Bifunctional Peptide-Based Opioid Agonist-Nociceptin Antagonist Ligands for Dual Treatment of Acute and Neuropathic Pain.

Herein, the opioid pharmacophore H-Dmt-d-Arg-Aba-ß-Ala-NH2 (7) was linked to peptide ligands for the nociceptin receptor. Combination of 7 and NOP ligands (e.g., H-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) led to binding affinities in the low nanomolar domain. In vitro, the hybrids behaved as agonists at the opioid receptors and antagonists at the nociceptin receptor. Intravenous administration of hybrid 13a (H-Dmt-d-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) to mice resulted in potent and long lasting antinociception in the tail-flick test, indicating that 13a was able to permeate the BBB. This was further supported by a cell-based BBB model. All hybrids alleviated allodynia and hyperalgesia in neuropathic pain models. Especially with respect to hyperalgesia, they showed to be more effective than the parent compounds. Hybrid 13a did not result in significant respiratory depression, in contrast to an equipotent analgesic dose of morphine. These hybrids hence represent a promising avenue toward analgesics for the dual treatment of acute and neuropathic pain.

Pharmacological kynurenine 3-monooxygenase enzyme inhibition significantly reduces neuropathic pain in a rat model.

Recent studies have highlighted the involvement of the kynurenine pathway in the pathology of neurodegenerative diseases, but the role of this system in neuropathic pain requires further extensive research. Therefore, the aim of our study was to examine the role of kynurenine 3-monooxygenase (Kmo), an enzyme that is important in this pathway, in a rat model of neuropathy after chronic constriction injury (CCI) to the sciatic nerve. For the first time, we demonstrated that the injury-induced increase in the Kmo mRNA levels in the spinal cord and the dorsal root ganglia (DRG) was reduced by chronic administration of the microglial inhibitor minocycline and that this effect paralleled a decrease in the intensity of neuropathy. Further, minocycline administration alleviated the lipopolysaccharide (LPS)-induced upregulation of Kmo mRNA expression in microglial cell cultures. Moreover, we demonstrated that not only indirect inhibition of Kmo using minocycline but also direct inhibition using Kmo inhibitors (Ro61-6048 and JM6) decreased neuropathic pain intensity on the third and the seventh days after CCI. Chronic Ro61-6048 administration diminished the protein levels of IBA-1, IL-6, IL-1beta and NOS2 in the spinal cord and/or the DRG. Both Kmo inhibitors potentiated the analgesic properties of morphine. In summary, our data suggest that in neuropathic pain model, inhibiting Kmo function significantly reduces pain symptoms and enhances the effectiveness of morphine. The results of our studies show that the kynurenine pathway is an important mediator of neuropathic pain pathology and indicate that Kmo represents a novel pharmacological target for the treatment of neuropathy.

A multi-target approach for pain treatment: dual inhibition of fatty acid amide hydrolase and TRPV1 in a rat model of osteoarthritis.

The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

Minocycline prevents dynorphin-induced neurotoxicity during neuropathic pain in rats.

Despite many advances, our understanding of the involvement of prodynorphin systems in the development of neuropathic pain is not fully understood. Recent studies suggest an important role of neuro-glial interactions in the dynorphin effects associated with neuropathic pain conditions. Our studies show that minocycline reduced prodynorphin mRNA levels that were previously elevated in the spinal and/or dorsal root ganglia (DRG) following sciatic nerve injury. The repeated intrathecal administration of minocycline enhanced the analgesic effects of low-dose dynorphin (0.15 nmol) and U50,488H (25-100 nmol) and prevented the development of flaccid paralysis following high-dose dynorphin administration (15 nmol), suggesting a neuroprotective effect. Minocycline reverts the expression of IL-1ß and IL-6 mRNA within the spinal cord and IL-1ß mRNA in DRG, which was elevated following intrathecal administration of dynorphin (15 nmol). These results suggest an important role of these proinflammatory cytokines in the development of the neurotoxic effects of dynorphin. Similar to minocycline, a selective inhibitor of MMP-9 (MMP-9 levels are reduced by minocycline) exerts an analgesic effect in behavioral studies, and its administration prevents the occurrence of flaccid paralysis caused by high-dose dynorphin administration (15 nmol). In conclusion, our results underline the importance of neuro-glial interactions as evidenced by the involvement of IL-1ß and IL-6 and the minocycline effect in dynorphin-induced toxicity, which suggests that drugs that alter the prodynorphin system could be used to better control neuropathic pain.

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.

Minocycline influences the anti-inflammatory interleukins and enhances the effectiveness of morphine under mice diabetic neuropathy.

A single streptozotocin (STZ) injection in mice can induce significant neuropathic pain along with an increase in plasma glucose levels and a decrease in body weight. Seven days after the administration of STZ, an upregulation of C1q-positive cells was observed. Additionally, interleukins (IL-1beta, IL-3, IL-4, IL-6, IL-9, IL12p70, IL-17); proteins of the tumor necrosis factor (TNF) family, e.g., IFNgamma and sTNF RII, were upregulated. Chronic administration of minocycline increases antinociceptive factors (IL-1alpha, IL-2, IL-10, sTNFRII) in diabetic mice. Minocycline also reduces the occurrence of neuropathic pain and significantly potentiates the antiallodynic and antihyperalgesic effects of morphine.

Neuronal and immunological basis of action of antidepressants in chronic pain - clinical and experimental studies.

The current knowledge of the pharmacological actions of the tricyclic antidepressants (TCAs) has slowly evolved through their over 40-year history. Chronic pain represents one of the most important public health problems, and antidepressants are an essential part of the therapeutic strategy in addition to classical analgesics. This article reviews the available evidence on the efficacy and safety of antidepressants in chronic pain conditions; namely, headaches, low back pain, fibromyalgia, cancer pain and especially neuropathic pain. TCAs are traditionally the main type of depression medication used to treat chronic pain. Recently, new antidepressants were introduced into clinical use, with a significant reduction in side effects and equivalent efficacy on mood disorders. These new drugs that are effective for chronic pain belong to the tetracyclic antidepressants (TeCAs) group (amoxapine, maprotiline), the serotonin and noradrenaline reuptake inhibitors (SNRIs) group (duloxetine, venlafaxine, milnacipran) and the atypical antidepressants group (bupropion, trazodone, mirtazapine, nefazodone). In this review, we present the available publications on TCAs (amitriptyline, doxepin, imipramine, desipramine, nortriptyline), TeCAs (amoxapine, maprotiline), selective serotonin reuptake inhibitors (SSRIs) (citalopram, fluoxetine, paroxetine), SNRIs (duloxetine, venlafaxine, milnacipran) and atypical antidepressants (bupropion) for the treatment of neuropathic pain. We also review analgesics acting as both opioid receptor agonists and also acting as aminergic reuptake inhibitors. Existing data are insufficient to conclude which of these new classes of antidepressants has the best clinical profile and will be the most effective in the treatment of neuropathic pain; in addition, a lower incidence of side effects should be considered. Increased experimental and translational research is a key for further improvement of the treatment of chronic pain with antidepressants. However, evidence from basic science is needed to improve our understanding of the mechanisms of action and their possible pharmacodynamic interactions.

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.

Effects of glycogen synthase kinase 3beta and cyclin-dependent kinase 5 inhibitors on morphine-induced analgesia and tolerance in rats.

Repeated administration of morphine is associated with the development of tolerance, yet the mechanism underlying this phenomenon is still poorly understood. Recent evidence implicating glycogen synthase kinase 3 (GSK3) in opioid receptor signaling pathways has prompted us to investigate its role in morphine tolerance. Administration of 10 mg/kg morphine i.p. to Wistar rats twice daily for 8 days resulted in complete tolerance to its analgesic effects as measured by the tail-flick test. When injections of morphine were preceded by intrathecal (i.t.) administration of either an inhibitor of GSK3 [(3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) or 6-bromoindirubin-3'oxime] or an inhibitor of cyclin-dependent kinase (Cdk), roscovitine, development of tolerance to morphine analgesia was completely abolished. In addition, a single i.t. injection of either kinase inhibitor was able to restore in a dose-dependent manner the analgesic effect of morphine in morphine-tolerant rats. None of the inhibitors in doses used in the present study had analgesic effects of their own nor an effect on the analgesic potency of morphine. Repeated i.t. administration of either inhibitor had caused an increase in abundance of GSK-3beta phosphorylated at Ser(9) in the dorsal lumbar part of the spinal cord of rats that were chronically treated with morphine. Furthermore, reversal of morphine tolerance by a single injection of either inhibitor was always associated with increased abundance of phospho-GSK3beta. In conclusion, our data indicate that chronic morphine treatment activates a highly efficient pathway by means of which Cdk5 regulates GSK3beta activity.

The effects of local pentoxifylline and propentofylline treatment on formalin-induced pain and tumor necrosis factor-alpha messenger RNA levels in the inflamed tissue of the rat paw.

UNLABELLED: We sought to determine whether local administration of pentoxifylline (PTF) or propentofylline (PPTF), which hinders cytokine production, influences pain threshold and formalin-induced pain behavior in rats or the level of tumor necrosis factor-alpha (TNF-alpha) messenger RNA (mRNA) concentrations in the inflamed paw tissue. PTF (0.5, 1, or 2 mg) and PPTF (1 or 2 mg) injected intraplantarly (i.pl.) had no significant effect on pain threshold. Injection of 0.1 mL of a 12% formalin solution subcutaneously into the dorsal surface of the left hindpaw induced pain behavior (47.6 +/- 4.6 incidents per 5 min), and PTF injected at doses of 1 and 2 mg/100 microL i.pl. before (but not after) formalin was effective in antagonizing (33.6 +/- 2.5 and 23.6 +/- 3.4 incidents per 5 min, respectively) formalin-induced pain behavior. A similar antagonistic effect was observed after PPTF treatment at a dose of 2 mg/100 microL; however, in contrast to PTF, at a later time point (85-90 min) after the formalin challenge, this effect was independent of the scheme of PPTF administration, before or after formalin. The effect of PTF on formalin-induced pain behavior did not parallel paw volume as measured by plethysmometer; however, PTF per se significantly increased the paw volume. Formalin injection significantly increased the TNF-alpha mRNA level in the inflamed tissue of the rat hind paw (150%). PTF administered before, but not after, formalin significantly antagonized (by approximately 40%) the observed increase in the level of TNF-alpha mRNA. Our study demonstrates and provides biochemical evidence that preemptive inhibition of proinflammatory cytokine synthesis by the use of PTF and PPTF, phosphodiesterase, and glial activation inhibitors is useful in antagonizing hyperalgesia in formalin-induced pain. Moreover, local administration of PTF may be a valuable approach to the treatment of inflammatory pain. IMPLICATIONS: This study demonstrates and provides biochemical evidence that preemptive inhibition of proinflammatory cytokine synthesis by local administration of pentoxifylline and propentofylline is useful in antagonizing hyperalgesia in formalin-induced pain. Moreover, local administration of pentoxifylline could be regarded as a valid approach to the treatment of inflammatory pain.

Endomorphin-2, deltorphin II and their analogs suppress formalin-induced nociception and c-Fos expression in the rat spinal cord.

In this study, we evaluated the effects of intrathecally administered agonists of mu- and delta-opioid receptor and their analogs on the pain-induced behavior and expression of c-Fos immunoreactivity in the spinal cord, elicited by intraplantar injection of 12% formalin to the hindpaw of the rat. Previous report from our laboratory and other author's study indicated that intrathecal administration of mu agonists morphine and endomorphin-2 and delta-opioid agonist deltorphin II produced a dose-dependent antinociceptive effects in acute and inflammatory pain. In this study, intrathecal injection of morphine (10 microg), endomorphin-2 (5 microg) and its analog Dmt-endomorphin-2 (10 microg) significantly decreased the formalin-induced pain behavior, and lowered a number of c-Fos positive neurons in the laminae I, II and III of the spinal cord by about 40%, 30% and 40%, respectively. Significant reduction of formalin-induced behavioral responses was also observed after i.th. administration of deltorphin II (15 microg) and its analog ile-deltorphin II (15 microg). Agonists of delta-opioid receptor significantly reduced a number of c-Fos positive neurons by about 28% and 40%, respectively. Analog of endomorphin-2 and analog of deltorphin II suppressed more potently expression of c-Fos in the dorsal horn of the spinal cord than the parent peptides. Our study indicates that new analogs of mu- and delta-opioid receptor exhibit strong antinociceptive potency similar or even higher than the parent peptides, and that their effect is positively correlated with the inhibition of c-Fos expression.

Formalin-induced pain and mu-opioid receptor density in brain and spinal cord are modulated by A1 and A2a adenosine agonists in mice.

The effects of adenosine analogues on pain have been shown to depend on the subtype receptor involved as well as on the nociceptive stimuli and on the route of administration. In the first experiment of the present study intraperitoneal administration of the A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) (0.015, 0.03, 0.09, 0.15, 0.21, 0.3 mg/kg) induced dose-dependent analgesia to formalin pain in both phases characterizing the test. The A(2a) receptor agonist 2-[p-2-(carbonyl-ethyl)-phenyethylamino]-5'-N-ethylcarboxaminoadenosine (CGS21680) (0.025, 0.05, 0.1, 0.15 mg/kg) significantly affected behavioral responses to formalin only during the early phase. In the second experiment the interaction between adenosine and the opioid system was investigated through both behavioral and neurochemical studies. The opioid antagonist naltrexone (0.1 mg/kg) did not affect the antinociception induced by CPA (0.21 mg/kg) and CGS21680 (0.05 mg/kg). Autoradiographic studies showed that formalin administration significantly modified mu-opioid receptor density in the superficial laminae of the spinal cord and in the paracentral thalamic nucleus, contralateral to the side of formalin injection. CPA and CGS21680 counteracted these effects induced by formalin. In conclusion the present study confirms and extends the role of A(1) and A(2a) adenosine receptors in the modulation of inflammatory pain and their interaction with the mu-opioid system, and suggests further investigation of these purinergic receptors from a therapeutic perspective.