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Indiscriminate activation of opioid receptors provides pain relief but also severe central and intestinal side effects. We hypothesized that exploiting pathological (rather than physiological) conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist that, because of its low acid dissociation constant, selectively activates peripheral μ-opioid receptors at the source of pain generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, heterotrimeric guanine nucleotide–binding protein (G protein) subunit dissociation by fluorescence resonance energy transfer, and adenosine 3′,5′-monophosphate inhibition in vitro. It produced injury-restricted analgesia in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation, constipation, or addiction potential.

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.

Typically, approach behaviour is displayed in the context of moving towards a desired goal, while avoidance behaviour is displayed in the context of moving away from threatening or novel stimuli. In the current research, we detected three sub-populations of C57BL/6J mice that spontaneously responded with avoiding, balancing or approaching behaviours in the presence of the same conflicting stimuli. While the balancing animals reacted with balanced responses between approach and avoidance, the avoiding or approaching animals exhibited inhibitory or advance responses towards one of the conflicting inputs, respectively. Individual differences in approach and avoidance motivation might be modulated by the normal variance in the level of functioning of different systems, such as endocannabinoid system (ECS). The present research was aimed at analysing the ECS involvement on approach and avoidance behavioural processes. To this aim, in the three selected sub-populations of mice that exhibited avoiding or balancing or approaching responses in an approach/avoidance Y-maze we analysed density and functionality of CB(1) receptors as well as enzyme fatty acid amide hydrolase activity in different brain regions, including the networks functionally responsible for emotional and motivational control. The main finding of the present study demonstrates that in both approaching and avoiding animals higher CB(1) receptor density in the amygdaloidal centro-medial nuclei and in the hypothalamic ventro-medial nucleus was found when compared with the CB(1) receptor density exhibited by the balancing animals. The characterization of the individual differences to respond in a motivationally based manner is relevant to clarify how the individual differences in ECS activity are associated with differences in motivational and affective functioning.

The olfactory bulbectomized (OB) rat, an animal model of chronic depression with comorbid anxiety, exhibits a profound dysregulation of the brain serotonergic signalling, a neurotransmission system involved in pain transmission and modulation. We here report an increased nociceptive response of OB rats in the tail flick test which is reverted after chronic, but not acute, administration of fluoxetine. Autoradiographic studies demonstrated down-regulation of 5-HT transporters ([ 3 H]citalopram binding) and decreased functionality of 5-HT 1A receptors (8-OH-DPAT-stimulated [ 35 S]GTPγS binding) in the dorsal horn of the lumbar spinal cord in OB rats. Acute administration of fluoxetine (5–40 mg/kg i.p.) did not modify tail flick latencies in OB rats. However, chronic fluoxetine (10 mg/kg/day s.c., 14 days; osmotic minipumps) progressively attenuated OB-associated thermal hyperalgesia, and a total normalization of the nociceptive response was achieved at the end of the treatment with the antidepressant. In these animals, autoradiographic studies revealed further down-regulation of 5-HT transporters and normalization in the functionality of 5-HT 1A receptors on the spinal cord. On the other hand, acute morphine (0.5–10 mg/kg s.c.) produced a similar analgesic effect in OB and sham and OB rats, and no changes were detected in the density ([ 3 H]DAMGO binding) and functionality (DAMGO-stimulated [ 35 S]GTPγS binding) of spinal μ-opioid receptors in OB rats before and after chronic fluoxetine. Our findings demonstrate the participation of the spinal serotonergic system in the increased thermal nociception exhibited by the OB rat and the antinociceptive effect of chronic fluoxetine in this animal model of depression.

Novel pain killers without adverse effects are urgently needed. Opioids induce central and intestinal side effects such as respiratory depression, sedation, addiction, and constipation. We have recently shown that a newly designed agonist with a reduced acid dissociation constant (pK a ) abolished pain by selectively activating peripheral μ-opioid receptors (MOR) in inflamed (acidic) tissues without eliciting side effects. Here, we extended this concept in that pK a reduction to 7.22 was achieved by placing a fluorine atom at the ethylidene bridge in the parental molecule fentanyl. The new compound (FF3) showed pH-sensitive MOR affinity, [ 35 S]-GTPγS binding, and G protein dissociation by fluorescence resonance energy transfer. It produced injury-restricted analgesia in rat models of inflammatory, postoperative, abdominal, and neuropathic pain. At high dosages, FF3 induced sedation, motor disturbance, reward, constipation, and respiratory depression. These results support our hypothesis that a ligand’s pK a should be close to the pH of injured tissue to obtain analgesia without side effects.

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.

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.

Lysophosphatidylinositol (LPI) is a biologically active lipid that produces a number of responses in cultured cells, and has been suggested to have neuroprotective properties in vivo. Some of the actions of LPI are mediated by G-protein coupled receptors, but it is not known whether G-protein coupled receptor-mediated responses can be seen in intact brain tissue. In consequence, in the present study, we investigated autoradiographically whether LPI increased the [(35)S]GTPγS binding level in brain tissue slices. In standard assay conditions, where as a positive control a robust response to cannabinoid receptor activation by the agonist ligand CP55,940 was seen, there was no increase in the autoradiographic density over basal produced by LPI. However, when the conditions were modified (incubation at 4°C rather than at 25°C, incubation time increased to 3 h, GDP concentration reduced from 2 to 0.1 mM), a significant increase in [(35)S]GTPγS autoradiographic density in response to 10 μM LPI was seen in the prefrontal cortex, hippocampus, and cortex at the level of the hippocampus, although the degree of increase was small and very variable. No significant increases were seen in the hypothalamus or cerebellum. It is concluded that LPI, in the right conditions, can activate a sufficient number of G-proteins in the rat prefrontal cortex and hippocampus to produce a response in the [(35)S]GTPγS autoradiographic assay of G-protein coupled receptor function.Lysophosphatidylinositol (LPI) is a biologically active lipid that produces a number of responses in cultured cells, and has been suggested to have neuroprotective properties in vivo. Some of the actions of LPI are mediated by G-protein coupled receptors, but it is not known whether G-protein coupled receptor-mediated responses can be seen in intact brain tissue. In consequence, in the present study, we investigated autoradiographically whether LPI increased the [(35)S]GTPγS binding level in brain tissue slices. In standard assay conditions, where as a positive control a robust response to cannabinoid receptor activation by the agonist ligand CP55,940 was seen, there was no increase in the autoradiographic density over basal produced by LPI. However, when the conditions were modified (incubation at 4°C rather than at 25°C, incubation time increased to 3 h, GDP concentration reduced from 2 to 0.1 mM), a significant increase in [(35)S]GTPγS autoradiographic density in response to 10 μM LPI was seen in the prefrontal cortex, hippocampus, and cortex at the level of the hippocampus, although the degree of increase was small and very variable. No significant increases were seen in the hypothalamus or cerebellum. It is concluded that LPI, in the right conditions, can activate a sufficient number of G-proteins in the rat prefrontal cortex and hippocampus to produce a response in the [(35)S]GTPγS autoradiographic assay of G-protein coupled receptor function.

Lysophosphatidylinositol (LPI) is a biologically active lipid that produces a number of responses in cultured cells, and has been suggested to have neuroprotective properties in vivo. Some of the actions of LPI are mediated by G-protein coupled receptors, but it is not known whether G-protein coupled receptor-mediated responses can be seen in intact brain tissue. In consequence, in the present study, we investigated autoradiographically whether LPI increased the [^sup 35^S]GTPγS binding level in brain tissue slices. In standard assay conditions, where as a positive control a robust response to cannabinoid receptor activation by the agonist ligand CP55,940 was seen, there was no increase in the autoradiographic density over basal produced by LPI. However, when the conditions were modified (incubation at 4°C rather than at 25°C, incubation time increased to 3 h, GDP concentration reduced from 2 to 0.1 mM), a significant increase in [^sup 35^S]GTPγS autoradiographic density in response to 10 μM LPI was seen in the prefrontal cortex, hippocampus, and cortex at the level of the hippocampus, although the degree of increase was small and very variable. No significant increases were seen in the hypothalamus or cerebellum. It is concluded that LPI, in the right conditions, can activate a sufficient number of G-proteins in the rat prefrontal cortex and hippocampus to produce a response in the [^sup 35^S]GTPγS autoradiographic assay of G-protein coupled receptor function.[PUBLICATION ABSTRACT]

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