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The non-selective activation of central and peripheral opioid receptors is a major shortcoming of currently available opioids. Targeting peripheral opioid receptors is a promising strategy to preclude side effects. Recently, we showed that fentanyl-derived μ-opioid receptor (MOR) agonists with reduced acid dissociation constants (pK a ) due to introducing single fluorine atoms produced injury-restricted antinociception in rat models of inflammatory, postoperative and neuropathic pain. Here, we report that a new double-fluorinated compound (FF6) and fentanyl show similar pK a , MOR affinity and [ 35 S]-GTPγS binding at low and physiological pH values. In vivo , FF6 produced antinociception in injured and non-injured tissue, and induced sedation and constipation. The comparison of several fentanyl derivatives revealed a correlation between pK a values and pH-dependent MOR activation, antinociception and side effects. An opioid ligand’s pK a value may be used as discriminating factor to design safer analgesics.

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model.

Novel painkillers are urgently needed. The activation of opioid receptors in peripheral inflamed tissue can reduce pain without central adverse effects such as sedation, apnoea, or addiction. Here, we use an unprecedented strategy and report the synthesis and analgesic efficacy of the standard opioid morphine covalently attached to hyperbranched polyglycerol (PG-M) by a cleavable linker. With its high-molecular weight and hydrophilicity, this conjugate is designed to selectively release morphine in injured tissue and to prevent blood-brain barrier permeation. In contrast to conventional morphine, intravenous PG-M exclusively activated peripheral opioid receptors to produce analgesia in inflamed rat paws without major side effects such as sedation or constipation. Concentrations of morphine in the brain, blood, paw tissue, and in vitro confirmed the selective release of morphine in the inflamed milieu. Thus, PG-M may serve as prototype of a peripherally restricted opioid formulation designed to forego central and intestinal side effects.

Background Mammalian arachidonic acid lipoxygenases (ALOXs) have previously been implicated in the pathogenesis of inflammatory disease, and pro- as well as anti-inflammatory activities have been reported. The human genome involves six functional ALOX genes and each of them encodes for a functionally distinct enzyme. ALOX15 is one of these isoforms and the majority of mammalian ALOX15 orthologs including mouse Alox15 convert arachidonic acid to its 12-hydroperoxy derivative. In contrast, human ALOX15 forms 15-hydroperoxy arachidonic acid instead. This difference in the catalytic properties of the two mammalian ALOX15 orthologs may be of biological relevance since arachidonic acid 15-lipoxygenating ALOX-isoforms exhibit an improved biosynthetic capacity for pro-resolving mediators. We recently generated Alox15 knock-in mice, which homozygously express a humanized Alox15 mutant (Leu353Phe) instead of the wildtype enzyme. These animals should be protected from the development of inflammatory symptoms in whole animal inflammation models if the biosynthesis of pro-resolving mediators plays a major role. Methods To explore whether functional humanization of mouse Alox15 might impact the pathogenesis of inflammatory diseases we tested Alox-KI mice in comparison with wildtype control animals in two whole animal inflammation models (dextran sodium sulfate induced colitis, Freund’s complete adjuvant induced paw edema). In these experiments we quantified the severity of inflammatory symptoms during the acute phase of inflammation and during the resolution period. Results We found that Alox15 knock-in mice are strongly protected from the development of inflammatory symptoms in the dextran sodium sulfate colitis model when the loss of body weight was used as major readout parameter. Quantification of the colon tissue oxylipidomes revealed that the colon concentrations of resolvin D5 were elevated in Alox15-KI mice and thus, this mediator might contribute to the protective effect induced by our genetic manipulation. However, other specialized pro-resolving mediators, such as maresin-2, neuroprotectin-1, and lipoxins, may not play a major role for the protective response. In the Freund’s complete adjuvant induced paw edema inflammation model no protective effect was observed. Conclusions Taken together, our data suggest that humanization of the reaction specificity of mouse Alox15 (Leu353Phe mutation) exhibits differential effects in two mouse inflammation models.

The use of opioid agonists acting outside the central nervous system (CNS) is a promising therapeutic strategy for pain control that avoids deleterious central side effects such as apnea and addiction. In human clinical trials and rat models of inflammatory pain, peripherally restricted opioids have repeatedly shown powerful analgesic effects; in some mouse models however, their actions remain unclear. Here, we investigated opioid receptor coupling to K + channels as a mechanism to explain such discrepancies. We found that GIRK channels, major effectors for opioid signalling in the CNS, are absent from mouse peripheral sensory neurons but present in human and rat. In vivo transgenic expression of GIRK channels in mouse nociceptors established peripheral opioid signalling and local analgesia. We further identified a regulatory element in the rat GIRK2 gene that accounts for differential expression in rodents. Thus, GIRK channels are indispensable for peripheral opioid analgesia, and their absence in mice has profound consequences for GPCR signalling in peripheral sensory neurons. Graphical Abstract GIRK channels are indispensable for peripheral opioid analgesia. The absence of GIRK channels from mouse dorsal root ganglion neurons questions the predictive validity of mice as a model organism for investigating peripheral GPCRmediated analgesia.

Touch base The sense of touch is mediated by the many mechanoreceptors present in the skin. Now, for first time in a vertebrate, a single molecule whose function is necessary for this primary transduction event has been identified. The membrane protein SLP3 is necessary for mechanoreceptor function in mice in vivo , and is also required for mechanosensitive channels to function. SLP3 also modulates ion channels previously implicated in touch sensation, so may be a subunit of the mammalian touch transducer. Touch and mechanical pain are first detected at our largest sensory surface, the skin. The cell bodies of sensory neurons that detect such stimuli are located in the dorsal root ganglia, and subtypes of these neurons are specialized to detect specific modalities of mechanical stimuli. Molecules have been identified that are necessary for mechanosensation in invertebrates but so far not in mammals. In Caenorhabditis elegans , mec-2 is one of several genes identified in a screen for touch insensitivity and encodes an integral membrane protein with a stomatin homology domain 1 . Here we show that about 35% of skin mechanoreceptors do not respond to mechanical stimuli in mice with a mutation in stomatin-like protein 3 (SLP3, also called Stoml3), a mammalian mec-2 homologue that is expressed in sensory neurons. In addition, mechanosensitive ion channels found in many sensory neurons do not function without SLP3. Tactile-driven behaviours are also impaired in SLP3 mutant mice, including touch-evoked pain caused by neuropathic injury. SLP3 is therefore indispensable for the function of a subset of cutaneous mechanoreceptors, and our data support the idea that this protein is an essential subunit of a mammalian mechanotransducer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neuropathic pain often arises from damage to peripheral nerves and is difficult to treat. Activation of opioid receptors in peripheral sensory neurons is devoid of respiratory depression, sedation, nausea, and addiction mediated in the brain, and ameliorates neuropathic pain in animal models. Mechanisms of peripheral opioid analgesia have therefore gained interest, but the role of G protein-coupled inwardly rectifying potassium (Kir3) channels, important regulators of neuronal excitability, remains unclear. Whereas functional Kir3 channels have been detected in dorsal root ganglion (DRG) neurons in rats, some studies question their contribution to opioid analgesia in inflammatory pain models in mice. However, neuropathic pain can be diminished by activation of peripheral opioid receptors in mouse models. Therefore, here we investigated effects of the selective μ-opioid receptor (MOR) agonist [D-Ala , -Me-Phe , Gly -ol]-enkephalin (DAMGO) on potassium conductance in DRG neurons upon a chronic constriction injury (CCI) of the sciatic nerve in mice. For verification, we also tested human embryonic kidney (HEK) 293 cells transfected with MOR and Kir3.2. Using patch clamp, we recorded currents at -80 mV and applied voltage ramps in high extracellular potassium concentrations, which are a highly sensitive measures of Kir3 channel activity. We found a significantly higher rate of HEK cells responding with potassium channel blocker barium-sensitive inward current (233 ± 51 pA) to DAMGO application in transfected than in untransfected group, which confirms successful recordings of inward currents through Kir3.2 channels. Interestingly, DAMGO induced similar inward currents (178 ± 36-207 ± 56 pA) in 15-20% of recorded DRG neurons from naïve mice and in 4-27% of DRG neurons from mice exposed to CCI, measured in voltage clamp or voltage ramp modes. DAMGO-induced currents in naïve and CCI groups were reversed by barium and a more selective Kir3 channel blocker tertiapin-Q. These data indicate the coupling of Kir3 channels with MOR in mouse peripheral sensory neuron cell bodies, which was unchanged after CCI. A comparative analysis of opioid-induced potassium conductance at the axonal injury site and peripheral terminals of DRG neurons could clarify the role of Kir3 channel-MOR interactions in peripheral nerve injury and opioid analgesia.

Background: Proopiomelanocortin (POMC)-derived beta-endorphin1-31 from immune cells can inhibit inflammatory pain. Here we investigated cytokine signaling pathways regulating POMC gene expression and beta-endorphin production in lymphocytes to augment such analgesic effects. Results: Interleukin-4 dose-dependently elevated POMC mRNA expression in naïve lymph node-derived cells in vitro, as determined by real-time PCR. This effect was neutralized by janus kinase (JAK) inhibitors. Transfection of Signal Transducer and Activator of Transcription (STAT) 1/3 but not of STAT6 decoy oligonucleotides abolished interleukin-4 induced POMC gene expression. STAT3 was phosphorylated in in vitro interleukin-4 stimulated lymphocytes and in lymph nodes draining inflamed paws in vivo. Cellular beta-endorphin increased after combined stimulation with interleukin-4 and concanavalin A. Consistently, in vivo reduction of inflammatory pain by passively transferred T cells improved significantly when donor cells were pretreated with interleukin-4 plus concanavalin A. This effect was blocked by naloxone-methiodide. Conclusion: Interleukin-4 can amplify endogenous opioid peptide expression mediated by JAK-STAT1/3 activation in mitogen-activated lymphocytes. Transfer of these cells leads to inhibition of inflammatory pain via activation of peripheral opioid receptors.

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5−/− mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5−/− animals tested previously in similar experimental setups.