatory responses through activation of the NLRP3 inflammasome (Zeng et al., 2019). Our recent studies confirmed the potential role of NLRP3 and IL-1b, critical components of inflammasome-3, in mediating the anti-inflammatory responses of KLS-13019 (Brenneman et al., 2022).
In the studies to be reported, high content fluorescent imaging of dissociated, embryonic day 19-DRG neurons were used to assess target depletion by siRNA in this model system that is relevant to CIPN (Guo et al., 2017). While siRNA knockdown strategies typically use mRNA levels, target immunoreactivity measures from a Western blot analysis or biological attenuation of function as a means of assessing target depletion, the present studies employed a combination of methodologies. With the utilization of high content imaging applied to the diversity and complexity inherent to primary sensory neuron morphology, the goal was to compare the neuronal knockdown of GPR55 immunoreactive spot target area and the attenuation of efficacy for anti-inflammatory actions produced by the two cannabinoids. Importantly, by utilizing the capability of high content imaging, the feasibility of distinguishing and comparing the responses of neurites and cell bodies of sensory neurons in regard to GPR55 immunoreactivity was realized. The overall goal was to assess the relationships between specific cellular locations of target depletion with that of the siRNA-mediated attenuation of anti-inflammatory efficacy produced by the cannabinoid compounds of interest.
In addition to the anti-inflammatory properties of KLS-13019 and CBD, the current studies also examined effects of these cannabinoids on paclitaxel-induced neurite retraction, as neuritic length and area are included in the array of neuronal parameters routinely assayed in high content imaging. Previous studies employing a variety of DRG preparations have indicated that paclitaxel produced significant decreases (> 50%) in neurite length in dorsal root ganglion cultures after 24–48 hours of treatment (Scutteri et al., 2006; Chen et al, 2015). Previous demonstrations of intervention of paclitaxel-induced toxicity and inhibition of neurite outgrowth utilized pre-treatment paradigms to demonstrate protection by test compounds. In the studies to be described, DRG cultures were pretreated with paclitaxel for 8 hours followed by 16-hour treatment with either CBD or KLS-13019, without the removal of paclitaxel. This strategy was aimed at evaluating possible cannabinoid-induced reversal of neurite retraction in DRG neurons produced by paclitaxel. In addition, because these cultures were pre-treated with GPR55 siRNA, it was an accompanying goal to determine if knockdown of this target produced an attenuation of cannabinoid-related reversal from paclitaxel-induced neurite retraction. Indeed, demonstration of the reversibility of both inflammation or neurite retraction from paclitaxel was confirmed with KLS-13019 treatment, but not with cannabidiol.