Pharmacology/Drug Development

This study evaluated a battery of pain-stimulated, pain-depressed, and pain-independent behaviors for preclinical pharmacological assessment of candidate analgesics in mice. Intraperitoneal injection of dilute lactic acid (IP acid) served as an acute visceral noxious stimulus to produce four pain-related behaviors in male and female ICR mice: stimulation of (1) stretching and (2) facial grimace, and depression of (3) rearing and (4) nesting. Additionally, nesting and locomotion in the absence of the noxious stimulus were used to assess pain-independent drug effects. These six behaviors were used to compare effects of two mechanistically distinct but clinically-effective positive controls (ketoprofen and oxycodone), and two negative controls that are not clinically approved as analgesics but produce either general motor depression (diazepam) or motor stimulation (amphetamine). We predicted that analgesics would alleviate all IP acid effects at doses that did not alter pain-independent behaviors, whereas negative controls would not. Consistent with this prediction, ketoprofen (0.1-32 mg/kg) produced the expected analgesic profile, whereas oxycodone (0.32-3.2 mg/kg) alleviated all IP acid effects except depression of rearing at doses lower than those that altered pain-independent behaviors. For the negative controls, diazepam (1-10 mg/kg) failed to block IP acid-induced depression of either rearing or nesting, and only decreased IP acid-stimulated behaviors at doses that also decreased pain-independent behaviors. Amphetamine (0.32-3.2 mg/kg) alleviated all IP acid effects, but only at doses that also stimulated locomotion. These results support utility of this model as a framework to evaluate candidate-analgesic effects in a battery of complementary pain-stimulated, pain-depressed, and pain-independent behavioral endpoints. Preclinical assays of pain and analgesia often yield false-positive effects with candidate analgesics. This study used two positive-control analgesics (ketoprofen, oxycodone) and two active negative controls (diazepam, amphetamine) to validate a strategy for distinguishing analgesics from non-analgesics by profiling drug effects in a battery of complementary pain-stimulated, pain-depressed, and pain-independent behaviors in male and female mice.

Enhanced signaling of the endocannabinoid system (eCB) through inhibition of the catabolic enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) has received increasing interest for development of candidate analgesics. This study compared effects of MAGL and FAAH inhibitors with effects of ∆9-tetrahydrocannabinol (THC) using a battery of pain-stimulated, pain-depressed, and pain-independent behaviors in male and female mice. Intraperitoneal injection of dilute lactic acid (IP acid) served as an acute visceral noxious stimulus to stimulate two behaviors (stretching, facial grimace) and depress two behaviors (rearing, nesting). Nesting and locomotion were also assessed in the absence of IP acid as pain-independent behaviors. THC and a spectrum of six eCB catabolic enzyme inhibitors ranging from MAGL- to FAAH-selective were assessed for effectiveness to alleviate pain-related behaviors at doses that did not alter pain-independent behaviors. The MAGL-selective inhibitor MJN110 produced the most effective antinociceptive profile, with 1.0 mg/kg alleviating IP acid effects on stretching, grimace, and nesting without altering pain-independent behaviors. MJN110 effects on IP acid-depressed nesting had a slow onset and long duration (40min to 6hr), were blocked by rimonabant, and tended to be greater in females. As inhibitors increased in FAAH-selectivity, antinociceptive effectiveness decreased. PF3845, the most FAAH-selective inhibitor, produced no antinociception up to doses that disrupted locomotion. THC decreased IP acid-stimulated stretching and grimace at doses that did not alter pain-independent behaviors; however, it did not alleviate IP acid-induced depression of rearing or nesting. These results support further consideration of MAGL-selective as candidate analgesics for acute inflammatory pain. This study characterized a spectrum of endocannabinoid (eCB) catabolic enzyme inhibitors ranging in selectivity from monoacylglycerol lipase- (MAGL-) selective to fatty acid amide hydrolase- (FAAH-) selective in a battery of pain-stimulated, pain-depressed, and pain-independent behaviors previously pharmacologically characterized in a companion paper. This battery provides a method for prioritizing candidate analgesics by effectiveness to alleviate pain-related behaviors at doses that do not alter pain-independent behaviors, with inclusion of pain-depressed behaviors increasing translational validity and decreasing susceptibility to motor-depressant false positives.

The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain; investigation into their mode of action is essential for ongoing clinical development. A3ARs on immune cells, and their activation during pathology, modulates cytokine release. Thus, immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing but unknown. Studies herein discovered that RagKO mice lacking T- and B-cells are insensitive to the anti-allodynic effects of A3AR agonists versus wild-type (WT) mice. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4+ T-cells (CD4+-T) from WT mice infiltrated the dorsal root ganglion (DRG) and restored A3AR agonist-mediated anti-allodynia in RagKO mice; CD4+-T from Adora3KO or Il10KO mice did not. Transfer of CD4+-T from WT, but not Il10KO, into Il10KO mice fully reinstated anti-allodynic effects of A3AR activation. Transfer of CD4+-T from WT, but not Il10KO, into Adora3KO mice fully reinstated anti-allodynic effects of A3AR activation. Notably, A3AR agonism reduced DRG neuron excitability when co-cultured with CD4+-T in an IL-10-dependent manner. A3AR actions on CD4+-T infiltrate in the DRG decreased phosphorylation of GluN2B-containing N-methyl-D-aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A3AR on CD4+-T cells to release of IL-10 is required and sufficient for A3AR agonists as therapeutics.

Despite the success of combined antiretroviral therapy (cART) in reducing viral load, a substantial portion of Human Immunodeficiency Virus (HIV)+ patients report chronic pain. The exact mechanism underlying this co-morbidity even with undetectable viral load remains unknown, but the transactivator of transcription (HIV-Tat) protein is of particular interest. Functional HIV-Tat protein is observed even in cerebrospinal fluid of patients who have an undetectable viral load. It is hypothesized that Tat protein exposure is sufficient to induce neuropathic pain-like manifestations via both activation of microglia and generation of oxidative stress. iTat mice conditionally expressed Tat protein in the central nervous system upon daily administration of doxycycline (100 mg/kg/d, i.p., up to 14 days). The effect of HIV-Tat protein exposure on the well-being of the animal was assessed using sucrose-evoked grooming and acute nesting behavior for pain-depressed behaviors, and the development of hyperalgesia assessed with warm-water tail-withdrawal and von Frey assays for thermal hyperalgesia and mechanical allodynia, respectively. Tissue harvested at select time points was used to assess ex vivo alterations in oxidative stress, astrocytosis and microgliosis, and blood-brain barrier integrity with assays utilizing fluorescence-based indicators. Tat protein induced mild thermal hyperalgesia but robust mechanical allodynia starting after 4 days of exposure, reaching a nadir after 7 days. Changes in nociceptive processing were associated with reduced sucrose-evoked grooming behavior without altering acute nesting behavior, and in spinal cord dysregulated free radical generation as measured by DCF fluorescence intensity, altered immunohistochemical expression of the gliotic markers, Iba-1 and GFAP, and increased permeability of the blood-brain barrier to the small molecule fluorescent tracer, sodium fluorescein, in a time-dependent manner. Pretreatment with the anti-inflammatory, indomethacin (1 mg/kg/d, i.p.), the antioxidant, methylsulfonylmethane (100 mg/kg/d i.p.), or the immunomodulatory agent, dimethylfumarate (100 mg/kg/d p.o.) thirty minutes prior to daily injections of doxycycline (100 mg/kg/d i.p.) over 7 days significantly attenuated the development of Tat-induced mechanical allodynia. Collectively, the data suggests that even acute exposure to HIV-1 Tat protein at pathologically relevant levels is sufficient to produce select neurophysiological and behavioral manifestations of chronic pain consistent with that reported by HIV-positive patients.