Andrea Grace Hohmann

Background and Purpose: Positive allosteric modulators (PAMs) engage sites distinct from the orthosteric CB ₁ cannabinoid receptor binding site. GAT211, a racemic ligand, contains GAT228 ( R ) and GAT229 ( S ) enantiomers. Whether enantiomer‐mediated differences in allosteric activation or positive allosteric modulation translate into differences in therapeutic efficacy or unwanted cannabimimetic effects remain unknown. Experimental Approach: We evaluated therapeutic efficacy of the pure CB ₁ allosteric agonist, GAT228 ( R ), and the PAM, GAT229 ( S ), in inflammatory pain induced by complete Freund's adjuvant (CFA) in male mice. We assessed whether these allosteric ligands would spare tolerance, memory deficits, motor impairment and respiratory depression associated with the orthosteric CB ₁ /CB ₂ agonist CP55,940. Key Results: GAT228 and GAT229 attenuated CFA‐induced mechanical hypersensitivity in a CB ₁ ‐dependent manner without producing tolerance or hypothermia associated with the orthosteric CB ₁ /CB ₂ agonist CP55,940. CP55,940 also produced memory deficits, motor impairment, and robust respiratory depression. GAT228 and GAT229 failed to do so. Unlike CP55,940, allosteric ligands did not produce motor impairment or notable respiratory depression in the presence or absence of CFA‐induced inflammation. Allosteric ligands increased novel object recognition memory, whereas CP55,940 decreased time spent exploring the novel object. Intraplantar CFA increased immobility time in the tail suspension test, indicative of pain‐related behavioural despair, which was normalized by allosteric ligands and exacerbated by CP55,940. Conclusion and Implication: CB ₁ allosteric ligands represent promising, safer therapeutic strategies for pain management compared to direct CB ₁ orthosteric agonists. Overall, this work contributes to offering a novel pathway for pain management that minimizes the risk of adverse effects.

Cannabidiol (CBD) is a non-psychoactive component of cannabis that has been studied as a potential therapy for chronic pain. CBD attenuates behavioral hypersensitivities in models of neuropathic pain, and promotes production of bioactive lipids (e.g., anandamide), altering lipid signaling. However, a lack of understanding of the mechanisms underlying the therapeutic effects of CBD has hindered development and application of CBD to mechanism-based therapies for pain in people. We asked whether the analgesics effects of CBD were dependent upon the enzyme NAPE-PLD. We used a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) to evaluate the acute and chronic antinociceptive effects of CBD and investigate its mechanisms. Pharmacological specificity was tested with antagonists targeting CB1, CB2, PPARγ, and PPARα receptors. Mechanisms were further examined using NAPE-PLD and GPR55 knockout mice. We also assessed repeated CBD dosing during both the development and maintenance of paclitaxel-induced CIPN in wild-type, GPR55 KO, and NAPE-PLD KO mice. CBD suppressed paclitaxel-induced behavioral hypersensitivities; these effects were attenuated by a PPARα and PPARγ antagonists, but not CB1 or CB2 antagonists. CBD reduced both the development and maintenance of neuropathic nociception in a model CIPN in wild-type mice, but these effects were absent in NAPE-PLD KO mice. By contrast, anti-allodynic efficacy of CBD was fully preserved in GPR55 KO mice. Pharmacological blockade of the PPARα receptor and genetic deletion of NAPE-PLD abolished the antinociceptive effects of CBD in a model of CIPN, suggesting a pivotal role for NAPE-PLD and PPAR receptors in CBD-mediated analgesia in chemotherapy-induced neuropathic pain.

Introduction: Cannabinoid CB ₂ agonists reduce stimulus-evoked behavioral hypersensitivities in preclinical pain models, but their ability to modulate spontaneous pain remains largely unexplored. Spontaneous pain has been assessed in rodents using a conditioned place preference (CPP) approach, given that the relief of pain is described as rewarding and results in the removal of an aversive pain state. LY2828360 is a CB ₂ agonist that failed in a clinical trial for osteoarthritis pain. Materials and Methods: We compared the impact of LY2828360 on evoked and spontaneous pain in a spared nerve injury (SNI) model using a within-subjects design in rats. First, we used an unbiased CPP approach to verify that an analgesic dose of gabapentin (GBP) (100 mg/kg, i.p.) produces CPP in rats with SNI, but not in sham-operated rats (Experiment 1). We then used a within-subjects design to ascertain whether LY2828360 (10 mg/kg i.p., chronic) would suppress both evoked and spontaneous pain in rats with SNI (Experiment 2). To assess spontaneous/affective pain behavior, we tested the ability of chronic dosing with LY2828360, in comparison to vehicle, to prevent GBP-induced CPP in the SNI model, as failure to develop CPP to GBP following treatment with an analgesic has been considered evidence of suppression of spontaneous pain. To assess evoked pain behavior, paw withdrawal thresholds were measured in the same rats used for CPP. Results: GBP produced CPP in rats with SNI, but not sham surgery, and suppressed SNI-induced mechanical hypersensitivity in Experiment 1. In Experiment 2, LY2828360 reliably suppressed mechanical hypersensitivity in the paw ipsilateral , but not contralateral to SNI. Furthermore, efficacy was sustained across repeated injections without development of tolerance. The same rats that showed suppression of mechanically-evoked responses following chronic LY282860 treatment did not develop CPP to GBP. However, rats with SNI that were tested in parallel and treated chronically with vehicle showed robust mechanical hypersensitivity but also did not develop CPP to GBP. Conclusion: These studies document that CB ₂ agonist-induced suppression of mechanically-evoked pain is highly robust and reproducible in the SNI model, whereas CPP, used to assess spontaneous pain, is vulnerable to disruption and requires rigorous controls to rule out alternative explanations (e.g., failure to learn).

Introduction: Xylazine is a veterinary sedative and widespread adulterant of illicit opioids, where it is commonly detected in combination with the potent synthetic µ opioid receptor (MOR) agonist fentanyl in fatal overdose. Methods: We used whole body plethysmography to evaluate the respiratory effects of xylazine in the presence and absence of fentanyl in awake C57BL/6 mice of both sexes. We asked whether suppression of breathing by xylazine-adulterated fentanyl resisted reversal with naloxone. We identified receptor mechanisms necessary to reverse effects of xylazine-adulterated fentanyl on breathing. Results: Xylazine rapidly and dose-dependently suppressed minute ventilation, respiratory frequency, and tidal volume, producing profound respiratory depression at low doses. These effects were dependent on α-2 adrenergic receptors (α2ARs) and were blocked by the α2AR antagonist atipamezole. Xylazine, combined with a dose of fentanyl with modest respiratory effects, suppressed breathing with greater efficacy than when administered alone. A dose of naloxone sufficient to reverse fentanyl-induced respiratory depression was ineffective in reversing the respiratory suppression induced by xylazine-adulterated fentanyl. By contrast, combinations of naloxone with atipamezole rapidly and fully reversed respiratory suppression induced by xylazine-adulterated fentanyl. The same dose of atipamezole, administered alone, produced significant, but markedly delayed reversal. Conclusion: Xylazine suppresses breathing via activation of α2ARs, an effect enhanced by coadministration with the MOR agonist fentanyl. Respiratory suppression inflicted by the mixture of xylazine and fentanyl resisted reversal by naloxone but was fully reversible by subsequent coadministration of both naloxone and atipamezole.

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating complication of cancer treatment. CB2 cannabinoid receptor activation reduces inflammation and is an attractive therapeutic target. Antibodies targeting G protein-coupled receptors (GPCRs), like CB2, offer high specificity and peripheral-restriction, thereby minimizing off-target activity. Here, we investigated the efficacy of first-in-class CB2-specific antibody agonists (AB110 and AB120) and an isotype control (AB100) on mechanical and cold hypersensitivity induced by paclitaxel in both tumor-free and mammary (4T1) tumor-bearing female mice. These CB2 antibody agonists exhibit biased G-α signaling and also reduce macrophage markers and pro-inflammatory cytokines in vitro . Paclitaxel produced behavioral hypersensitivities to mechanical and cold stimulation, which were reduced by AB110 and AB120 for approximately 48 hours post-injection in female mice. Repeated daily dosing did not lead to tolerance to the anti-allodynic effects. Prophylactic treatment with AB110 and AB120 during paclitaxel treatment delayed, but did not prevent, the development of paclitaxel-induced behavioral hypersensitivities after termination of dosing with antibody agonists. AB100 had no effect under any conditions. The anti-allodynic effects of AB120 were absent in CB2 knockout mice, confirming pharmacological specificity via CB2 receptors. Furthermore, AB120 remained effective in paclitaxel-treated tumor-bearing mice. Neither AB110 nor AB120 affected locomotor activity in otherwise naïve mice. The cytotoxic activity of paclitaxel on 4T1 tumor cell line was maintained in the presence of CB2 antibody agonists in vitro . Overall, our results suggest that CB2-specific antibody agonists are promising candidates for treating CIPN, providing lasting pain relief without tolerance, off target effects or unwanted CB1-mediated motor side effects.

Cannabinoid CB ₂ agonists reduce stimulus-evoked behavioral hypersensitivities in preclinical pain models, but their ability to modulate spontaneous pain remains unexplored. Spontaneous pain has been assessed in rodents using the conditioned place preference (CPP) approach, given that the relief of pain is described as rewarding and results in negative reinforcement (i.e. removal of an aversive pain state). LY2828360 is a CB2 agonist that failed in a clinical trial for osteoarthritis pain. We compared impact of LY2828360 on evoked and spontaneous pain using a spared nerve injury (SNI) model in rats. First, we verified that an analgesic dose of gabapentin (100 mg/kg i.p.) produces CPP in rats with SNI, but not in sham-operated rats, consistent with a previous report (Griggs et al. 2015). We then used a within-subjects design to ascertain whether the CB ₂ agonist LY2828360 (10 mg/kg i.p., chronic) would suppress both evoked and spontaneous pain in rats with SNI. To assess evoked pain behavior, mechanical paw withdrawal thresholds were measured and revealed that LY2828360 reliably suppressed mechanical hypersensitivity in the paw ipsilateral , but not contralateral to SNI. Furthermore, efficacy was sustained across repeated injections without development of tolerance. To assess spontaneous pain behavior, we tested the ability of LY2828360 to prevent gabapentin-induced CPP in the SNI model, as failure to develop CPP to gabapentin following treatment with an analgesic has been considered evidence of suppression of spontaneous pain. The same rats that showed suppression of mechanically-evoked responses following chronic LY282860 treatment did not develop CPP to gabapentin. However, rats that were tested in parallel and treated chronically with vehicle showed robust mechanical hypersensitivity, but also did not develop CPP to gabapentin. These studies document that CB ₂ agonist-induced suppression of mechanically evoked pain is highly robust and reproducible, whereas CPP, used to assess spontaneous pain, is vulnerable to disruption and requires rigorous controls to rule out alternative explanations (e.g. failure to learn).

Currently, mechanism-based inactivators (MBIs) are the only available therapeutic option to target γ-aminobutyric acid aminotransferase (GABA-AT). However, off-target activity against homologous enzymes has posed major challenges for the clinical use of MBIs. For example, CPP-115, a MBI of GABA-AT that completed a Phase I clinical trial, also inactivates ornithine aminotransferase (OAT). Here, we present an OAT-specific inactivation mechanism for CPP-115, enabled by the dynamic motion of a key reactive intermediate. Based on this, to selectively reduce the off-target activity, a proof-of-concept molecule that regulates the intermediate conformational flexibility was designed and synthesized. The resulting inactivator achieved the greatly enhanced GABA-AT selectivity over OAT and demonstrated therapeutic efficacy in an inflammatory pain animal model. Our strategy in this study, targeting dynamics of a reactive intermediate based on precise mechanistic understanding, could serve as a generalizable design principle for fine-tuning the selectivity of MBIs, particularly for other aminotransferases.

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a common and debilitating side effect of paclitaxel treatment. Pharmacological strategies that enhance inhibitory tone, via inhibition of γ-aminobutyric acid aminotransferase (GABA-AT), the enzyme which degrades endogenous GABA, provides analgesic benefit in preclinical studies. ( S )-MeCPP-115 is a novel selective GABA-AT inactivator designed from CPP-115 to minimize off-target activity. Whether ( S )-MeCPP-115 shows efficacy in preclinical pain models is unknown. Methods: In vitro assays of cell viability were conducted to ascertain whether ( S )-MeCPP-115 interfered with antitumor activity of paclitaxel or produced cytotoxicity in normal cells. Male mice received paclitaxel to induce chemotherapy-induced peripheral neuropathy in vivo . In mice with established paclitaxel-induced mechanical hypersensitivity, ( S )-MeCPP-115 was administered via acute and chronic administration using intraperitoneal (i.p.) or intrathecal (i.t.) dosing strategies. Mechanical sensitivity was assessed in all mice with an electronic von Frey analgesiometer before and after paclitaxel and pharmacological treatments. Locomotor activity was also measured in the same subjects to assess possible motor impairment. Results: In MTT assays, ( S )-MeCPP-115 did not alter the cytotoxic activity of paclitaxel in 4T1 breast cancer cells and did not produce cytotoxicity in non-tumor HEK293 cells. ( S )-MeCPP-115 reduced paclitaxel-induced mechanical hypersensitivity after i.p. and i.t. administration. Therapeutic efficacy was maintained with repeated dosing without development of tolerance. ( S )-MeCPP-115 did not alter paw withdrawal thresholds in mice that received the Cremophor-based vehicle in lieu of paclitaxel following acute or chronic dosing. Systemic treatment with ( S )-MeCPP-115 was well tolerated, whereas i.t. administration produced only minimal locomotor effects. Conclusion: ( S )-MeCPP-115 did not interfere with the ability of paclitaxel to produce tumor cell cytotoxicity in vitro and did not produce cytotoxicity in non-tumor cells. Systemic and intrathecal administration of ( S )-MeCPP-115 produced robust suppression of mechanical hypersensitivity in a mouse model of paclitaxel-induced CIPN without major adverse effects. Selective GABA-AT inhibition represents a promising therapeutic approach for suppressing paclitaxel-induced mechanical hypersensitivity. Further preclinical characterization of the therapeutic profile of ( S )-MeCPP-115 is warranted.