Bifunctional Opioid/Melanocortin Peptidomimetics for Use in Neuropathic Pain: Variation in the Type and Length of the Linker Connecting the Two Pharmacophores.

Based on the mechanism of neuropathic pain induction, a new type of bifunctional hybrid peptidomimetics was obtained for potential use in this type of pain. Hybrids consist of two types of pharmacophores that are connected by different types of linkers. The first pharmacophore is an opioid agonist, and the second pharmacophore is an antagonist of the pronociceptive system, i.e., an antagonist of the melanocortin-4 receptor. The results of tests in acute and neuropathic pain models of the obtained compounds have shown that the type of linker used to connect pharmacophores had an effect on antinociceptive activity. Peptidomimetics containing longer flexible linkers were very effective at low doses in the neuropathic pain model. To elucidate the effect of linker lengths, two hybrids showing very high activity and two hybrids with lower activity were further tested for affinity for opioid (mu, delta) and melanocortin-4 receptors. Their complexes with the target receptors were also studied by molecular modelling. Our results do not show a simple relationship between linker length and affinity for particular receptor types but suggest that activity in neuropathic pain is related to a proper balance of receptor affinity rather than maximum binding to any or all of the target receptors.

Multifunctional Opioid-Derived Hybrids in Neuropathic Pain: Preclinical Evidence, Ideas and Challenges.

When the first- and second-line therapeutics used to treat neuropathic pain (NP) fail to induce efficient analgesia-which is estimated to relate to more than half of the patients-opioid drugs are prescribed. Still, the pathological changes following the nerve tissue injury, i.a. pronociceptive neuropeptide systems activation, oppose the analgesic effects of opiates, enforcing the use of relatively high therapeutic doses in order to obtain satisfying pain relief. In parallel, the repeated use of opioid agonists is associated with burdensome adverse effects due to compensatory mechanisms that arise thereafter. Rational design of hybrid drugs, in which opioid ligands are combined with other pharmacophores that block the antiopioid action of pronociceptive systems, delivers the opportunity to ameliorate the NP-oriented opioid treatment via addressing neuropathological mechanisms shared both by NP and repeated exposition to opioids. Therewith, the new dually acting drugs, tailored for the specificity of NP, can gain in efficacy under nerve injury conditions and have an improved safety profile as compared to selective opioid agonists. The current review presents the latest ideas on opioid-comprising hybrid drugs designed to treat painful neuropathy, with focus on their biological action, as well as limitations and challenges related to this therapeutic approach.

Blockade of IL-18 signaling diminished neuropathic pain and enhanced the efficacy of morphine and buprenorphine.

Currently, the low efficacy of antinociceptive drugs for the treatment of neuropathic pain is a major therapeutic problem. Here, we show the potential role of interleukin (IL)-18 signaling in this phenomenon. IL-18 is an important molecule that performs various crucial functions, including the alteration of nociceptive transmission in response to neuropathic pain. We have studied the changes in the mRNA and protein levels (qRT-PCR and Western blot analysis, respectively) of IL-18, IL-18-binding protein (IL-18BP) and the IL-18 receptor (IL-18R) over time in rats following chronic constriction injury (CCI) of the sciatic nerve. Our study demonstrated that the spinal levels of IL-18BP were slightly downregulated at days 7 and 14 in the rats subjected to CCI. In contrast, the IL-18 and IL-18R mRNA expression and protein levels were elevated in the ipsilateral spinal cord on days 2, 7 and 14. Moreover, in rats exposed to a single intrathecal administration of IL-18BP (50 and 100 ng) 7 or 14 days following CCI, symptoms of neuropathic pain were attenuated, and the analgesia pursuant to morphine and buprenorphine (0.5 and 2.5 µg) was enhanced. In summary, the restoration of the analgesic activity of morphine and buprenorphine via the blockade of IL-18 signaling suggests that increased IL-18 pathway may account for the decreased analgesic efficacy of opioids for neuropathic pain.

The multiplicity of spinal AA-5-HT anti-nociceptive action in a rat model of neuropathic pain.

There is considerable evidence to support the role of anandamide (AEA), an endogenous ligand of cannabinoid receptors, in neuropathic pain modulation. AEA also produces effects mediated by other biological targets, of which the transient receptor potential vanilloid type 1 (TRPV1) has been the most investigated. Both, inhibition of AEA breakdown by fatty acid amide hydrolase (FAAH) and blockage of TRPV1 have been shown to produce anti-nociceptive effects. Recent research suggests the usefulness of dual-action compounds, which may afford greater anti-allodynic efficacy. Therefore, in the present study, we examined the effect of N-arachidonoyl-serotonin (AA-5-HT), a blocker of FAAH and TRPV1, in a rat model of neuropathic pain after intrathecal administration. We found that treatment with AA-5-HT increased the pain threshold to mechanical and thermal stimuli, with highest effect at the dose of 500nM, which was most strongly attenuated by AM-630, CB2 antagonist, administration. The single action blockers PF-3845 (1000nM, for FAAH) and I-RTX (1nM, for TRPV1) showed lower efficacy than AA-5-HT. Moreover AA-5-HT (500nM) elevated AEA and palmitoylethanolamide (PEA) levels. Among the possible targets of these mediators, only the mRNA levels of CB2, GPR18 and GPR55, which are believed to be novel cannabinoid receptors, were upregulated in the spinal cord and/or DRG of CCI rats. It was previously reported that AA-5-HT acts in CB1 and TRPV1-dependent manner after systemic administration, but here for the first time we show that AA-5-HT action at the spinal level involves CB2, with potential contributions from GRP18 and/or GPR55 receptors.

Blockade of Toll-Like Receptors (TLR2, TLR4) Attenuates Pain and Potentiates Buprenorphine Analgesia in a Rat Neuropathic Pain Model.

Accumulating evidence indicates that microglial TLR2 and TLR4 play a significant role in nociception. Experiments were conducted to evaluate the contribution of TLR2 and TLR4 and their adaptor molecules to neuropathy and their ability to amplify opioid effectiveness. Behavioral tests (von Frey's and cold plate) and biochemical (Western blot and qRT-PCR) analysis of spinal cord and DRG tissue were conducted after chronic constriction injury (CCI) to the sciatic nerve. Repeated intrathecal administration of LPS-RS (TLR2 and TLR4 antagonist) and LPS-RS Ultrapure (TLR4 antagonist) attenuated allodynia and hyperalgesia. Biochemical analysis revealed time-dependent upregulation of mRNA and/or protein levels of TLR2 and TLR4 and MyD88 and TRIF adaptor molecules, which was paralleled by an increase in IBA-1/CD40-positive cells under neuropathy. LPS-RS and LPS-RS Ultrapure similarly influenced opioid analgesia by enhancing the effectiveness of buprenorphine but not morphine. Summing up, in light of their upregulation over the course of pain, both TLR2 and TLR4 may indeed play a significant role in neuropathy, which could be linked to the observed activation of IBA-1/CD40-positive cells. Blockade of TLR2 and TLR4 produced analgesia and enhanced buprenorphine's effectiveness, which suggests that they may be a putative target for future pharmacological pain relief tools, especially for opioid rotation, when the effect of morphine is tolerated.

Anandamide, Acting via CB2 Receptors, Alleviates LPS-Induced Neuroinflammation in Rat Primary Microglial Cultures.

Microglial activation is a polarized process divided into potentially neuroprotective phenotype M2 and neurotoxic phenotype M1, predominant during chronic neuroinflammation. Endocannabinoid system provides an attractive target to control the balance between microglial phenotypes. Anandamide as an immune modulator in the central nervous system acts via not only cannabinoid receptors (CB1 and CB2) but also other targets (e.g., GPR18/GPR55). We studied the effect of anandamide on lipopolysaccharide-induced changes in rat primary microglial cultures. Microglial activation was assessed based on nitric oxide (NO) production. Analysis of mRNA was conducted for M1 and M2 phenotype markers possibly affected by the treatment. Our results showed that lipopolysaccharide-induced NO release in microglia was significantly attenuated, with concomitant downregulation of M1 phenotypic markers, after pretreatment with anandamide. This effect was not sensitive to CB1 or GPR18/GPR55 antagonism. Administration of CB2 antagonist partially abolished the effects of anandamide on microglia. Interestingly, administration of a GPR18/GPR55 antagonist by itself suppressed NO release. In summary, we showed that the endocannabinoid system plays a crucial role in the management of neuroinflammation by dampening the activation of an M1 phenotype. This effect was primarily controlled by the CB2 receptor, although functional cross talk with GPR18/GPR55 may occur.

Expression profiling of genes modulated by minocycline in a rat model of neuropathic pain.

BACKGROUND: The molecular mechanisms underlying neuropathic pain are constantly being studied to create new opportunities to prevent or alleviate neuropathic pain. The aim of our study was to determine the gene expression changes induced by sciatic nerve chronic constriction injury (CCI) that are modulated by minocycline, which can effectively diminish neuropathic pain in animal studies. The genes associated with minocycline efficacy in neuropathic pain should provide insight into the etiology of neuropathic pain and identify novel therapeutic targets. RESULTS: We screened the ipsilateral dorsal part of the lumbar spinal cord of the rat CCI model for differentially expressed genes. Out of 22,500 studied transcripts, the abundance levels of 93 transcripts were altered following sciatic nerve ligation. Percentage analysis revealed that 54 transcripts were not affected by the repeated administration of minocycline (30 mg/kg, i.p.), but the levels of 39 transcripts were modulated following minocycline treatment. We then selected two gene expression patterns, B1 and B2. The first transcription pattern, B1, consisted of 10 mRNA transcripts that increased in abundance after injury, and minocycline treatment reversed or inhibited the effect of the injury; the B2 transcription pattern consisted of 7 mRNA transcripts whose abundance decreased following sciatic nerve ligation, and minocycline treatment reversed the effect of the injury. Based on the literature, we selected seven genes for further analysis: Cd40, Clec7a, Apobec3b, Slc7a7, and Fam22f from pattern B1 and Rwdd3 and Gimap5 from pattern B2. Additionally, these genes were analyzed using quantitative PCR to determine the transcriptional changes strongly related to the development of neuropathic pain; the ipsilateral DRGs (L4-L6) were also collected and analyzed in these rats using qPCR. CONCLUSION: In this work, we confirmed gene expression alterations previously identified by microarray analysis in the spinal cord and analyzed the expression of selected genes in the DRG. Moreover, we reviewed the literature to illustrate the relevance of these findings for neuropathic pain development and therapy. Further studies are needed to elucidate the roles of the individual genes in neuropathic pain and to determine the therapeutic role of minocycline in the rat neuropathic pain model.

The glutamatergic system as a target for neuropathic pain relief.

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. The understanding of glutamatergic transmission in the nervous system has been greatly expanded with the discovery and investigation of the family of ionotropic and metabotropic glutamate receptors (mGluRs). Metabotropic glutamate receptors are localized at nerve terminals, postsynaptic sites and glial cells and thus, they can influence and modulate the action of glutamate at different levels in the synapse. Moreover, there is substantial evidence of glial participation in glutamate nociceptive processes and neuropathic pain. Metabotropic glutamate receptors have been shown to play a role in neuropathic pain, which is one of the most troublesome illnesses because the therapy is still not satisfactory. Recently, the development of selective mGluR ligands has provided important tools for further investigation of the role of mGluRs in the modulation of chronic pain processing. This paper presents a review of the literature of glutamate receptors in neuropathic pain and the role of glia in these effects. Specifically, pharmacological interventions aimed at inhibiting group I mGluRs and/or potentiating group II and III mGluR-mediated signalling is discussed. Moreover, we introduce data about the role of glutamate transporters. They are responsible for the level of glutamate in the synaptic cleft and thus regulate the effects of all three groups of mGluRs and, in consequence, the activity of this system in nociceptive transmission. Additionally, the question of how the modulation of the glutamatergic system influences the effectiveness of analgesic drugs used in neuropathic pain therapy is addressed.

Novel bifunctional hybrid compounds designed to enhance the effects of opioids and antagonize the pronociceptive effects of nonopioid peptides as potent analgesics in a rat model of neuropathic pain.

ABSTRACT: The purpose of our work was to determine the role of nonopioid peptides derived from opioid prohormones in sensory hypersensitivity characteristics of neuropathic pain and to propose a pharmacological approach to restore the balance of these endogenous opioid systems. Nonopioid peptides may have a pronociceptive effect and therefore contribute to less effective opioid analgesia in neuropathic pain. In our study, we used unilateral chronic constriction injury (CCI) of the sciatic nerve as a neuropathic pain model in rats. We demonstrated the pronociceptive effects of proopiomelanocortin- and proenkephalin-derived nonopioid peptides assessed by von Frey and cold plate tests, 7 to 14 days after injury. The concentration of proenkephalin-derived pronociceptive peptides was increased more robustly than that of Met-enkephalin in the ipsilateral lumbar spinal cord of CCI-exposed rats, as shown by mass spectrometry, and the pronociceptive effect of one of these peptides was blocked by an antagonist of the melanocortin 4 (MC4) receptor. The above results confirm our hypothesis regarding the possibility of creating an analgesic drug for neuropathic pain based on enhancing opioid activity and blocking the pronociceptive effect of nonopioid peptides. We designed and synthesized bifunctional hybrids composed of opioid (OP) receptor agonist and MC4 receptor antagonist (OP-linker-MC4). Moreover, we demonstrated that they have potent and long-lasting antinociceptive effects after a single administration and a delayed development of tolerance compared with morphine after repeated intrathecal administration to rats subjected to CCI. We conclude that the bifunctional hybrids OP-linker-MC4 we propose are important prototypes of drugs for use in neuropathic pain.

Agonist-dependent attenuation of mu-opioid receptor-mediated G-protein activation in the dorsal root ganglia of neuropathic rats.

Besides generally accepted lower analgesic potencies of opioids in neuropathic pain, our recent pharmacological reports have demonstrated that the effectiveness of the micro-opioid receptor (MOR) agonists in neuropathy might depends upon the chemical/structural property of these compounds (alkaloid vs. peptides). Such findings prompted us to investigate the changes in MOR mRNA expression (estimated by PCR) as well as MOR functional activity (examined by [(35)S]GTPgammaS binding) in the dorsal horn of the spinal cord and the dorsal root ganglia (DRG) of neuropathic rats at different time points after sciatic nerve ligation. We found that the spinal MOR mRNA level and agonist-stimulated [(35)S]GTPgammaS binding were not affected by nerve injury. In contrast, down-regulation of MOR mRNA in the ipsilateral side of DRG developed 3 (approximately 63% reduction) and 14 (approximately 89% reduction) days after the ligation. The decrease was paralleled with pronounced reduction in the stimulation of [(35)S]GTPgammaS binding by morphine (approximately 37-39%). Thus, neuropathy-induced specific dysfunction of MOR to activate G-protein together with changes in the MOR synthesis might be related, at least in part, to diminish analgesic efficacy of morphine in neuropathic pain. Interesting observations from current studies are linked to endomorphins (EMs), which do not affect the G protein stimulation of MOR after nerve ligation. This intriguing property of EMs, together with previously reported high analgesic efficacy of these compounds indicate that chemically/structurally different MOR agonists, particularly morphine versus EMs, may differentially interact with receptors causing distinct pharmacological effects in chronic pain.

Changes induced by formalin pain in central alpha1-adrenoceptor density are modulated by adenosine receptor agonists.

We aimed to elucidate the role of alpha(1)-adrenoceptors in adenosine analgesia in the formalin test. Formalin was injected into the hind paw of male CD-1 mice after injection of adenosine A(1) or A(2a) receptor agonists, CPA, [N(6)-cyclopentyladenosine], and CGS21680 [2-p-(2-carboxyethyl)-phenylethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride]. In the behavioral experiment, alpha(1)-adrenoceptors were blocked by an alpha(1)-adrenoceptor antagonist prazosin, 0.01 mg/kg i.p., and the time mice spent paw licking was recorded for the early (0-15 min) and late (15-60 min) phase of formalin pain. In the neurochemical experiments, mice were killed 15 or 45 min after formalin injection. The density of alpha(1)-adrenoceptors was assessed in various brain areas and in the lumbar spinal cord by [(3)H]prazosin autoradiography. Adenosine agonists produced analgesia in both phases of formalin pain, while prazosin showed a tendency to pronociceptive action in the late phase, and antagonized the effect of CGS21680. After formalin injection, alpha(1)-adrenoceptor density was elevated in some brain areas, mainly in the late phase (some contralateral amygdaloid and ipsilateral thalamic nuclei) and depressed in others (early phase in the ipsilateral spinal cord and late phase in both ipsi- and contralateral sensorimotor cortex). Elevation of alpha(1)-adrenoceptor density, which may be interpreted as a defensive response, did not develop in several cases of CPA-pretreated mice. This suggests that the analgesic effect of adenosine A(1) receptor activation renders the defensive response unnecessary. The depression of alpha(1)-adrenoceptors may suggest development of hypersensitivity in a given structure, and this was antagonized by CGS21680, suggesting the role of A(2a) receptors in control of inflammatory formalin pain.

Neuropathic Pain Dysregulates Gene Expression of the Forebrain Opioid and Dopamine Systems.

Disturbances in the function of the mesostriatal dopamine system may contribute to the development and maintenance of chronic pain, including its sensory and emotional/cognitive aspects. In the present study, we assessed the influence of chronic constriction injury (CCI) of the sciatic nerve on the expression of genes coding for dopamine and opioid receptors as well as opioid propeptides in the mouse mesostriatal system, particularly in the nucleus accumbens. We demonstrated bilateral increases in mRNA levels of the dopamine D1 and D2 receptors (the latter accompanied by elevated protein level), opioid propeptides proenkephalin and prodynorphin, as well as delta and kappa (but not mu) opioid receptors in the nucleus accumbens at 7 to 14 days after CCI. These results show that CCI-induced neuropathic pain is accompanied by a major transcriptional dysregulation of molecules involved in dopaminergic and opioidergic signaling in the striatum/nucleus accumbens. Possible functional consequences of these changes include opposite effects of upregulated enkephalin/delta opioid receptor signaling vs. dynorphin/kappa opioid receptor signaling, with the former most likely having an analgesic effect and the latter exacerbating pain and contributing to pain-related negative emotional states.

Novel hybrid compounds, opioid agonist+melanocortin 4 receptor antagonist, as efficient analgesics in mouse chronic constriction injury model of neuropathic pain.

When the nerve tissue is injured, endogenous agonist of melanocortin type 4 (MC4) receptor, α-MSH, exerts tonic pronociceptive action in the central nervous system, contributing to sustaining the neuropathic pain state and counteracting the analgesic effects of exogenous opioids. With the intent of enhancing opioid analgesia in neuropathy by blocking the MC4 activation, so-called parent compounds (opioid agonist, MC4 antagonist) were joined together using various linkers to create novel bifunctional hybrid compounds. Analgesic action of four hybrids was tested after intrathecal (i.t.) administration in mouse models of acute and neuropathic pain (chronic constriction injury model, CCI). Under nerve injury conditions, one of the hybrids, UW3, induced analgesia in 1500 times lower i.t. dose than the opioid parent (ED50: 0.0002 nmol for the hybrid, 0.3 nmol for the opioid parent) and in an over 16000 times lower dose than the MC4 parent (ED50: 3.33 nmol) as measured by the von Frey test. Two selected hybrids were tested for analgesic properties in CCI mice after intravenous (i.v.) and intraperitoneal (i.p.) administration. Opioid receptor antagonists and MC4 receptor agonists diminished the analgesic action of these two hybrids studied, though the extent of this effect differed between the hybrids; this suggests that linker is of key importance here. Further results indicate a significant advantage of hybrid compounds over the physical mixture of individual pharmacophores in their analgesic effect. All this evidence justifies the idea of synthesizing a bifunctional opioid agonist-linker-MC4 antagonist compound, as such structure may bring important benefits in neuropathic pain treatment.

Attenuation of morphine tolerance by minocycline and pentoxifylline in naive and neuropathic mice.

We have previously demonstrated that glial inhibitors reduce the development of allodynia and hyperalgesia, potentiating the effect of a single morphine dose in a neuropathic pain model. This study explores the effects of two glial activation inhibitors, minocycline and pentoxifylline, on the development of tolerance to morphine in naive and chronic constriction injury (CCI)-exposed mice. Administration of morphine to naive (20 mg/kg; i.p.) and CCI-exposed mice (40 mg/kg; i.p.) twice daily resulted in tolerance to its anti-nociceptive effect after 6 days. Injections of morphine were combined with minocycline (30 mg/kg, i.p.) or pentoxifylline (20 mg/kg, i.p.) administered as two preemptive doses before first morphine administration in naive or pre-injury in CCI-exposed mice, and repeated twice daily 30 min before each morphine administration. With treatment, development of morphine tolerance was delayed by 5 days (from 6 to 11 days), as measured by the tail-flick test in naive and by tail-flick, von Frey, and cold plate tests in CCI-exposed mice. Western blot analysis of CD11b/c and GFAP protein demonstrated that minocycline and pentoxifylline, at doses delaying development of tolerance to morphine analgesia, significantly diminished the morphine-induced increase in CD11b/c protein level. We found that repeated systemic administration of glial inhibitors significantly delays development of morphine tolerance by attenuating the level of this microglial marker under normal and neuropathic pain conditions. Our results support the idea that targeting microglial activation during morphine therapy/treatment is a novel and clinically promising method for enhancing morphine's analgesic effects, especially in neuropathic pain.

Functional characterization of a novel opioid, PZM21, and its effects on the behavioural responses to morphine.

BACKGROUND AND PURPOSE: The concept of opioid ligands biased towards the G protein pathway with minimal recruitment of ß-arrestin-2 is a promising approach for the development of novel, efficient, and potentially nonaddictive opioid therapeutics. A recently discovered biased µ-opioid receptor agonist, PZM21, showed analgesic effects with reduced side effects. Here, we aimed to further investigate the behavioural and biochemical properties of PZM21. EXPERIMENT APPROACH: We evaluated antinociceptive effects of systemic and intrathecal PZM21 administration. Its addiction-like properties were determined using several behavioural approaches: conditioned place preference, locomotor sensitization, precipitated withdrawal, and self-administration. Also, effects of PZM21 on morphine-induced antinociception, tolerance, and reward were assessed. Effects of PZM21 on striatal release of monoamines were evaluated using brain microdialysis. KEY RESULTS: PZM21 caused long-lasting dose-dependent antinociception. It did not induce reward- and reinforcement-related behaviour; however, its repeated administration led to antinociceptive tolerance and naloxone-precipitated withdrawal symptoms. Pretreatment with PZM21 enhanced morphine-induced antinociception and attenuated the expression of morphine reward. In comparison to morphine, PZM21 administration induced a moderate release of dopamine and a robust release of 5-HT in the striatum. CONCLUSIONS AND IMPLICATIONS: PZM21 exhibited antinociceptive efficacy, without rewarding or reinforcing properties. However, its clinical application may be restricted, as it induces tolerance and withdrawal symptoms. Notably, its ability to diminish morphine reward implies that PZM21 may be useful in treatment of opioid use disorders.

Tapentadol - A representative of a new class of MOR-NRI analgesics.

Tapentadol is a centrally acting analgesic with a dual mode of action as a µ-opioid receptor (MOR) agonist and a noradrenaline reuptake inhibitor (NRI). It was initially approved by the US Food and Drug Administration in November 2008 for the treatment of moderate-to-severe acute pain in adult patients, and in August 2011, for chronic pain in an prolonged release form in the same population. Due to its limited protein binding capacity, the absence of active metabolites and significant microsomal enzyme induction or inhibition, tapentadol has a limited potential for drug-drug interactions. It appears to be well-tolerated and effective in the treatment of moderate-to severe acute and chronic pain. Owing to its dual mechanism of action, it is hypothesized to be good option in the treatment of both nociceptive and neuropathic pain.

Alterations in the Activity of Spinal and Thalamic Opioid Systems in a Mice Neuropathic Pain Model.

Clinical studies have reported lower effectivity of opioid drugs in therapy of neuropathic pain. Therefore, to determine the changes in endogenous opioid systems in this pain more precisely, we have studied the changes in the pain-related behavior on days 1, 14, and 28 following a chronic constriction injury (CCI) to the sciatic nerve in mice. In parallel, we have studied the changes of µ-(MOP), δ-(DOP) and κ-(KOP) receptors, proenkephalin (PENK) and prodynorphin (PDYN) mRNA levels, as well as GTPγS binding of opioid receptors on the ipsi- and contralateral parts of the spinal cord and thalamus on the 14th day following CCI, as on this day the greatest manifestation of pain-related behavior was observed. On ipsilateral spinal cord, the decrease in MOP/DOP/KOP receptor and increase in PDYN/PENK mRNA expression was observed. In thalamus, MOP/DOP/KOP receptor expression decreased contralaterally. On ipsilateral side, there were no changes in PDYN/PENK or DOP/KOP receptor expression, but MOP mRNA decreased. The spinal GTPγS binding of MOP/DOP/KOP receptor ligands decreased on the ipsilateral side, yet the effect was less pronounced for DOP receptor ligands. In thalamus, a decrease was observed on the contralateral side for all opioid receptor ligands, especially for DOP ligand. A less pronounced decrease in GTPγS binding of spinal DOP ligands may indicate a weaker stimulation of ascending nociceptive pathways, which could explain the absence of decreased activity of DOP receptor ligands in neuropathy. These findings may suggest that drugs with a higher affinity for the DOP receptor will perform better in neuropathic pain.

Peripheral antinociceptive effects of exogenous and immune cell-derived endomorphins in prolonged inflammatory pain.

Endomorphins (EMs) are endogenous selective mu-opioid receptor agonists. Their role in inflammatory pain has not been fully elucidated. Here we examine peripheral antinociception elicited by exogenously applied EM-1 and EM-2 and the contribution of EM-containing leukocytes to stress- and corticotropin-releasing factor (CRF)-induced antinociception. To this end, we applied behavioral (paw pressure) testing, radioligand binding, immunohistochemistry, and flow cytometry in rats with unilateral hindpaw inflammation induced with Freund's adjuvant. EMs injected directly into both hindpaws produced antinociception exclusively in inflamed paws. This was blocked by locally applied mu-receptor-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) but not kappa-receptor-selective (nor-binaltorphimine) antagonists. Delta-receptor antagonists (naltrindole and N,N-diallyl-Tyr-Aib-Aib-Phe-Leu) did not influence EM-1-induced but dose-dependently decreased EM-2-induced antinociception. Antibodies against beta-endorphin, methionine-enkephalin, or leucine-enkephalin did not significantly change EM-2-induced antinociception. Both EMs displaced binding of [3H]-[D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to mu-receptors in dorsal root ganglia (DRG). Using [3H]-naltrindole or [(125)I]-[D-Pen2,5]-enkephalin, no detectable delta-binding was found in DRG of inflamed hindlimbs. Numerous beta-endorphin-containing and fewer EM-1- and EM-2-containing leukocytes were detected in subcutaneous tissue of inflamed paws. Leukocyte-depleting serum decreased the number of immigrating opioid-containing immune cells and attenuated swim stress- and CRF-induced antinociception in inflamed paws. Both forms of antinociception were strongly attenuated by anti-beta-endorphin and to a lesser degree by anti-EM-1 and anti-EM-2 antibodies injected into inflamed paws. Together, exogenously applied and immune cell-derived EMs alleviate prolonged inflammatory pain through selective activation of peripheral opioid receptors. Exogenous EM-2 in addition to mu-receptors also activates peripheral delta-receptors, which does not involve actions via other opioid peptides.

Minocycline and pentoxifylline attenuate allodynia and hyperalgesia and potentiate the effects of morphine in rat and mouse models of neuropathic pain.

Recent research has shown that microglial cells which are strongly activated in neuropathy can influence development of allodynia and hyperalgesia. Here we demonstrated that preemptive and repeated i.p., administration (16 h and 1 h before injury and then after nerve ligation twice daily for 7 days) of minocycline (15; 30; 50 mg/kg), a potent inhibitor of microglial activation, significantly attenuated the allodynia (von Frey test) and hyperalgesia (cold plate test) measured on day 3, 5, 7 after chronic constriction injury (CCI) in rats. Moreover, the 40% improvement of motor function was observed. In mice, i.p., administration of minocycline (30 mg/kg) or pentoxifylline (20 mg/kg) according to the same schedule also significantly decreased allodynia and hyperalgesia on day 7 after CCI. Antiallodynic and antihyperalgesic effect of morphine (10 mg/kg; i.p.) was significantly potentiated in groups preemptively and repeatedly injected with minocycline (von Frey test, 18 g versus 22 g; cold plate test, 13 s versus 20 s in rats and 1.2 g versus 2.2 g; 7.5 s versus 10 s in mice; respectively) or pentoxifylline (1.3 g versus 3 g; 7.6 s versus 15 s in mice; respectively). Antiallodynic and antihyperalgesic effect of morphine (30 microg; i.t.) given by lumbar puncture in mice was also significantly potentiated in minocycline-treated group (1.2 g versus 2.2 g; 7.5 s versus 11 s; respectively). These findings indicate that preemptive and repeated administration of glial inhibitors suppresses development of allodynia and hyperalgesia and potentiates effects of morphine in rat and mouse models of neuropathic pain.

Local peripheral antinociceptive effects of 14-O-methyloxymorphone derivatives in inflammatory and neuropathic pain in the rat.

Antinociception achieved after peripheral administration of opioids has opened a new approach to the treatment of severe and chronic pain. Additionally, opioid analgesics with restricted access to the central nervous system could improve safety of opioid drugs used in clinical practice. In the present study, peripheral components of antinociceptive actions of 6-amino acid-substituted derivatives of 14-O-methyloxymorphone were investigated after local intraplantar (i.pl.) administration in rat models of inflammatory and neuropathic pain. Their antinociceptive activities were compared with those of morphine, the classical mu-opioid receptor agonist. Intraplantar administration of morphine and the 6-amino acid derivatives produced dose-dependent reduction of formalin-induced flinching of the inflamed paw, without significant effect on the paw edema. Local i.pl. administration of the new derivatives in rats with neuropathic pain induced by sciatic nerve ligation produced antiallodynic and antihyperalgesic effects; however, the antinociceptive activity was lower than that observed in inflammatory pain. In both models, the 6-amino acid derivatives and morphine at doses that produced analgesia after i.pl. administration were systemically (s.c.) much less active indicating that the antinociceptive action is due to a local effect. Moreover, the local opioid antinociceptive effects were significantly attenuated by naloxone methiodide, a peripherally acting opioid receptor antagonist, demonstrating that the effect was mediated by peripheral opioid receptors. The present data indicate that the peripherally restricted 6-amino acid conjugates of 14-O-methyloxymorphone elicit antinociception after local administration, being more potent in inflammatory than in neuropathic pain. Opioid drugs with peripheral site of action can be an important target for the treatment of long lasting pain.