Animal Studies

The combination of corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs) has been commonly used for inflammation and chronic articular pain in the clinic. Nonetheless, the long-term administration of both medications might result in osteonecrosis of the knee due to repeated injections of steroids and side effects in the gastrointestinal and cardiovascular systems. To overcome these unmet medical needs, we designed a microsphere-microcrystal-gel delivery system for intra-articular injection. Dexamethasone (DEX)-loaded microspheres (DMs) were optimized by Plackett-Burman and Taguchi orthogonal designs to extend their retention time in the knee joint. Celecoxib (CLX) microcrystals (CMs) were manufactured using an ultrasonic method to improve solubility and bioavailability. Moreover, a green solvent-free method was employed to crosslink and synthesize a novel poloxamer 407/Gantrez® S97-based gel system (GZF), which can undergo the sol-gel transition at lower concentrations. Then, DM and CM were loaded by GZF to form intra-articular injectable gels (DM/CM/Gel). The in vitro release of DEX and CLX showed a fast phase in 24 h followed by a controlled release of ∼8 d. Both blank microspheres and GZF gels displayed great biocompatibility against RAW264.7 macrophages. The most suitable dosages of 5 nM DEX and 125 nM CLX in the formulation were chosen because of their significant effects against macrophage inflammation with a lower administrative amount. An In vivo animal evaluation showed that DM/CM/Gel suppressed the release of inflammatory cytokines (TNF-α and IL-6) after 21 d of treatment. In addition, a histological evaluation revealed that DM/CM/Gel interrupted the progression of cartilage surface denudation and matrix loss. Therefore, DM/CM/Gel provides a prospective strategy for reforming traditional therapy for chronic articular disease.

The μ-opioid receptors belong to the family of G protein-coupled receptors (GPCRs), and their activation triggers a cascade of intracellular relays with the final effect of analgesia. Classical agonists of this receptor, such as morphine, are the main targets in the treatment of both acute and chronic pain. However, the dangerous side effects, such as respiratory depression or addiction, significantly limit their widespread use. The allosteric centers of the receptors exhibit large structural diversity within particular types and even subtypes. Currently, a considerable interest is aroused by the modulation of μ-opioid receptors. The application of such a technique may result in a reduction in the dose or even discontinuation of classical opiates, thus eliminating the side effects typical of this class of drugs. Our aim is to obtain a series of 1-aryl-5,6(1)dioxo-2,3-dihydroimidazo[1,2-]imidazole derivatives and provide more information about their activity and selectivity on OP3 (MOP, human mu opioid receptor). The study was based on an observation that some carbonyl derivatives of 1-aryl-2-aminoimidazoline cooperate strongly with morphine or DAMGO in sub-threshold doses, producing similar results to those of normal active doses. To elucidate the possible mechanism of such enhancement, we performed a few in vitro functional tests (involving cAMP and β-arrestin recruitment) and a radioligand binding assay on CHO-K1 cells with the expression of the OP3 receptor. One of the compounds had no orthosteric affinity or intrinsic activity, but inhibited the efficiency of DAMGO. These results allow to conclude that this compound is a negative allosteric modulator (NAM) of the human μ-opioid receptor.

Multi-receptor targeting has been proposed as a promising strategy for the development of opioid analgesics with fewer side effects. Cebranopadol and AT-121 are prototypical bifunctional ligands targeting the nociceptin/orphanin FQ peptide receptor (NOP) and µ-opioid receptor (MOP) that elicit potent analgesia in humans and nonhuman primates, respectively. Cebranopadol was reported to produce typical MOP-related side effects such as respiratory depression and reward, whereas AT-121 appeared to be devoid of these liabilities. However, the molecular basis underlying different side effect profiles in opioid analgesics remains unknown. Here, we examine agonist-induced receptor phosphorylation and G protein signaling profiles of a series of chemically diverse mixed MOP/NOP agonists, including cebranopadol and AT-121. We found that these compounds produce strikingly different MOP phosphorylation profiles. Cebranopadol, AT-034 and AT-324 stimulated extensive MOP phosphorylation, whereas AT-201 induced selective phosphorylation at S375 only. AT-121, on the other hand, did not promote any detectable MOP phosphorylation. Conversely, none of these compounds was able to elicit strong NOP phosphorylation and low NOP receptor phosphorylation correlated with partial agonism in a GIRK-channel assay. Our results suggest a close correlation between MOP receptor phosphorylation and side effect profile. Thus, bifunctional MOP/NOP opioid ligands combining low efficacy G protein signaling at both NOP and MOP with no detectable receptor phosphorylation appear to be devoid of side-effects such as respiratory depression, abuse liability or tolerance development, as with AT-121.

Kappa-opioid receptor (KOR) agonists have been studied as potential treatments for pain, pruritus, and substance-use disorders, but prototypical KOR agonists produce side-effects like dysphoria and sedation. Atypical KOR agonists that exhibit G-protein biased signaling at the KOR have been reported to produce therapeutic-like effects with fewer or reduced side-effects relative to prototypical KOR agonists. In the current report, behavioral profiles were determined using a behavioral scoring system that was modified to quantify drug-induced behaviors in nonhuman primates (NHPs). Profiles were determined for a prototypical and two biased KOR agonists, alone and combined with the mu-opioid receptor (MOR) agonist, oxycodone. Five adult male rhesus monkeys implanted with intravenous catheters were administered a range of doses of the KOR agonist, U50-488H (0.01-0.1 mg/kg) and the biased KOR agonists, nalfurafine (0.0001-0.001 mg/kg) and triazole 1.1 (0.32-1.0 mg/kg), alone and combined with the MOR agonist, oxycodone (0.01-0.32 mg/kg). In addition, the largest triazole 1.1 dose tested (1.0 mg/kg) was administered in time-course determinations (0-56 min), alone and combined with oxycodone (0.1 mg/kg). U50-488H and nalfurafine produced sedative-like and motor-impairing effects. Triazole 1.1 had a milder side-effect profile, in some instances producing sedative-like effects but to a lesser degree compared with the other KOR agonists, particularly for lip droop and rest/sleep posture. All KOR agonists reduced oxycodone-induced scratch, but nalfurafine produced behavior-disrupting and sedative-like effects when combined with oxycodone that were not observed with triazole 1.1. The duration of triazole 1.1's behavioral effects was relatively short, dissipating entirely by 56 min. Our results suggest that KOR agonists with comparable pharmacology to triazole 1.1 may be useful therapeutics with reduced side-effect profiles, and the mechanisms conferring these benefits may be attributed to factors other than G-protein bias.

Neuropathic pain is a debilitating chronic condition that remains difficult to treat. There is a high priority to identify novel non-opioid-based therapeutic targets as long-term use of opioids is problematic due to its severe side effects and strong abuse potential. Our lab recently discovered G-protein coupled receptor 160 (GPR160) has a role in neuropathic pain. Gpr160 was upregulated in the dorsal horn of the spinal cord (DH-SC) on the side of nerve injury relative to the uninjured side in mice. Blocking GPR160 using siRNA or a neutralizing antibody (Ab) reversed and prevented pain hypersensitivity. In addition, our lab deorphanized GPR160 as the receptor for cocaine- and amphetamine-regulated transcript peptide (CARTp). We showed that an intrathecal (i.th.) injection of CARTp in naive mice caused mechano-hypersensitivity that was dependent on GPR160. CARTp-induced gene expression is relatively unexplored and the mechanism by which CARTp/GPR160 signaling promotes chronic pain is not well known. Therefore, our objective was to perform an unbiased RNA transcriptomics analysis to identify which genes were altered at the time of CARTp-mediated peak pain in the DH-SC. We found that Nucleotide-binding oligomerization domain-containing protein 2 (Nod2) expression was increased upon i.th. injection of CARTp and its expression decreased after CARTp was co-administered with GPR160 Ab, pointing to a potential interaction between CARTp/GPR160 and NOD2. NOD2 is a cytosolic pattern recognition receptor that is involved in activating the immune system in response to pathogens. Moreover, a recent study linked NOD2 to the development of neuropathic pain. This led to our hypothesis that CARTp/GPR160 causes mechano-hypersensitivity through NOD2 and NOD2 has a functional role in neuropathic pain. C57BL/6 (WT) and NOD2 mice received an i.th. injection of CARTp and mechano-allodynia was assessed. The WT mice developed mechano-allodynia by 30min and persisted for 5hrs. In contrast, mechano-allodynia was attenuated for 4hrs in NOD2 mice, suggesting CARTp/GPR160 induces mechano-hypersensitivity through NOD2. Furthermore, we found that NOD2 has a functional role in a chronic constriction injury (CCI) model of neuropathic pain. WT mice started developing mechano-allodynia on day 3 (D3) after CCI, reached a maximum by D7, and persisted until D14. However, NOD2 mice did not develop mechano-allodynia until D10, indicating that NOD2 is involved in the development of CCI neuropathic pain. Overall, our results provide a potential mechanistic insight on how CARTp causes mechano-sensitivity and NOD2 has a functional role in CCI-mediated neuropathic pain.

Neuropathic pain is a chronic disorder resulting from damage to the afferent nerve fibers or central pain pathways and is often a complication in pathological conditions such as diabetes, shingles, multiple sclerosis, and stroke. The opioid epidemic has elucidated the need for more efficacious treatments for neuropathic pain. In 2019 alone, nearly 1.6 million people were diagnosed with an opioid use disorder and 48,000 people died from a synthetic opioid overdose. Despite the addictive properties, opioids are still the most frequently prescribed pain medication, even for chronic neuropathic pain. Heterotrimeric G-proteins consisting of the α, β, and γ subunits convey extracellular signals sensed by G-protein coupled receptors (GPCRs) to intracellular effectors. The Gβ5 subunit is a divergent member of the G-protein β subunit family as it does not bind to traditional γ subunits. Instead, Gβ5 complexes with the R7 subfamily of the regulators of G-protein signaling (R7-RGS) containing 4 members: RGS6, 7, 9 and 11. The Gβ5/R7-RGS complex acts as a GTPase accelerating protein (GAP) for G-protein αi/o (Gαi/o) subunits. Previous studies have established the integral role of R7-RGS proteins in pain transmission via their interactions with Gαi/o-coupled receptors including opioid and metabotropic gamma-aminobutyric acid (GABA-B) receptors. Our lab has shown the lack of Gβ5 in sensory ganglia diminishes mechanical, thermal, and chemical nociception. However, the conditional knockout of Gβ5 in Rgs7 expressing neurons reduces only mechanical nociception. This Gβ5/RGS7-dependent mechanical nociception relies on GABA-B receptor signaling as indicated by the rescue of mechanical nociception in Rgs7-Cre; Gnb5 fl/fl mice after treatment with 2-hydroxysaclofen, a GABA-B antagonist. We also established that Rgs9 expressing neurons regulate thermal nociception via a Gβ5-dependent pathway as assayed by the hotplate test in Rgs9-Cre; Gnb5 fl/fl mice. The purpose of this project has been to understand the molecular role of each R7-RGS member in the regulation of pain transmission. First, we confirmed co-localization between the Gnb5 transcript and all four R7-RGS mRNA transcripts in murine dorsal root ganglia (DRG) using the RNAscope HiPlex assay, a novel in situ hybridization technique. We then established the co-localization patterns between each R7-RGS member and various pain related receptors including Mrgprd, Trpa1, and Trpv1. Our RNAscope results support the behavioral tests since Rgs7 transcripts highly co-express with Mrgprd, a mechanical nociceptor, while Rgs9 transcripts most frequently co-express with Trpv1, a thermosensitive receptor. These results suggest that each R7-RGS member might regulate unique types of nociception. We have also shown that Rgs11 transcripts co-localize with Trpv1 and Trpa1 receptor transcripts which indicates Rgs11 might regulate the chemical nociception as tested by capsaicin and mustard oil administration in the eye-wipe test. Next, we aim to study the possible roles of Rgs6 and Rgs11 in regulating chemical nociception using conditional Gβ5 knockout mice mediated by Rgs6-cre and Rgs11-cre, respectively.

Illicitly manufactured fentanyl is fueling the current overdose epidemic, and non-fentanyl mu-opioid receptor (MOR) agonists are emerging in street drug markets worldwide. The etonitazene analog, isotonitazene, is one example of a non-fentanyl MOR agonist linked to overdose deaths. Little is known about the biological effects of isotonitazene in humans or animal models. To this end, we examined the pharmacodynamics, pharmacokinetics, and metabolism of isotonitazene in rats. Male rats were fitted with surgically implanted intravenous (i.v.) catheters and subcutaneous (s.c.) temperature transponders under ketamine/xylazine anesthesia. One week later, rats received s.c. isotonitazene (3, 10, or 30 μg/kg) or its vehicle, and blood samples (0.3 mL) were collected via catheters at 15, 30, 60, 120, 240 min post-injection. Plasma was assayed for isotonitazene and its metabolites by liquid chromatography tandem mass spectrometry. Pharmacodynamic effects – including hot plate latency, catalepsy score, and body temperature – were assessed at each blood withdrawal. Isotonitazene produced dose-dependent increases in hot plate latency (ED50=4.2 μg/kg) and catalepsy (ED50=8.7 μg/kg), while 30 μg/kg produced marked hypothermia. Isotonitazene concentrations in plasma rose linearly with increasing dose, Cmax (0.5 to 6.6 ng/mL) was achieved within 15 min, and drug half-life ranged from 40 to 60 min. Isotonitazene metabolites were detectable but below the level of quantification. Analgesia, catalepsy, and hypothermia were correlated with mean isotonitazene concentrations. Radioligand binding assays revealed that N-desethyl isotonitazene displays higher affinity at MOR (Ki=2.2 nM) than the parent compound (Ki=15.8 nM). Our findings reveal that isotonitazene is a MOR agonist that is ~1000-fold more potent than morphine (ED50=4.2 mg/kg) as an analgesic agent. Plasma concentrations of isotonitazene are in the low ng/mL range, whereas metabolites are found in even lesser amounts. Although the N-desethyl metabolite of isotonitazene displays high affinity at MOR, extremely low levels are formed in vivo. The ultra-high potency of isotonitazene presents challenges for forensic detection and likely poses grave risk to users who are inadvertently exposed to the drug.