Anti-Inflammatory Properties of KLS-13019: a Novel GPR55 Antagonist for Dorsal Root Ganglion and Hippocampal Cultures

KLS-13019, a novel devised cannabinoid-like compound, was explored for anti-inflammatory actions in dorsal root ganglion cultures relevant to chemotherapy-induced peripheral neuropathy (CIPN). Time course studies with 3 µM paclitaxel indicated > 1.9-fold increases in immunoreactive (IR) area for cell body GPR55 after 30 min as determined by high content imaging. To test for reversibility of paclitaxel-induced increases in GPR55, cultures were treated for 8 h with paclitaxel alone and then a dose response to KLS-13019 added for another 16 h. This “reversal” paradigm indicated established increases in cell body GPR55 IR areas were decreased back to control levels. Because GPR55 had previously reported inflammatory actions, IL-1β and NLRP3 (inflammasome-3 marker) were also measured in the “reversal” paradigm. Significant increases in all inflammatory markers were produced after 8 h of paclitaxel treatment alone that were reversed to control levels with KLS-13019 treatment. Accompanying studies using alamar blue indicated that decreased cellular viability produced by paclitaxel treatment was reverted back to control levels by KLS-13019. Similar studies conducted with lysophosphatidylinositol (GPR55 agonist) in DRG or hippocampal cultures demonstrated significant increases in neuritic GPR55, NLRP3 and IL-1β areas that were reversed to control levels with KLS-13019 treatment. Studies with a human GPR55-β-arrestin assay in Discover X cells indicated that KLS-13019 was an antagonist without agonist activity. These studies indicated that KLS-13019 has anti-inflammatory properties mediated through GPR55 antagonist actions. Together with previous studies, KLS-13019 is a potent neuroprotective, anti-inflammatory cannabinoid with therapeutic potential for high efficacy treatment of neuropathic pain.


Introduction
Neuropathic pain is a pathological condition characterized by abnormal pain sensations, including 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 chemotherapyinduced 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). Three key mechanisms have been proposed in the development of CIPN: mitochondrial dysfunction, loss of Ca homeostasis, and oxidative stress (Celsi et al. 2009;Han and Smith 2013;Canta et al. 2015). 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). Furthermore, various models of neuropathic pain have demonstrated changes in hippocampal functioning including memory deficits (Tyrtyshnaia and Manzhulo 2020) and impaired long-term potentiation (Kodama et al. 2007).
Recently, there has been a resurgence in interest in the potential medical utility of the cannabis plant and its constituents, particularly in the treatment of pain. Cannabidiol (CBD) is a non-psychoactive component of Cannabis sativa that is neuroprotective, with reported effects on reducing pain and allodynia in various models (Milost et al. 2020). We have recently reported that CBD prevented the development of paclitaxel-induced mechanical sensitivity in mice in vivo (Ward et al. 2011(Ward et al. , 2014(Ward et al. , 2017. Additionally, we and others have demonstrated that CBD and other nonpsychoactive cannabinoids are effective in animal models of spinal cord injury-associated neuropathic pain (Li et al. 2018). However, CBD has limitations in terms of potency, efficacy, safety, and oral bioavailability (Brenneman et al. 2018;Foss et al. 2021). With the recognition that optimization of CBD was warranted, the development of analogues of CBD was undertaken with the eventual emergence of KLS-13019 (Kinney et al. 2016), a novel compound that has been shown to be effective in both the prevention and reversal of allodynia in a mouse model of CIPN (Foss et al. 2021).
While a previous report indicated that both CBD and KLS-13019 were effective in preventing allodynia, only KLS-13019 was shown to be effective in reversing allodynia that was established in mice treated with paclitaxel, a chemotherapeutic agent recognized to reproducibly produce allodynia in mice. Using identical treatment paradigms, these pivotal background studies clearly indicated CBD was not effective in reversing allodynia, although morphine tested under the same conditions did result in high efficacy reversal of mechanical allodynia. Earlier mechanistic studies of effects on KLS-13019 and CBD focused on neuroprotective effects produced through mitochondrial NCX-1, a Na-Ca exchanger that regulates calcium levels important to preventing neuronal injury (Ryan et al. 2009). Pharmacological blockade and siRNA-mediated reduction of mNCX-1 were shown to effectively inhibit the neuroprotective effects of both CBD and KLS-13019 in rat dorsal root ganglion cultures (Brenneman et al. 2019). Thus, it was concluded that short-term (3-5 h) neuroprotection from paclitaxel toxicity in DRG neurons produced by CBD and KLS-13013 was mediated through mNCX-1. However, a longer term treatment with paclitaxel clearly produced an important difference in the responses between the two compounds on reversing allodynia in the mouse CIPN model. Because of the known importance of neuroinflammation in CIPN (Fumagalli et al. 2021), the focus of our studies pivoted to mediators of inflammation. As with the neuroprotective effects mediated by regulation of mNCX-1, the strategy for selecting target candidates relevant to inflammation was based on cannabidiol 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). Recently, Okine et al. (2020) have demonstrated positive effects of GPR55 receptor antagonism in a rodent model of formalininduced inflammatory pain.
Thus, with the focus of our mechanistic studies shifting to inflammation, consideration of GPR55 and the inflammasome-3 were addressed experimentally in DRG and hippocampal cultures. Previous studies had suggested that GPR55 played an important role in pain modulation (Schuelert and McDougall 2011;Staton et al. 2008). Previous data had suggested a proinflammatory role for GPR55 in innate immunity (Chiurchiu et al. 2015). Furthermore, based on these previous reports and our own preliminary data which indicated that paclitaxel elicited increases in GPR55 immunoreactive area in DRG cultures, the present studies were undertaken. In addition, an emerging concept was that chemotherapeutic agents (including paclitaxel) promote inflammatory responses through activation of the NLRP3 inflammasome (Zeng et al. 2019). Therefore, the potential role of NLRP3, a critical component of inflammasome-3, was also examined.
In the studies to be described, paclitaxel and the lysophosphatidylinositol arachidonate (LPIA), an endogenous agonist of GPR55 (Yamashita et al. 2013), were shown to produce a rapid and robust increase in GPR55 immunoreactive area in DRG cultures. These elicited increases in GPR55 were completely prevented and reversed by co-treatment with KLS-13019. Furthermore, increases in proinflammatory IL-1β and NLRP3 immunoreactive area were also responsive to KLS-13019 treatment, bringing their increased immunoreactive area levels back to that of control cultures. Together, the proposed rationale for KLS-13019 in mediating anti-inflammatory actions resides in a GPR55 antagonist outcome that can block the inflammatory actions of both paclitaxel and LPIA in rat DRG cultures.

Materials
Alamar blue and nerve growth factor were obtained from Invitrogen (Eugene, OR). Paclitaxel was obtained from Teva Pharmaceuticals USA (Sellersville, PA) as a 6 mg/ml solution containing 527 mg polyoxyl 35 castor oil, 2 mg citric acid, and 49.7% dehydrated alcohol/ml. CY-09 (SML2465) and cannabidiol were obtained from MilliporeSigma.
The synthesis of KLS-13019 has been described previously in detail (Kinney et al. 2016). Verification of the structural identity for KLS-13019 was determined by 1 H NMR, 13 C NMR, HMBC, HSQC, COSY, NOESY, LC/ UV, and LC/MS. The purity of KLS-13019 was 98.6% as determined by LC/MS.

Culture Models
Dissociated dorsal root ganglia (DRG) cultures derived from embryonic day 18 rats were employed as the primary assay system to explore the anti-inflammatory mechanism of action for KLS-13019 in the context of reversing established inflammation associated with paclitaxel treatment. In brief, rat DRG were obtained commercially through Brain Bits (Springfield, IL) and cultures prepared according to methods described previously (Brenneman et al. 2019). Tissue was dissociated with a papain-based kit from Worthington Biochemical Corporation (Lakewood, NJ). The DRG cells were plated at low density (10,000 cells/ well) in a 96-well format and maintained in a serum-free medium consisting of Neurobasal Medium supplemented with B27, GlutaMAX (Gibco), and 25 ng/ml nerve growth factor. Poly-D-lysine-coated plates (BD Biosciences, Franklin Lakes, NJ) were employed for this culture system. Prior to the initiation of experiments between days 5 and 9 in vitro, a complete change of medium was performed in a working volume of 100 µL.
Anti-inflammatory actions of KLS-13019 were also studied in hippocampal cultures that were prepared by methods previously described (Brenneman et al. 2018). In brief, dissociated hippocampal cultures derived from embryonic day 18 rats were employed as a second test system to assess responses in a central nervous system preparation that exhibited the expression of GPR55, a drug-target candidate of KLS-13019. The primary purpose of using this culture system was to measure inflammatory responses produced by lysophosphatidylinositol arachidonate (LPIA), a recognized endogenous agonist of GPR55 (Gangadharan et al. 2013). Hippocampal tissue was obtained commercially through Brain Bits (Springfield, IL). Tissue was dissociated with a papain-based kit from Worthington Biochemical Corporation (Lakewood, NJ). The hippocampal neurons were platted at low density (10,000 cells/well) in a 96-well format and maintained in a serum-free medium consisting of Neurobasal Medium supplemented with B27 and GlutaMAX (Gibco). Poly-L-lysine-coated plates (BD Biosciences, Franklin Lakes, NJ) were used because of the preferential adherence and survival of neurons on this matrix support. Prior to the initiation of experiments between days 11 and 21 in vitro, a complete change of medium was performed in a working volume of 100 µL.

Culture Treatments and Inflammation
The primary purpose of these studies was to assess two relevant culture models for their GPR55-related responses to inflammation and then to test if our novel cannabinoid (KLS-13019) had anti-inflammatory actions on cultured neurons. Of clinical relevance to neuropathic pain, DRG cultures were treated with 3 µM paclitaxel, a chemotherapeutic agent with known inflammatory properties (Staff et al. 2020). The toxic level of paclitaxel (3 µM) used for all the present studies was based on both clinically relevant serum concentrations and a previously determined toxic concentration (3 µM) that also produced increased levels of reactive oxygen species as detected with 6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. KLS-13019 was dissolved in dimethyl sulfoxide (DMSO) to obtain a 1 mM stock solution and then serially diluted with sterile Dulbecco's phosphate-buffered saline (DPBS) from Gibco (Grand Island, NY). At the highest concentration tested (1 µM), the concentration of DMSO was ≤ 0.1%. To study the potential effect of KLS-13019 in reversing paclitaxel-induced inflammation, DRG cultures were pre-treated with either 3 µM paclitaxel or 1 nM LPIA to establish inflammatory responses with increases in IL-1β and NLRP3 immunoreactive (IR) spot area (inflammasome-3 marker) being demonstrated. After the establishment of an inflammatory response, KLS-13019 was added to the cultures for an additional 16 h, with the paclitaxel remaining on the cultures. At the conclusion of the 16-h treatment period, cultures were fixed and assays as described in the following immunofluorescent assay section. This sequence of 8-h paclitaxel treatment followed by KLS-13019 treatment will be referred to as the "reversal" paradigm for DRG cultures throughout these studies.
In the present studies that utilized DRG tissue from a commercial source, replicate plates from separate preparations were conducted for all treatment conditions to establish reproducibility and experimental variance. Whenever possible, the results from two separate replicate preparations were combined for all data on the basis of similar control responses. In some cases where controls differed significantly between preparations, representative experiments were utilized for presentation, as combining the data was not feasible. In all cases, the number of determinations and the number of wells analyzed are shown in each figure legend to indicate the mean values and standard errors for each treatment group and for each assay employed. Using this approach, the key pharmacological potency of KLS-13019 was found to be similar and sub-nanomolar in the case of all the diverse assays employed throughout the study. Importantly, within each assay, the efficacy responses of KLS-13019 in reversing paclitaxel-or LPIA-mediated changes were equivalent to that of control values.
The primary purpose of the hippocampal studies that investigated the responses produced by treatment with lysophosphatidylinositol arachidonate (LPIA), a recognized endogenous agonist of GPR55, was to elicit changes in inflammatory responses that were targets for reduction by KLS-13019. Based on preliminary experiments, it was determined that 1 nM LPIA produced the maximal increases in GPR55 after 2 h of incubation. To assess hippocampal cultures with a similar "reversal" paradigm for inflammation as that conducted for DRG, a shorter period of treatment (8 h) was utilized. Hippocampal cultures were treated with LPIA (1 nM) to establish inflammatory responses as measured with IL-1β and NLRP3 for 4 h. After sister cultures were assayed for viability with alamar blue and fixed, other cultures in the same experiment were treated with KLS-13019 to test for possible reversal of the inflammatory responses during an additional 4-h treatment. This later treatment with KLS-13019 was done without the removal of the LPIA. At the conclusion of this second 4-h treatment period, the cultures were assayed for viability and then fixed for immunofluorescence assays of inflammation-related targets.

Immunofluorescent Assays
To assess the effects of various KLS-13019 treatments, immunofluorescent methods were used to measure neuronal responses in both DRG and hippocampal cultures. The goals for these assays included (1) identification of neuronal structures with antibodies to type III beta tubulin; (2) to assess the immunoreactive spot area of selected molecular targets (IL-1β, GPR55, and NLRP3) with their respective primary antibodies and distinctively labeled secondary antibodies; and (3) to compare the relative responses of the molecular targets in both neuronal cell bodies and neurites. Prior to fixation, the growth medium was removed and the wells were rinsed one time with 100 µL DPBS (37 °C). This warm rinse is particularly important to maintain structural stability of neurites. After removal of the DPBS, cultures were fixed for 20 min at room temperature with 50 µL/well of 3.5% formaldehyde (Sigma-Aldrich: 252,549) in warm (37 °C) DPBS that contained 5.5 µg/mL of Hoechst 33,342 dye (Invitrogen: H3570) to label cell nuclei. After removal of the fixative, the cultures were rinsed twice with 100 µL of DPBS and then a permeabilization-blocking buffer containing 5% normal goat serum and 0.3% Triton X-100 in DPBS was added to the cultures for 10 min. After removal of the blocking buffer, the cultures were rinsed twice with 100 µL of DPBS and then primary antibodies were added for 1-h incubation at room temperature. Neurons were identified with antiserum to type III beta tubulin (tuj 1) to measure changes in all neuronal structure parameters. The primary antiserum employed was a rabbit polyclonal obtained from Sigma-Aldrich (T2200) and used at 1:250 dilution. The secondary antibody was an Alexa Fluor 488-conjugated Fab fragment of goat anti-rabbit IgG obtained from Life Technologies (A11070) used at 1:600 for 30 min. After the secondary antibody treatment, cultures were rinsed 3 times with 100 µL of DPBS before performing high content fluorescent analysis. For storage, the wells were placed in 100 µL of sterile DPBS, with the plates wrapped in aluminum foil and maintained at 4 °C. For the detection of inflammatory markers, the following primary antibodies were used: IL-1β (PA5-88,078); NLRP3 (PA5-79,740); and for GPR55 (ab203663). All primary antibodies for IL-1β and NLRP3 were obtained from Life Technologies. The GPR55 antibody was obtained from Abcam. All primary antibodies were diluted at 1:250 and all secondary antibodies were used at 1:600. The secondary antibodies were obtained from Life Technologies. The following Alexa Fluor dyes labeled the secondary antibodies: Alexa Fluor 488 (A11070), 555 (A32732), 687 (A32733), and 750 nm (A21039).

High Content Image Analysis
The immunofluorescent assays were conducted on the Cell Insight CX5 high content imaging system (Thermo Fisher Scientific). The system is based on an inverted microscope that automatically focuses and scans fields of individual culture wells using a motorized stage at predetermined field locations. Fluorescent images from individual fields (895 µm × 895 µm were obtained with a 10 × (0.30NA) Olympus objective and Photometrics X1 CCD camera, with analysis by HCS Studio 2.0 Software. The light source was LED with a solid-state five-color light engine used with filter sets that had the following excitation/emission: 386/440, 485/521, 560/607, 650/694, and 740/809). With this capability, multiple fluorescent assays in a single well were conducted. Images were acquired in a low-resolution mode (4 × 4 binning). Image analyses for neuronal cell bodies and neurites were performed with the Cellomics Neuronal Profiling BioApplication. For analysis of neurons, objects were identified as cells if they had valid nuclei and cell body measures based on size, shape, and average intensity. Acceptable ranges were determined in preliminary studies to ensure that aggregated cells and non-cellular objects were excluded from the analysis.
For both the DRG and hippocampal cultures, the goals were to examine the immunoreactive spot areas for all the analytes of neurons only. Because the neuronal morphology was different between the two cultures, unique size and shape parameters for cell bodies and neuritic arbors were empirically determined for each culture type in preliminary studies. Once these parameters were determined, the analyses for each culture type were used throughout their respective experiments. Important to these analyses, an essential goal was to compare the immunoreactive spot areas for all analytes in both cell bodies and neurites. Type III beta tubulin immunoreactivity was used to identify the neurons for each culture type (Brenneman et al. 2019). For DRG cultures, twenty predetermined fields of view were sampled in each of six replicate wells per plate, with two replicate plates from different cellular preparations used for each assay. This extensive sampling of the low-density cultures was conducted as some fields contained 1-3 neurons while other fields contained complex networks of 10 or more neurons. The age of the DRG cultures at the time of analysis was 8-10 days after plating. Because primary cultures exhibit a variety of neuronal phenotypes and a range of morphological complexities, extensive sampling was employed to obtain an average neuronal response with the inflammatory markers. For measuring parameters of type III beta tubulin immunoreactivity and spot analysis for the inflammatory markers, the Cellomics Neuronal Profiling Bioapplication was used that combined spot analysis on neurons that resided within this bioapplication. For analysis of spot immunoreactivity with this bioapplication, a key parameter was the empirical establishment of fluorescent thresholding that permitted the use of a dynamic range that optimized the measurement of fluorescent differences among the treatment groups as well as distinguishing the fluorescent signal from the background. This thresholding level was set based on our previous experience with antibody-based assays and the smallest distinguishable size of immunoreactive spots (radius:1.5 µ) under our imaging conditions. With this algorithm, the immunoreactive area was a relative measure that was characterized by an effective computerized spot analysis in a rapid screening mode. Importantly, the same imaging parameters for neurons from all treatment groups were employed for the DRG studies. The key comparisons in these studies were aimed at measuring the changes in the immunoreactive area that were associated with KLS-13019 treatment. Due to the observed differences in the cellular distribution among the analytes, the cellular locations of all inflammatory markers in DRG were determined, thus distinguishing the relative changes between cell bodies and neurites. This capability and experimental focus were obligatory aspects of measuring the inflammatory markers by image analysis. Because dissociated hippocampal cultures exhibited more abundant neurons and had a more extensive neuritic arbor than the DRG cultures, ten predetermined fields of view were sampled in each of six to eight replicate wells per treatment group for each plate, with two replicate plates from different cellular preparations used for each assay. The fluorescent thresholding parameters for the immunoreactive spot analysis for hippocampal cultures were similar to that employed for the DRG cultures. Again, the goal of the studies was to obtain measures of the relative changes in the immunoreactive area for each of the analytes after KLS-13019 treatment. In contrast to the DRG cultures, the experiment with hippocampal cultures was conducted 14-20 days after plating. As in the case of DRG cultures, field sampling was extensive in order to obtain an assessment of the average neuronal response to the pharmacological treatments. For each experiment, the values are the mean from 6 wells with 10 fields per well being analyzed for each treatment group. An estimate of 400-600 neurons were assessed for each treatment. In all cases, the results were expressed as the immunoreactive area of each of the analytes per neuron.
Since CBD had played a prominent role in the discovery and mechanistic history of KLS-13019, preliminary studies also were conducted in hippocampal cultures to investigate the possible activity of this cannabinoid on the GPR55 immunoreactive area after treatment with 1 nM LPIA. Treatment with 10 µM CBD for 6 h had no detectable effect on LPIA-induced increases in GPR55 IR area in either neurites or cell bodies of hippocampal neurons (data not shown). Furthermore, CBD treatment produced only low efficacy protection (40% of maximum) from LPIA-mediated decreases in cellular viability as assessed by the alamar blue assay. Since these screening studies with CBD and GPR55 were ineffective, further studies in DRG cultures were not performed.

Mitochondrial GPR55 Localization
Exploratory studies were conducted on day 21 hippocampal cultures to assess the possible localization of GPR55 within some mitochondria. These studies were undertaken because of the central importance of GPR55 in the inflammatory responses reported in the present study as well as our findings that both DRG and hippocampal cultures respond to increases produced by the GPR55 agonist: LPIA. The primary antibody used as a marker for mitochondria was GT6310 for cytochrome c oxidase 4 [COX4] (Life Technologies). GT6310, used at 1:300, is a mouse monoclonal antibody that was made to a recombinant fragment between amino acid 1-169 in COX4. The secondary antibody (A-21037) for the COX4 assay was goat anti-mouse labeled with Alexa Fluor 750 (Life Technologies) that was used at 1:1000. For measuring parameters of type III beta tubulin immunoreactivity and spot analysis for COX4, the Cellomics Neuronal Profiling Bioapplication was used that combined spot analysis on neurons that resided within this bioapplication. Conditions for detecting neurons and GPR55 were identical to that described previously. For analysis of spot immunoreactivity with this bioapplication, a key parameter was the empirical establishment of fluorescent thresholding which was based on our previous experience with antibodybased assays and the size of the spots which were set at the lowest limit for spot radius (1.5 µ). Under these imaging parameters, the spot overlap function was set a 100% in order to detect spot overlap with the highest stringency.
The goal of these studies was to use the highest exclusionary conditions to estimate if such colocalizations may exist within the limits of detection for this system. With the use of pseudo color imaging, 100% overlapped spots were reported with a blue color, while COX4-positive spots alone were reported as yellow and GPR55-positive spots alone were reported red.

Viability Assay
At the conclusion of some experiments, sister cultures to those assessed by image analysis were evaluated first with the viability dye alamar blue. Because of our interest in mitochondria, we noted with interest the suggestion (Iuchi et al. 2019) that the alamar blue dye "mainly" consisted of an assessment of succinate dehydrogenase reductive activity, an enzyme that participates in both the electron transport chain and the citric acid cycle. The preferred dye was alamar blue as this assay often was conducted first and then the dye washed off the cultures for subsequent fixation and follow-up immunocytochemical assays. On every plate, wells without cells were used to provide a blank reading that was used to subtract background fluorescence. For the alamar blue viability assay, 10 ul of the dye was added directly to the culture well that contained 100 ul of nutrient medium. Incubation times with the dye ranged from 2 to 4 h. Fluorescence was measured at an excitation of 530 nm and an emission of 590 on a Cytofluor plate reader.

β-arrestin Assay
A commercial assay for β-arrestin was obtained from Eurofins that provided a means of testing human GPR55 in a cell line (93-024C2) that had a background of CHO-K1. In this assay, agonist-induced activation of GPR55 stimulated binding of β-arrestin to the ProLink-tagged GPCR and forces complementation of two enzyme fragments that resulted in the formation of an active β-galactosidase enzyme. The Pro-Link fragment of β-gal was a low-affinity enzyme donor that was stably expressed with β-arrestin tagged with an enzyme acceptor. This interaction leads to an increase in enzyme activity that was measured using chemiluminescent Path-Hunter detection reagents. For our application, the GPR55 agonist lysophosphatidylinositol (LPI) was tested from 0.1 nM to 30 µM to increase the relative luminescent signal relative to that of control. Incubations were conducted for 90 min at 37 °C in 5% CO 2 . Lysophosphatidylinositol (LPI) produced a robust and reproducible signal at 10 µM in this transfected system. For the GPR55 antagonism assay, KLS-13019 concentrations ranging from 0.1 nM to 30 µM were pre-incubated with the cells for 10 min prior to the addition of 16 µM LPI. Concentrations of KLS-13019 ranging from 0.1 nM to 30 µM were tested alone for possible effects of agonistic activity on β-arrestin. The data indicated that KLS-13019 had no detectible agonist activity in the β-arrestin assay.

Statistical Analysis
All statistical comparisons were made by ANOVA, with the normality of values tested by the Shapiro-Wilk test followed by a multiple comparison of means test with the Holm-Sidak method as performed through SigmaPlot 14. All EC50 and IC50 values were generated by the curve-fitting procedure provided by the 4-parameter logistic analysis.

Results
The goals of these studies included an exploration of a potentially novel mediator (GPR55) of inflammatory responses to paclitaxel treatment of dorsal root ganglion cultures and the testing of our lead compound (KLS-13019) for anti-inflammatory actions. Since the initial interest was to establish an association of a chemotherapeutic agent (paclitaxel) with inflammation, a time course of changes in the immunoreactive (IR) area of a putative proinflammatory target (GPR55) was measured in dorsal root ganglion cultures after treatment with paclitaxel. In Fig. 1a, a comparison of GPR55 IR area was made in cell bodies and neurites as measured by high content imaging in neurons that were identified with antibodies to type III beta tubulin. These initial studies indicated that significant increases in GPR55 IR area were apparent in the cell bodies after 15 min of incubation and plateauing after 30 min (p < 0.01). The paclitaxelinduced increases in cell bodies were 1.9-fold greater than that observed in control cultures. No further increases in cell body GPR55 were observed up to 8 h of incubation. In contrast, the area of GPR55 in neurites after treatment with 3 µM paclitaxel remained unchanged throughout the 8-h time course. While these effects of paclitaxel were relevant to our interest to chemotherapy-induced peripheral neuropathy (CIPN), further exploration of GPR55 responses was conducted with a recognized agonist (Gangadharan et al. 2013) of GPR55: lysophosphatidylinositol arachidonate (LPIA). Based on preliminary experiments of concentration-effect responses of LPIA to determine maximally effective conditions, a time course of 1 nM LPIA on GPR55 IR area was conducted with the rat DRG cultures. As shown in Fig. 1b, significant increases in GPR55 IR area were observed in neurites after 30 min of incubation, with an apparent plateau in the response observed between 30 min and 8 h of incubation. The LPIA-induced increases in neuritic GPR55 were 40% greater than that of control cultures. In contrast to the responses of paclitaxel shown in Fig. 1a, 1nM LPIA produced no significant increases in GPR55 IR area in the cell bodies of the DRG during the 8-h time course. While the time course of both paclitaxel and LPIA produced significant changes in GPR55 in the DRG neurons, the responses between cell bodies and neurites were consistently different between these two regions of the neurons that were dependent on the nature of the compound used to elicit the GPR55 increases. In addition, the magnitude of the GPR55 IR area response in cell bodies after paclitaxel was much greater (1.9-fold) than that observed in the neurites after LPIA treatment (40%). Although the cellular responses between the two agents were different, the conditions of image analysis parameters involving cell body and neurite demarcations for DRG neurons were the same between the two series of time courses. Together, these data indicated that significant increases in GPR55 IR area were rapidly produced (30 min) in DRG neurons after treatment with either a GPR55 agonist or the chemotherapeutic agent paclitaxel.
A second major goal of these studies was to test if changes in GPR55 IR area were reversible after treatment with KLS-13019, a novel compound that has been shown to reverse mechanical allodynia in paclitaxel-treated mice (Foss et al. 2021). To test for reversibility of paclitaxelinduced increases in cell body GPR55 IR area, DRG cultures were pre-treated for 8 h with 3 µM paclitaxel and then the cultures were co-treated for an additional 16 h with various concentrations of KLS-13019 in the continuing presence of paclitaxel. As shown in Fig. 2a, treatment with KLS-13019 for 16 h produced a concentration-dependent decrease in GPR55 cell body area back to control levels. The IC50 for this effect was shown to be 117 ± 56 pM. In addition, the effect of the 8-h pre-treatment with paclitaxel alone is shown with the reference line for GPR55 IR area in cell bodies. These studies indicated that a complete reversal of the increases in GPR55 IR area was demonstrated with KLS-13019 treatment. While the demonstration of reversibility of the increased GPR55 was of significance, cell viability also was measured in cultures from these experiments as shown in Fig. 2b. Using the same treatment paradigm as that for the GPR55 responses, KLS-13019 treatment was shown to reverse back to control levels the paclitaxel-induced decreases in cell viability as measured with alamar blue. Thus, paclitaxel treatment alone for 8 h produced a 40% decrease in cellular viability in the DRG cultures that could be restored to control levels by 16-h treatment with KLS-13019 with an EC50 of 200 ± 46 pM.
To address the possibility that proinflammatory markers may be playing a role in the responses of DRG cultures to paclitaxel and LPIA, additional analytes for IR area for IL-1β (proinflammatory cytokine) and NLRP3 (inflammasome-3 component) were conducted. As shown in Fig. 3a, the time course of paclitaxel effects on IL-1β and NLRP3 IR area in cell bodies was compared. For IL-1β IR area (closed circles), significant (P < 0.05) increases were observed after 15 min of 3 µM paclitaxel treatment that reached an observed peak at 30 min. The amount of increase in IL-1β was 74% over that observed at the beginning of the incubation. Similarly The level of GPR55 area in control cultures is shown with the dashed reference line. Each point is the mean of 12 culture wells comprised of 20 fields from two replicate experiments. The standard errors of data points were ≤ 6% of the mean. The IC50 was determined from a 4-parameter logistic analysis. b Cell viability was measured with alamar blue using the same "reversal "treatment paradigm described above. The EC50 of increases in cell viability in DRG cultures are shown after treatment with KLS-13019 after a 24-h treatment period. The reference lines indicated cultures responses of control (dashed line) and 8-h treatment of paclitaxel (dot-dash) as in a. Each point is the mean of 6 determinations from a representative experiment. The standard error of each data point was ≤ 5-8% of the mean. The EC50 was determined from a 4-parameter logistic analysis for NLRP3 (open circles), significant increases (P < 0.001) from control levels were observed from 30 min to 8 h of 3 µM paclitaxel treatment. Each point is the mean of 6 well determinations obtained from 20 fields per well from a representative experiment. For IL-1β (closed circles), significant increases (P < 0.01) from control values were observed from 15 min to 8 h of paclitaxel treatment as shown by the symbol "^." Each point is the mean of 10 well determinations from 20 fields per well from replicate experiments. b The time course of 1 nM lysophosphatidylinositol arachidonate (LPIA) treatment of DRG cultures. All data were obtained from high content analyses of fluorescent neuronal images. Asterisk: for neuritic NLRP3 IR area (open circles), significant increases (P < 0.01) from control levels were observed from 15 to 30 min of 1 nM LPIA treatment. Each point is the mean of 8 well determinations obtained from 20 fields per well from replicate experiments. "^" For neuritic IL-1β immunoreactive area (closed circles), significant increases (P < 0.01) from control values were observed from 15 min to 8 h of paclitaxel treatment. Each point is the mean of 12 well determinations from 20 fields per well from replicate experiments after 1 nM LPIA treatment of DRG cultures. As shown in Fig. 3b, treatment with LPIA produced significant increases in neuritic IL-1β after 15 min of incubation (P < 0.017). A longer duration of treatment with LPIA did produce further increases in IL-1β to a peak at 1 h with significant decreases observed after 2 h of treatment. Similarly, LPIA (1 nM) produced increases in neuritic NLRP3 that peaked at 30 min, with more prolonged incubation producing an attenuation of observed increases produced by LPIA. Thus, both paclitaxel and LPIA had effects on both these proinflammatory markers, although the cellular location of these increases was different for the predominant effects between the two compounds, and the transient nature of the increases was apparent with LPIA, but not paclitaxel.
Like the studies on GPR55, exploration of the antiinflammatory actions of KLS-13019 was continued by measuring the effect of this compound on IR areas of both IL-1β and NLRP3 utilizing the "reversal" paradigm for DRG cultures. In this series of experiments, cultures were pre-incubated with 3 µM paclitaxel for 8 h after which the changes in inflammatory marker IR areas were measured. As shown by the 8-h dotted reference line of paclitaxel treatment alone in Fig. 4a, a significant increase in IL-1β was detected in the cell bodies of DRG neurons. With the reversal paradigm employed, treatment with KLS-13019 was initiated in cultures that had already been treated for 8 h with paclitaxel. The duration of the KLS-13019 treatment was for 16 h without removing the paclitaxel. The goal of this experiment was to test if a previously established inflammatory response from IL-1β could be returned to control levels with the KLS-13019 treatment, without removing the paclitaxel. As shown in Fig. 4a, a concentration-dependent decrease in IL-1β was observed in cell bodies, with an IC50 of 156 ± 45 pM. At a concentration of 1 nM KLS-13019, the mean level of IL-1β was not different from that of the control value in cell bodies. Similar studies as KLS-13019-mediated reversal studies also were conducted by measuring NLRP3 as shown in Fig. 4b. As with IL-1β, the 16-h treatment with KLS-13019 resulted in a concentration-dependent decrease in paclitaxel-induced increases in NLRP3 with an IC50 (140 ± 72 pM), similar to that measured with IL-1β. Together, these studies support the conclusion that KLS-13019 reversibly intervened in an established inflammatory response in cultured DRG neurons previously treated with paclitaxel during a 24-h test period.

GPR55 and Hippocampal Cultures
To further explore GPR55-mediated responses, a series of experiments were conducted in dissociated hippocampal cultures, with a particular interest in the reversibility of LPIA-mediated changes in a CNS preparation that is recognized to express GPR55. Utilizing a similar, but shorter (8 h) duration of treatment schedules, LPIA was used to   Fig. 5d. Together, the studies conducted in hippocampal neurons produced similar responses on GPR55 and inflammatory responses to that observed on neurons in DRG cultures. Importantly, KLS-13019 exhibited high potency and high efficacy anti-inflammatory actions in hippocampal cultures in addition to the observed effects on sensory neurons from DRG. To further explore the potential role of the NLRP3 inflammasome-3 in LPIA-related in hippocampal cultures, an inhibitor (CY-09) for the assembly of the inflammasome-3 complex was tested for the ability to block LPIAmediated increases in NLRP3 during a 6-h test period. Utilizing high content imaging, the increase in IR area of NLRP3 in neurites elicited by 1 nM LPIA was completely blocked by pre-treatment with 10 µM CY-09 as shown in Fig. 6a. In addition, the control levels of NLRP3 were not affected by CY-09 treatment alone. In the same cultures assayed for NLRP3 spot area, cell viability was measured prior to fixation to monitor possible outcomes as assessed with the alamar blue assay. As shown in Fig. 6b, treatment with 1 nM LPIA alone for 6 h resulted in a decrease to 58% of control values in the viability assay. Importantly, pre-treatment of hippocampal cultures with CY-09 prevented the loss of cellular viability produced by 1 nM LPIA. Together, these studies indicate that an inhibitor of inflammasome assembly (CY-09) can effectively block LPIA-mediated effects on increases in neuritic NLRP3 area and decreases in LPIA-mediated cellular viability. Because LPIA is a potent agonist of GPR55, these studies suggest that this orphan GPCR is proinflammatory with a capability of producing reversable decreases in both cellular viability and neuroinflammation.
To provide further evidence of KLS-13019 as a GPR55 antagonist, additional studies were conducted with a Discov-erX assay system with human GPR55 from which increases in β-arrestin produced by 16 µM LPI were completely blocked by 30 µM KLS-13019 (open triangles) in this model system (Fig. 7). In addition, as shown in the open circles, treatment with KLS-13019 alone produced no increases in β-arrestin, consistent with this compound having no agonist activity on human GPR55 in this model system. As a positive control, ML-193, a recognized high potency GPR55 antagonist, also blocked LPI-mediated increases in β-arrestin, consistent with this compound being a GPR55 antagonist.
Based on reports that mitochondria can co-localize with the NRLP3 inflammasome (Kelley et al. 2019), 3-week-old hippocampal cultures were analyzed by high content imaging for the presence of GPR55 IR spot area and COX4 IR spot area in type III beta tubulin-positive neurons. A spot colocalization algorithm within a neuronal profiling bioapplication was used to screen spots for GPR55 and COX4 that were 100% overlapping. Identification of such co-localized spots was displayed utilizing the pseudo color blue, with spots showing less than 100% overlap being displayed in red for GPR55 and yellow for COX4. A prominent area spot overlap of GPR55 and COX4 on a proximal neurite is highlighted in the left panel of Fig. 8. These data suggested that within the measurement limitations of these two IR spots of ≤ 1.5 µ radii, complete overlap could be detected. These data are consistent with the conclusion that GPR55 can be localized to mitochondria in sub-population hippocampal neurons.

Discussion
The precedent finding which led to the present studies was that treatment with KLS-13019, a novel cannabinoid, produced a complete reversal of mechanical allodynia in a mouse model of paclitaxel-induced peripheral neuropathy. This mouse model of CIPN employed chronic paclitaxel treatment for 1 week followed by the demonstration of mechanical allodynia after 11 days. A reversal paradigm used a 3-day treatment schedule with orally administered KLS-13019 followed by an assessment of mechanical allodynia again that was compared to pre-paclitaxel responses. This reversal finding was characterized by its dose-dependency that was complete in its efficacy in comparison to control responses. Since this response to chronic treatment was so robust and complete, a consideration of another contributing mechanism besides mitochondrial NCX-1 (Brenneman The standard error of each data point was ≤ 5-8% of the mean. The IC50 was determined from a 4-parameter logistic analysis. d Cell viability was measured with alamar blue using the same "reversal "treatment program described above. The EC50 of increases in cell viability in DRG cultures are shown after treatment with KLS-13019 after the 8-h treatment period. The reference lines indicated culture responses of control (dashed line) and a 4-h treatment with LPIA (dot-dash) alone to establish a cell viability response prior to treatment with KLS-13019. Each point is the mean of 8 determinations from a representative experiment. The standard error of each data point was ≤ 3-6% of the mean. The EC50 was determined from a 4-parameter logistic analysis et al. 2019) was undertaken in the dissociated DRG cultures, as the neuroprotective action of NCX-1 against paclitaxel toxicity previously reported was discovered under only acute (3-5 h) treatment conditions in which both KLS-13019 and CBD were equally efficacious and mechanistically dependent on NCX-1.
Because of the recognized complexity of CIPN involving both acute (hourly) and chronic (daily and weekly) components, an additional mechanism for KLS-13019 was explored that was based on three findings from the literature: (1) the emerging preclinical studies which suggested that neuroinflammation may be playing important roles in CIPN (Fumagalli et al. 2021); (2) evidence that an atypical cannabinoid receptor (GPR55) has a role in mediating neuropathic pain in a mouse model of CIPN (Staton et al. 2008); and (3) GPR55 receptor antagonism recently being reported to have positive effects in a rodent model of formalin-induced inflammatory pain (Okine et al. 2020).
Our investigation of GPR55 began with the exploration of paclitaxel-induced changes in GPR55 immunoreactive area in dissociated DRG neurons as shown in the time course of Fig. 1a. Since this analysis was conducted with high content imaging, it was possible to distinguish between increases that were detected in cell bodies and those that occurred in neurites of DRG cultures. These studies indicated that this putative proinflammatory GPCR was increased within 15-30 min of treatment with a clinically relevant amount (3 µM) of paclitaxel. The concept that emerged from this finding is that GPR55 may be among multiple sentinel mechanisms that are induced after exposure to proinflammatory drugs or endogenous agonists. Subsequent studies with one of these GPR55 agonists (lysophosphatidylinositol arachidonate [LPIA]) also produced an increase in GPR55, but the cellular localization of the elevations of this receptor was in the neurites, without apparent increases observed in the cell bodies. While the reason for this GPR55 localization difference between paclitaxel and LPIA is not yet apparent,  Fig. 6 a The effect of the inflammasome-3 assembly inhibitor Cy-09 is shown on LPIA-mediated increases in NLRP3 in hippocampal cultures after 6 h of treatment on day 16 in culture. Cultures were pretreated with 10 µM CY-09 for 30 min prior to treatment with 1 nM LPIA. At termination, cultures were fixed and analyzed by high content imaging of spot IR area for NLRP3 in neurons. Significant increases (P < 0.001) in neuritic NLRP3 area were observed in comparison to all other groups. Each bar is the mean of 8 well determination made from 10 fields per well from a representative experiment. The error bar is the SEM. b In the same cultures as utilized in a, prior to fixation for high content analysis, the cultures were assayed for cellular viability with alamar blue. Treatment with 1 nM LPIA produced a significant decrease in this viability assay that was 58 ± 3% of that observed for control. The LPIA-mediated decreases in cellular viability were prevented by pre-treatment with 10 µM CY09. Treatment with CY09 alone produced no significant difference from control  -193 (open triangles). ML-193 is a known GPR55 antagonist (Kurano et al. 2021). Each point is the mean of 6 determinations in a representative experiment from duplicate studies. The error par is the standard error it is possible that multiple or different processes could be elicited by the two substances. For example, it is known that paclitaxel inhibits antegrade axonal transport (LaPointe et al. 2013) and such an inhibition could slow the transport of induced GPR55 from the cell bodies to the neurites. In addition, the antegrade velocity of mitochondrial transport is significantly decreased by paclitaxel in a concentration-dependent process (Smith et al. 2016). Alternatively, because paclitaxel has been shown to mediate priming of the NLRP3 inflammasome activation through TLR4 (toll-like receptor-4) receptors in bone marrow-derived macrophages (Son et al. 2019), it cannot be excluded that multiple receptors could be involved in explaining the differential cellular location of IL-1β and NLRP3 increases after paclitaxel treatment. Although the mechanism(s) of receptor induction and receptor complexity/specificity remain to be established, these data clearly show that GPR55 is rapidly and potently inducible in DRG neurons by multiple substances. Of importance to a potential interaction with KLS-13019 is the observation that the induced increases GPR55 produced by paclitaxel could be prevented by this drug candidate as well as increases produced by LPIA in DRG neurites. This confluence of reversable actions produced by KLS-13019 may have a common origin: GPR55 antagonism by the drug candidate, although it is not known if paclitaxel has either a direct or indirect action at GPR55. This mechanism for KLS-13019 was further supported by the demonstrated GPR55 antagonism action in the β-arrestin effects shown in Fig. 7.
The observation of the present study wherein an endogenous agonist and a drug can increase the expression of a GPCR is an atypical, albeit a defining response. Plasma membrane GPCRs most often either remain unchanged in receptor density or are down-regulated after prolonged treatment with their agonist (Rajagopal and Shenoy 2018). In the present study, within 30 min both a GPR55 agonist and a chemotherapeutic drug both produced increases in GPR55. The present findings were characterized by several observations: (1) evidence was presented that GPR55 could be detected in neurites and cell bodies of hippocampal and DRG neurons; (2) GPR55 could be detected in mitochondria; and (3) a sub-population of GPR55 immunoreactive spots could be co-localized with mitochondria as determined within resolution limits by high content imaging of COX4 (a mitochondrial marker) and GPR55 in hippocampal neurons. While reports of other mitochondrial GPCRs have begun to appear, this remains an emerging area of pharmacology (Nezhady et al. 2020). Of potential pertinence to the present study, CB1 is among the GPCRs that have been reported in mitochondria. Furthermore, mitochondrial CB1 has been shown to be upregulated after traumatic brain injury, indicating that the expression of this cannabinoid receptor can also undergo an upregulation in response to a pathological stress (Xu et al. 2016). Of further interest, β-arrestin, a recognized Fig. 8 Immunofluorescence of GPR55 in day 21 hippocampal cultures as analyzed by a spot analysis from high content imaging after a 4-h treatment with 1 nM LPIA. A spot co-localization algorithm within the neuronal profiling bioapplication was used to screen spot IR area for GPR55 and COX4 that were 100% overlapping. Identification of such co-localized spots displayed utilizing pseudo color blue, with spots showing less than 100% overlap being displayed in red for GPR55 and yellow for COX4. A prominent area of blue spot overlap of GPR55 and COX4 on a proximal neurite is highlighted in the left panel magnification of the high content imaging. The calibration bar is 20 µ mediator of GPR55 signal transduction, has been shown to be localized on and within neuronal mitochondria (Suofu et al. 2017). Together, our data suggest that the mitochondrial GPR55 is both a target of paclitaxel in DRG neurons and perhaps a physiological mediator of inflammation in both hippocampal and DRG neurons that would be predicted to enhance the sentinel function for inflammation by producing an induction of the GPR55 receptor density. With the sentinel function as a working hypothesis, an upregulation of GPR55 may act as an amplification mechanism to further an inflammatory response, regardless if paclitaxel interactions are either directly or indirectly associated with this GPCR. In general, the present example may be suggestive of a broader concept that the location of a GPCR within an organelle has significant implications for differential pathophysiological functions distinct from that of homologous receptors located at the plasma membrane.
With a goal of linking the observed action of paclitaxel and LPIA to proinflammatory effectors, measurements of IL-1β and NLRP3 were measured in the DRG cultures. Cytokines have been associated with inflammatory pain and both are recognized mediators of CIPN in animal models (Staff et al. 2020). Of interest, a recent report has shown that LPI also can induce the secretion of the proinflammatory cytokines (IL-6 and TNF-α) from macrophages (Kurano et al. 2021). In the present study, the time course of paclitaxel-mediated increases of two mediators of inflammation (NLRP3 and IL-1β) and that of LPIA-mediated increases were similar, regardless of their localization in the neurons. While the sources of the inflammatory marker increases were presumed to be attributed to neuronal biosynthesis and processing, a contribution by non-neuronal cells also needs to be explored. Several studies have indicated a significant role of satellite glial cells in CIPN (Boyette-Davis et al. 2015;Makker et al. 2017). The impact of glial and immune cells needs further complementary studies in the context of high content analyses. While these studies are essential to our understanding of the complexities of inflammation, non-neuronal cells were beyond the scope of the present studies. The action of KLS-13019 in regulating IL-1β and NLRP3 in the reversal experiments depicted in Fig. 4a, b indicated a reversion back to control levels within 16 h of KLS-13019 treatment. The fact that KLS-13019-mediated effects on cellular viability was also observed within this reversal paradigm, indicating that a protective role was also produced (Fig. 2b). Together, these high content data are consistent with KLS-13019-mediated effects on recognized proinflammatory and pain mediators that can be detected in DRG neurons that may be consistent with a return to pre-paclitaxel homeostasis. While the proinflammatory mediators measured by high content imaging in the present study provided the initial observations, the significance of these findings would be substantially enhanced with the application of other analytical measures that include gene expression with RT-qPCR and possibly complementary measures of released inflammatory mediators by ELISA. However, it should be cautioned that these other techniques do not provide for a regional analysis of neuronal components, and therefore other techniques may not be able to detect those changes as observed from the high content analyses that are either intrinsic or predominant to either neurites or cell bodies.
An emerging concept is that chemotherapeutic agents (including paclitaxel) promote inflammatory responses through activation of the NLRP3 inflammasome (Zeng et al. 2019;Son et al. 2019). Although paclitaxel is believed to be among the substances that can drive "priming" for signalmediated events needed for the activation of inflammasomemediated assembly in macrophages, our findings indicate that this action of paclitaxel may be similar in DRG neurons. Importantly, a novel finding of the present disclosure is that paclitaxel treatment can also increase the expression of GPR55 in DRG cultures. Thus, an alternative explanation for paclitaxel-mediated "priming" is herein described: the concept is that GPR55 is a mediator of paclitaxel-induced "priming" of NLRP3 inflammasome assembly. With this finding, GPR55 is a "priming" signal for inflammasome assembly.
Our new data suggest that GPR55 is a sufficient, although not likely exclusive, priming signal in DRG cultures to mediate all of the increases in IL-1β, which is known to be elevated through the action of the NLRP3 inflammasome-3. Because paclitaxel can also produce toxic increases in mitochondrial calcium and elevated reactive oxygen species in sensory neurons, we have concluded that these actions of paclitaxel on the activation of the inflammasome complex are major actions produced by paclitaxel in sensory neurons. Thus, two roles of paclitaxel have been revealed in DRG neurons: (1) increases in the expression of GPR55; and (2) increases in ROS and calcium in the mitochondria (Canta et al. 2015). The rationale for KLS-13019 mediating anti-inflammatory actions produced by paclitaxel is that this agent is a GPR55 antagonist that can block the GPR55-mediated "priming" of NLRP3 inflammasome assembly. In the present disclosure, the KLS-13019-mediated antagonism of GPR55 actions is supported through effects observed on decreasing levels of IL-1β and NLRP3 in DRG neurons. The present disclosure presents evidence that KLS-13019 is completely effective in reducing paclitaxel-and LPIA-mediated increases in these inflammatory markers back to that of control levels.
As a result of the recognized importance of sensory neurons in DRG to transmitting peripheral nociception (Krames 2014) and the reported accumulation of paclitaxel and mitochondrial reactive oxygen species in DRG from models of CIPN (Duggett et al. 2016), the preparation of choice for the in vitro studies to study protection and antiinflammation was dissociated cultures from rat DRG. The inclusion of studies on hippocampal cultures was intended primarily to explore the scope of the anti-inflammatory actions of KLS-13019 in CNS-related neurons as well. However, KLS-13019 may have important therapeutic actions relevant to neuropathic pain in the hippocampus as suggested by previously recognized impairment of hippocampal functioning in animal models of peripheral nerve injury and neuropathic pain (Kodama et al. 2007;Tyrtyshnaia and Manzhulo 2020). Of particular relevance, hippocampal cultures also express both mNCX-1 and GPR55, the two KLS-13019-related drug-target candidates that mediate protection and anti-inflammatory responses, respectively. In this present study, the effects of LPIA on increased GPR55 immunoreactive area and elevations of NLRP3 and IL-1β were also observed in hippocampal cultures. Importantly, similar to the findings obtained in DRG cultures, KLS-13019 was able to both prevent and reverse the effects produced by LPIA in hippocampal neurons. Furthermore, the importance of the inflammasome-3 was extended with the hippocampal cultures to demonstrate that the effects of LPIA-mediated increases in NLRP3 spots area in neurons were prevented with a recognized inhibitor (CY09) of inflammasome-3 assembly (Jiang et al. 2017). In addition, the decreases in cellular viability produced by LPIA were shown to be prevented by pre-treatment with CY09, suggesting the importance of the inflammasome in producing decreases in GPR55-mediated decreases in cellular viability (see Fig. 6b). Together, these studies suggest a sentinel function for GPR55 in both DRG and hippocampal neurons, extending the importance of this receptor as an early warning mechanism of some inflammatory mediators.
These studies support a pronociceptive, proinflammatory role for GPR55 that mediates acute and chronic effects in DRG. The GPR55 target is believed to be complementary to our previous studies with the NCX-1 target which exhibited acute regulation of mitochondrial calcium levels by extrusion of excess calcium in DRG (Brenneman et al. 2019). Our data suggest a bimodal pharmacological effect of KLS-13019 that can both increase the viability of sensory neurons exposed to paclitaxel and antagonize GPR55 that can mediate neuroinflammatory and sensory neuron damage that may contribute to neuropathic pain.
Author Contribution Douglas Brenneman designed and conducted all experiments using primary neuronal cultures and wrote the draft of the manuscript. William Kinney and Mark McDonnell chemically designed and provided the purified, structurally verified KLS-13019. Pingei Zhao conducted the β-arrestin assays and authored Fig. 7. Mary Abood provided intellectual input on GPR55 and second messenger systems relevant to study design. Sara Jane Ward provided intellectual input on study design pertaining to inflammation. Editing contributions were made by Brenneman, McDonnell, and Ward. All authors read and approved the final manuscript.
Funding These studies were supported by Grants from the National Institute on Drug Abuse (R41DA044898); (5P30DA013429-20); (RO1DA045698); and the National Institute of Neurological Disorders and Stroke (R42NS120548) of the National Institutes of Health.
Data Availability Data analyzed in these studies are available from the corresponding author upon reasonable request.

Declarations
Competing Interests Drs Brenneman, Kinney and McDonnell are inventors of KLS-13019 and hold international patents on this technology.

Ethics Approval and Consent to Participants
No animal or human subject data were in this work. Animal tissues for primary cultures were purchased from a commercial source.

Informed Consent
No human subjects were in these studies.