A TLR7/8 agonist increases efficacy of anti-fentanyl vaccines in rodent and porcine models.

Opioid use disorders (OUD) and overdose are public health threats worldwide. Widespread access to highly potent illicit synthetic opioids such as fentanyl is driving the recent rise in fatal overdoses. Vaccines containing fentanyl-based haptens conjugated to immunogenic carrier proteins offer a long-lasting, safe, and cost-effective strategy to protect individuals from overdose upon accidental or deliberate exposure to fentanyl and its analogs. Prophylactic or therapeutic active immunization with an anti-fentanyl vaccine induces the production of fentanyl-specific antibodies that bind the drug in the blood and prevent its distribution to the brain, which reduces its reinforcing effects and attenuates respiratory depression and bradycardia. To increase the efficacy of a lead anti-fentanyl vaccine, this study tested whether the incorporation of synthetic toll-like receptor (TLR) 4 and TLR7/8 agonists as vaccine adjuvants would increase vaccine efficacy against fentanyl challenge, overdose, and self-administration in either rats or Hanford miniature pigs. Formulation of the vaccine with a nucleolipid TLR7/8 agonist enhanced its immunogenicity and efficacy in preventing fentanyl-induced respiratory depression, analgesia, bradycardia, and self-administration in either rats or mini-pigs. These studies support the use of TLR7/8 adjuvants in vaccine formulations to improve their clinical efficacy against OUD and potentially other substance use disorders (SUD).

A trypanosome-derived immunotherapeutics platform elicits potent high-affinity antibodies, negating the effects of the synthetic opioid fentanyl.

Poorly immunogenic small molecules pose challenges for the production of clinically efficacious vaccines and antibodies. To address this, we generate an immunization platform derived from the immunogenic surface coat of the African trypanosome. Through sortase-based conjugation of the target molecules to the variant surface glycoprotein (VSG) of the trypanosome surface coat, we develop VSG-immunogen array by sortase tagging (VAST). VAST elicits antigen-specific memory B cells and antibodies in a murine model after deploying the poorly immunogenic molecule fentanyl as a proof of concept. We also develop a single-cell RNA sequencing (RNA-seq)-based computational method that synergizes with VAST to specifically identify memory B cell-encoded antibodies. All computationally selected antibodies bind to fentanyl with picomolar affinity. Moreover, these antibodies protect mice from fentanyl effects after passive immunization, demonstrating the ability of these two coupled technologies to elicit therapeutic antibodies to challenging immunogens.

Pre-clinical safety and toxicology profile of a candidate vaccine to treat oxycodone use disorder.

Opioid use disorders (OUD) and overdose represent a public health threat, resulting in thousands of deaths annually worldwide. Vaccines offer a promising treatment for OUD and potentially the prevention of fatal overdoses. The Oxy(Gly)4-sKLH Conjugate Vaccine, Adsorbed (Oxy(Gly)4-sKLH) has shown promising pre-clinical efficacy at reducing the behavioral and pharmacological effects of oxycodone. To support its clinical evaluation, a GLP toxicology study was performed to address the safety of Oxy(Gly)4-sKLH. Sprague Dawley rats were vaccinated with either aluminum adjuvant (alum) or vaccine adsorbed on alum. Low and high doses of Oxy(Gly)4-sKLH, equivalent to a 1X or 47X human dose, respectively, were administered every two weeks for a total of four vaccinations. Both vaccine doses induced high antibody titers. Vaccine-related toxicity was assessed postmortem in one experimental group after receiving the fourth immunization of the vaccine's high dose. For the remaining experimental groups, rats were challenged with 1.5 mg/kg/day s.c. oxycodone for 7 days after the fourth vaccination to assess whether concurrent exposure to oxycodone in vaccinated animals resulted in toxicity. All rats, except a subset of the aluminum control and the high dose vaccine groups, were sacrificed following oxycodone exposure. These subsets were allowed a four weeks recovery period prior to euthanasia. In this study, no Oxy(Gly)4-sKLH-related hematology, clinical chemistry, urinalysis, body weight, organ weight, or anatomic pathology toxicological findings were observed. These results demonstrate that the Oxy(Gly)4-sKLH vaccine is well tolerated, is immunogenic even at low doses, and does not produce undesired side effects in rats.

The monosodium iodoacetate model of osteoarthritis pain in the rat.

Osteoarthritis (OA) is one form of degenerative joint disease characterized by progressive loss of articular cartilage, decreased function and is frequently accompanied by chronic pain. Given the success of arthroplasty as a treatment for late-stage OA, there is considerable interest in developing therapies pertaining to the management of pain associated with OA as well as therapies designed to slow or reverse the progression of the disease. To this end, establishment of relevant animal models that are amenable to testing novel therapies is of considerable value to the scientific community. Here, we describe a model of OA-related pain in which progressive joint destruction is induced by injection of monosodium iodoacetate into the articular space of the knee of the rat. Further, we describe three different methods to measure pain-related behaviors in this model: hind limb weight bearing, primary mechanical hyperalgesia, and hind limb grip strength.

Quantitative trait locus and computational mapping identifies Kcnj9 (GIRK3) as a candidate gene affecting analgesia from multiple drug classes.

AIMS: Interindividual differences in analgesic drug response complicate the clinical management of pain. We aimed to identify genetic factors responsible for variable sensitivity to analgesic drugs of disparate neurochemical classes. METHODS AND RESULTS: Quantitative trait locus mapping in 872 (C57BL/6x129P3)F2 mice was used to identify genetic factors contributing to variability in the analgesic effect of opioid (morphine), alpha2-adrenergic (clonidine), and cannabinoid (WIN55,212-2) drugs against thermal nociception. A region on distal chromosome 1 showing significant linkage to analgesia from all three drugs was identified. Computational (in silico) genetic analysis of analgesic responses measured in a panel of inbred strains identified a haplotype block within this region containing the Kcnj9 and Kcnj10 genes, encoding the Kir3.3 (GIRK3) and Kir4.1 inwardly rectifying potassium channel subunits. The genes are differentially expressed in the midbrain periaqueductal gray of 129P3 versus C57BL/6 mice, owing to cis-acting genetic elements. The potential role of Kcnj9 was confirmed by the demonstration that knockout mice have attenuated analgesic responses. CONCLUSION: A single locus is partially responsible for the genetic mediation of pain inhibition, and genetic variation associated with the potassium channel gene, Kcnj9, is a prime candidate for explaining the variable response to these analgesic drugs.

Distinct populations of spinal cord lamina II interneurons expressing G-protein-gated potassium channels.

Noxious stimuli are sensed and carried to the spinal cord dorsal horn by A delta and C primary afferent fibers. Some of this input is relayed directly to supraspinal sites by projection neurons, whereas much of the input impinges on a heterogeneous population of interneurons in lamina II. Previously, we demonstrated that G-protein-gated inwardly rectifying potassium (GIRK) channels are expressed in lamina II of the mouse spinal cord and that pharmacologic ablation of spinal GIRK channels selectively blunts the analgesic effect of high but not lower doses of intrathecal mu-opioid receptor (MOR) agonists. Here, we report that GIRK channels formed by GIRK1 and GIRK2 subunits are found in two large populations of lamina II excitatory interneurons. One population displays relatively large apparent whole-cell capacitances and prominent GIRK-dependent current responses to the MOR agonist [D-Ala2,N-MePhe4,Gly-ol5] -enkephalin (DAMGO). A second population shows smaller apparent capacitance values and a GIRK-dependent response to the GABA(B) receptor agonist baclofen, but not DAMGO. Ultrastructural analysis revealed that GIRK subunits preferentially label type I synaptic glomeruli, suggesting that GIRK-containing lamina II interneurons receive prominent input from C fibers, while receiving little input from A delta fibers. Thus, excitatory interneurons in lamina II of the mouse spinal cord can be subdivided into different populations based on the neurotransmitter system coupled to GIRK channels. This important distinction will afford a unique opportunity to characterize spinal nociceptive circuitry with defined physiological significance.

Compartment-dependent colocalization of Kir3.2-containing K+ channels and GABAB receptors in hippocampal pyramidal cells.

G-protein-coupled inwardly rectifying K+ channels (Kir3 channels) coupled to metabotropic GABAB receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABAB receptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABA(B1). Consistent with this observation, a high degree of coclustering of Kir3.2 and GABA(B1) was revealed around excitatory synapses by the highly sensitive SDS-digested freeze-fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K+ channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites.

Spinal G-protein-gated potassium channels contribute in a dose-dependent manner to the analgesic effect of mu- and delta- but not kappa-opioids.

Opioids can evoke analgesia by inhibiting neuronal targets in either the brain or spinal cord, and multiple presynaptic and postsynaptic inhibitory mechanisms have been implicated. The relative significance of presynaptic and postsynaptic inhibition to opioid analgesia is essentially unknown, as are the identities and relevant locations of effectors mediating opioid actions. Here, we examined the distribution of G-protein-gated potassium (GIRK) channels in the mouse spinal cord and measured their contribution to the analgesia evoked by spinal administration of opioid receptor-selective agonists. We found that the GIRK channel subunits GIRK1 and GIRK2 were concentrated in the outer layer of the substantia gelatinosa of the dorsal horn. GIRK1 and GIRK2 were found almost exclusively in postsynaptic membranes of putative excitatory synapses, and a significant degree of overlap with the mu-opioid receptor was observed. Although most GIRK subunit labeling was perisynaptic or extrasynaptic, GIRK2 was found occasionally within the synaptic specialization. Genetic ablation or pharmacologic inhibition of spinal GIRK channels selectively blunted the analgesic effect of high but not lower doses of the mu-opioid receptor-selective agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin. Dose-dependent contributions of GIRK channels to the analgesic effects of the -opioid receptor-selective agonists Tyr-D-Ala-Phe-Glu-Val-Val-Gly amide and [D-Pen(2,5)]-enkephalin were also observed. In contrast, the analgesic effect of the agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate hydrate was preserved despite the absence of GIRK channels. We conclude that the activation of postsynaptic GIRK1 and/or GIRK2-containing channels in the spinal cord dorsal horn represents a powerful, albeit relatively insensitive, means by which intrathecal mu- and -selective opioid agonists evoke analgesia.

Spinal G-protein-gated K+ channels formed by GIRK1 and GIRK2 subunits modulate thermal nociception and contribute to morphine analgesia.

G-protein-gated potassium (K+) channels are found throughout the CNS in which they contribute to the inhibitory effects of neurotransmitters and drugs of abuse. Recent studies have implicated G-protein-gated K+ channels in thermal nociception and the analgesic action of morphine and other agents. Because nociception is subject to complex spinal and supraspinal modulation, however, the relevant locations of G-protein-gated K+ channels are unknown. In this study, we sought to clarify the expression pattern and subunit composition of G-protein-gated K+ channels in the spinal cord and to assess directly their contribution to thermal nociception and morphine analgesia. We detected GIRK1 (G-protein-gated inwardly rectifying K+ channel subunit 1) and GIRK2 subunits, but not GIRK3, in the superficial layers of the dorsal horn. Lack of either GIRK1 or GIRK2 was correlated with significantly lower expression of the other, suggesting that a functional and physical interaction occurs between these two subunits. Consistent with these findings, GIRK1 knock-out and GIRK2 knock-out mice exhibited hyperalgesia in the tail-flick test of thermal nociception. Furthermore, GIRK1 knock-out and GIRK2 knock-out mice displayed decreased analgesic responses after the spinal administration of higher morphine doses, whereas responses to lower morphine doses were preserved. Qualitatively similar data were obtained with wild-type mice after administration of the G-protein-gated K+ channel blocker tertiapin. We conclude that spinal G-protein-gated K+ channels consisting primarily of GIRK1/GIRK2 complexes modulate thermal nociception and mediate a significant component of the analgesia evoked by intrathecal administration of high morphine doses

Hyperalgesia and blunted morphine analgesia in G protein-gated potassium channel subunit knockout mice.

Our aim was to determine whether G protein-gated potassium (Kir3) channels contribute to thermonociception and morphine analgesia. Western blotting was used to probe for the presence of Kir3.1, Kir3.2, Kir3.3, and Kir3.4 subunits in the mouse brain and spinal cord. Hot-plate paw-lick latencies for wild-type, Kir3.2 knockout, Kir3.3 knockout, and Kir3.4 knockout mice were measured at 52 degrees C and 55 degrees C, following the s.c. injection of either saline or 10 mg/kg morphine. Paw-lick latencies for Kir3.4 knockout mice were similar to those of wild-type mice, consistent with the restricted expression pattern of Kir3.4 subunit in the mouse brain. In contrast, Kir3.2 knockout and Kir3.3 knockout mice displayed hyperalgesia at both temperatures tested, and both Kir3.2 knockout and Kir3.3 knockout mice displayed shorter paw-lick latencies following morphine administration, with Kir3.2 knockout mice exhibiting the more dramatic phenotype. Kir3.2/Kir3.3 double knockout mice displayed a greater degree of hyperalgesia than either the Kir3.2 knockout or Kir3.3 knockout mice, while performing similarly to Kir3.2 knockout mice following morphine administration. We conclude that G protein-gated potassium channels containing Kir3.2 and/or Kir3.3 play a significant role in responses to moderate thermal stimuli. Furthermore, the activation of Kir3 channels containing the Kir3.2 subunit contributes to the analgesia evoked by a moderate dose of morphine. As such, receptor-independent Kir3 channel agonists may represent a novel and selective class of analgesic agent.

Contribution of the Kir3.1 subunit to the muscarinic-gated atrial potassium channel IKACh.

The muscarinic-gated atrial potassium (I(KACh)) channel contributes to the heart rate decrease triggered by the parasympathetic nervous system. I(KACh) is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits, although Kir3.4 homomultimers have also been proposed to contribute to this conductance. While Kir3.4 homomultimers evince many properties of I(KACh), the contribution of Kir3.1 to I(KACh) is less well understood. Here, we explored the significance of Kir3.1 using knock-out mice. Kir3.1 knock-out mice were viable and appeared normal. The loss of Kir3.1 did not affect the level of atrial Kir3.4 protein but was correlated with a loss of carbachol-induced current in atrial myocytes. Low level channel activity resembling recombinant Kir3.4 homomultimers was observed in 40% of the cell-attached patches from Kir3.1 knock-out myocytes. Channel activity typically ran down quickly, however, and was not recovered in the inside-out configuration despite the addition of GTP and ATP to the bath. Both Kir3.1 knock-out and Kir3.4 knock-out mice exhibited mild resting tachycardias and blunted responses to pharmacological manipulation intended to activate I(KACh). We conclude that Kir3.1 confers properties to I(KACh) that enhance channel activity and that Kir3.4 homomultimers do not contribute significantly to the muscarinic-gated potassium current.

G-protein-gated potassium channels containing Kir3.2 and Kir3.3 subunits mediate the acute inhibitory effects of opioids on locus ceruleus neurons.

Acute opioid administration causes hyperpolarization of locus ceruleus (LC) neurons. A G-protein-gated, inwardly rectifying potassium (GIRK/K(G)) conductance and a cAMP-dependent cation conductance have both been implicated in this effect; the relative contribution of each conductance remains controversial. Here, the contribution of K(G) channels to the inhibitory effects of opioids on LC neurons was examined using mice that lack the K(G) channel subunits Kir3.2 and Kir3.3. Resting membrane potentials of LC neurons in brain slices from Kir3.2 knock-out, Kir3.3 knock-out, and Kir3.2/3.3 double knock-out mice were depolarized by 15-20 mV relative to LC neurons from wild-type mice. [Met](5)enkephalin-induced hyperpolarization and whole-cell current were reduced by 40% in LC neurons from Kir3.2 knock-out mice and by 80% in neurons from Kir3.2/3.3 double knock-out mice. The small opioid-sensitive current observed in LC neurons from Kir3.2/3.3 double knock-out mice was virtually eliminated with the nonselective potassium channel blockers barium and cesium. We conclude that the acute opioid inhibition of LC neurons is mediated primarily by the activation of G-protein-gated potassium channels and that the cAMP-dependent cation conductance does not contribute significantly to this effect.