Transcriptomic signature, bioactivity and safety of a non-hepatotoxic analgesic generating AM404 in the midbrain PAG region.

Safe and effective pain management is a critical healthcare and societal need. The potential for acute liver injury from paracetamol (ApAP) overdose; nephrotoxicity and gastrointestinal damage from chronic non-steroidal anti-inflammatory drug (NSAID) use; and opioids' addiction are unresolved challenges. We developed SRP-001, a non-opioid and non-hepatotoxic small molecule that, unlike ApAP, does not produce the hepatotoxic metabolite N-acetyl-p-benzoquinone-imine (NAPQI) and preserves hepatic tight junction integrity at high doses. CD-1 mice exposed to SRP-001 showed no mortality, unlike a 70% mortality observed with increasing equimolar doses of ApAP within 72 h. SRP-001 and ApAP have comparable antinociceptive effects, including the complete Freund's adjuvant-induced inflammatory von Frey model. Both induce analgesia via N-arachidonoylphenolamine (AM404) formation in the midbrain periaqueductal grey (PAG) nociception region, with SRP-001 generating higher amounts of AM404 than ApAP. Single-cell transcriptomics of PAG uncovered that SRP-001 and ApAP also share modulation of pain-related gene expression and cell signaling pathways/networks, including endocannabinoid signaling, genes pertaining to mechanical nociception, and fatty acid amide hydrolase (FAAH). Both regulate the expression of key genes encoding FAAH, 2-arachidonoylglycerol (2-AG), cannabinoid receptor 1 (CNR1), CNR2, transient receptor potential vanilloid type 4 (TRPV4), and voltage-gated Ca2+ channel. Phase 1 trial (NCT05484414) (02/08/2022) demonstrates SRP-001's safety, tolerability, and favorable pharmacokinetics, including a half-life from 4.9 to 9.8 h. Given its non-hepatotoxicity and clinically validated analgesic mechanisms, SRP-001 offers a promising alternative to ApAP, NSAIDs, and opioids for safer pain treatment.

Distinct antinociceptive and conditioned behavioral effects are produced by individual cannabinoids and a cannabis-derived mixture.

Cannabinoids have been proposed as therapeutics for pain mitigation. Therefore, the antihyperalgesic effects of a proprietary cannabis-derived mixture, Non-Euphoric Phytocannabinoid Elixir #14 (NEPE14), were examined in a persistent Complete Freund's Adjuvant (CFA)-induced model of inflammatory pain. The acute antinociceptive and operant behavioral effects of NEPE14 were then compared with single cannabinoid preparations of Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-THC, the synthetic cannabinoid (-)-CP 55,940 (CP), and cannabidiol (CBD). The THC isomers and CP were also administered with cannabinoid-type-1 receptor (CB1R) antagonist, AM251, and NEPE14 was administered in combination with THC or CP. To induce inflammation, CFA or saline was administered into the paw of male and female Wistar rats. After injections, mechanical hypersensitivity was assessed with Von Frey filaments, and thermal hyperalgesia with a thermal probe. Nine Sprague Dawley rats were also trained to respond under a fixed-ratio 30 schedule for food reinforcers during a 60-min session. Response rates were recorded during the session and warm-water tail-withdrawal latency post session. In CFA-administered rats, mechanical and thermal paw-withdrawal thresholds significantly decreased compared to vehicle, indicating hyperalgesia. Both i.p. (6.6-20.7 ml/kg) and o.m. (30-300 µL) NEPE14 significantly reduced the mechanical and thermal hyperalgesia. In contrast, neither NEPE14 (3.7-20.7 mL/kg i.p., 100-1000 µL o.m.) nor CBD (10-100 mg/kg) significantly decreased response rates or increased tail-withdrawal latency. Acute Δ9-THC, Δ8-THC (1-5.6 mg/kg), and CP (0.032-0.18 mg/kg) significantly and dose-dependently decreased overall response rate and increased tail-withdrawal latency compared to vehicle. AM251 significantly antagonized the rate-decreasing effects of THC, and CP, as well as the antinociceptive effects of CP. Combinations of NEPE14 with Δ9-THC or CP were not significantly different from these cannabinoids alone. In summary, while NEPE14 significantly reduced CFA-induced hyperalgesia, it was more similar to CBD than Δ9-THC, Δ8-THC, and CP for significantly reducing thermal nociception and disrupting conditioned behavior.

Sex-specific biobehavioral regulation of persistent inflammatory pain by alcohol.

BACKGROUND: Although a large percentage of chronic pain patients consume alcohol to manage their pain, there is a significant gap in knowledge regarding the mechanisms underlying the antinociceptive effects of alcohol. METHODS: To determine the longitudinal analgesic effects of alcohol, we utilized the complete Freund's adjuvant (CFA) model of inflammatory pain in adult female and male Wistar rats. Both somatic and negative motivational aspects of pain were measured using the electronic von Frey (mechanical nociception) system, thermal probe test (thermal nociception), and mechanical conflict avoidance task (pain avoidance-like behavior). Tests were conducted at baseline and 1 and 3 weeks following intraplantar CFA or saline administration. At both time points post-CFA, animals were treated with each of three doses of alcohol (intraperitoneal; 0, 0.5, and 1.0 g/kg) over separate days in a Latin square design. RESULTS: Alcohol produced dose-dependent mechanical analgesia and antihyperalgesia in females but only antihyperalgesia in males. Although alcohol continued to attenuate CFA-induced decreases in both thermal and mechanical nociceptive thresholds between 1 and 3 weeks post-CFA, it appeared less effective at increasing thresholds 3 weeks after CFA induction. CONCLUSIONS: These data suggest that individuals may develop tolerance to alcohol's ability to alleviate both somatic and negative motivational symptoms of chronic pain over time. We also discovered sex-specific neuroadaptations in protein kinase A-dependent phosphorylation of GluR1 subunits and extracellular signal-regulated kinase (ERK 1/2) phosphorylation in nociceptive brain centers of animals receiving an alcohol challenge 1 week post-CFA. Together, these findings illustrate a sex-specific regulation of behavioral and neurobiological indices of persistent pain by alcohol.

Agmatine reduces alcohol drinking and produces antinociceptive effects in rodent models of alcohol use disorder.

Alcohol use disorder (AUD) is a chronic, relapsing disorder characterized by an escalation of drinking and the emergence of negative affective states over time. Within this framework, alcohol may be used in excessive amounts to alleviate withdrawal-related symptoms, such as hyperalgesia. Future effective therapeutics for AUD may need to exhibit the ability to reduce drinking as well as to alleviate co-morbid conditions such as pain, and to take mechanistic sex differences into consideration. Agmatine is an endogenous neuromodulator that has been previously implicated in the regulation of reward and pain processing. In the current set of studies, we examined the ability of agmatine to reduce escalated ethanol drinking in complementary models of AUD where adult male and female mice and rats were made dependent via chronic, intermittent ethanol vapor exposure (CIE). We also examined the ability of agmatine to modify thermal and mechanical sensitivity in alcohol-dependent male and female rats. Agmatine reduced alcohol drinking in a dose-dependent fashion, with somewhat greater selectivity in alcohol-dependent female mice (versus non-dependent female mice), but equivalent efficacy across male mice and both groups of male and female rats. In mice and female rats, this efficacy did not extend to sucrose drinking, indicating some selectivity for ethanol reinforcement. Female rats made dependent on alcohol demonstrated significant hyperalgesia symptoms, and agmatine produced dose-dependent antinociceptive effects across both sexes. While additional mechanistic studies into agmatine are necessary, these findings support the broad-based efficacy of agmatine to treat co-morbid excessive drinking and pain symptoms in the context of AUD.

Pramipexole treatment attenuates mechanical hypersensitivity in male rats experiencing chronic inflammatory pain.

Opioids are commonly prescribed for pain despite growing evidence of their low efficacy in the treatment of chronic inflammatory pain and the high potential for misuse. There is a clear need to investigate non-opioid alternatives for the treatment of pain. In the present study, we tested the hypothesis that acute and repeated dopamine agonist treatment would attenuate mechanical hypersensitivity in male Long-Evans rats experiencing chronic inflammatory pain. We used two clinically available therapeutics, l-DOPA (precursor of dopamine biosynthesis) and pramipexole (dopamine D2/3 receptor agonist), to examine the functional role of dopamine signaling on mechanical hypersensitivity using an animal model of chronic inflammatory pain (complete Freund's adjuvant, CFA). We found that both acute and repeated pramipexole treatment attenuated hyperalgesia-like behavior in CFA-treated animals but exhibited no analgesic effects in control animals. In contrast, there was no effect of acute or repeated l-DOPA treatment on mechanical hypersensitivity in either CFA- or saline-treated animals. Notably, we discovered some extended effects of l-DOPA and pramipexole on decreasing pain-like behavior at three days and one week post-drug treatment. We also examined the effects of pramipexole treatment on glutamatergic and presynaptic signaling in pain- and reward-related brain regions including the nucleus accumbens (NAc), dorsal striatum (DS), ventral tegmental area (VTA), cingulate cortex (CC), central amygdala (CeA), and periaqueductal gray (PAG). We found that pramipexole treatment decreased AMPA receptor phosphorylation (pGluR1845) in the NAc and DS but increased pGluR1845 in the CC and CeA. A marker of presynaptic vesicle release, pSynapsin, was also increased in the DS, VTA, CC, CeA, and PAG following pramipexole treatment. Interestingly, pramipexole increased pSynapsin in the NAc of saline-treated animals, but not CFA-treated animals, suggesting blunted presynaptic vesicle release in the NAc of CFA-treated animals following pramipexole treatment. To examine the functional implications of impaired presynaptic signaling in the NAc of CFA animals, we used ex vivo electrophysiology to examine the effects of pramipexole treatment on the intrinsic excitability of NAc neurons in CFA- and saline-treated animals. We found that pramipexole treatment reduced NAc intrinsic excitability in saline-treated animals but produced no change in NAc intrinsic excitability in CFA-treated animals. These findings indicate alterations in dopamine D2/3 receptor signaling in the NAc of animals with a history of chronic pain in association with the anti-hyperalgesic effects of pramipexole treatment.

Alcohol amplifies cingulate cortex signaling and facilitates immobilization-induced hyperalgesia in female rats.

Complex Regional Pain Syndrome (CRPS) is a musculoskeletal pain condition that often develops after limb injury and/or immobilization. Although the exact mechanisms underlying CRPS are unknown, the syndrome is associated with central and autonomic nervous system dysregulation and peripheral hyperalgesia symptoms. These symptoms also manifest in alcoholic neuropathy, suggesting that the two conditions may be pathophysiologically accretive. Interestingly, people assigned female at birth (AFAB) appear to be more sensitive to both CRPS and alcoholic neuropathy. To better understand the biobehavioral mechanisms underlying these conditions, we investigated a model of combined CRPS and alcoholic neuropathy in female rats. Animals were pair-fed either a Lieber-DeCarli alcohol liquid diet or a control diet for ten weeks. CRPS was modeled via unilateral hind limb cast immobilization for seven days, allowing for the other limb to serve as a within-subject control for hyperalgesia measures. To investigate the role of circulating ovarian hormones on pain-related behaviors, half of the animals underwent ovariectomy (OVX). Using the von Frey procedure to record mechanical paw withdrawal thresholds, we found that cast immobilization and chronic alcohol drinking separately and additively produced mechanical hyperalgesia observed 3 days after cast removal. We then examined neuroadaptations in AMPA GluR1 and NMDA NR1 glutamate channel subunits, extracellular signal-regulated kinase (ERK), and cAMP response element-binding protein (CREB) in bilateral motor and cingulate cortex across all groups. Consistent with increased pain-related behavior, chronic alcohol drinking increased GluR1, NR1, ERK, and CREB phosphorylation in the cingulate cortex. OVX did not alter any of the observed effects. Our results suggest accretive relationships between CRPS and alcoholic neuropathy symptoms and point to novel therapeutic targets for these conditions.

Neurobiological aspects of pain in the context of alcohol use disorder.

Alcohol is an effective and widely utilized analgesic. However, the chronic use of alcohol can actually facilitate nociceptive sensitivity over time, a condition known as hyperalgesia. Excessive and uncontrollable alcohol drinking is also a hallmark feature of alcohol use disorder (AUD). Both AUD and chronic pain are typically accompanied by negative affective states that may underlie reinforcement mechanisms contributing to AUD maintenance or progression. Frequent utilization of alcohol to relieve pain in individuals suffering from AUD or other chronic pain conditions may thus represent a powerful negative reinforcement construct. This chapter will describe ties between alcohol-mediated pain relief and potential exacerbation of AUD. We describe neurobiological systems engaged in alcohol analgesia as well as systems recruited in the development and maintenance of AUD and hyperalgesia. Although few effective therapies exist for either chronic pain or AUD, the common interaction of these conditions will likely lead the way for promising new discoveries of more effective and even simultaneous treatment of AUD and co-morbid hyperalgesia. An abundance of neurobiological findings from multiple laboratories has implicated a potentiation of central amygdala (CeA) signaling in both pain and AUD, and these data also suggest that attenuation of stress-related systems (including corticotropin-releasing factor, vasopressin, and glucocorticoid receptor activity) would be particularly effective and comprehensive therapeutic strategies targeting the critical intersection of somatic and motivational mechanisms driving AUD, including alcohol-induced hyperalgesia.