Insights into the Current and Possible Future Use of Opioid Antagonists in Relation to Opioid-Induced Constipation and Dysbiosis.

Opioid receptor agonists, particularly those that activate µ-opioid receptors (MORs), are essential analgesic agents for acute or chronic mild to severe pain treatment. However, their use has raised concerns including, among others, intestinal dysbiosis. In addition, growing data on constipation-evoked intestinal dysbiosis have been reported. Opioid-induced constipation (OIC) creates an obstacle to continuing treatment with opioid analgesics. When non-opioid therapies fail to overcome the OIC, opioid antagonists with peripheral, fast first-pass metabolism, and gastrointestinal localized effects remain the drug of choice for OIC, which are discussed here. At first glance, their use seems to only be restricted to constipation, however, recent data on OIC-related dysbiosis and its contribution to the appearance of several opioid side effects has garnered a great of attention from researchers. Peripheral MORs have also been considered as a future target for opioid analgesics with limited central side effects. The properties of MOR antagonists counteracting OIC, and with limited influence on central and possibly peripheral MOR-mediated antinociception, will be highlighted. A new concept is also proposed for developing gut-selective MOR antagonists to treat or restore OIC while keeping peripheral antinociception unaffected. The impact of opioid antagonists on OIC in relation to changes in the gut microbiome is included.

Pro-Inflammatory Cytokines: Potential Links between the Endocannabinoid System and the Kynurenine Pathway in Depression.

Substance use/abuse is one of the main causes of depressive symptoms. Cannabis and synthetic cannabinoids in particular gained significant popularity in the past years. There is an increasing amount of clinical data associating such compounds with the inflammatory component of depression, indicated by the up-regulation of pro-inflammatory cytokines. Pro-inflammatory cytokines are also well-known to regulate the enzymes of the kynurenine pathway (KP), which is responsible for metabolizing tryptophan, a precursor in serotonin synthesis. Enhanced pro-inflammatory cytokine levels may over-activate the KP, leading to tryptophan depletion and reduced serotonin levels, which can subsequently precipitate depressive symptoms. Therefore, such mechanism might represent a possible link between the endocannabinoid system (ECS) and the KP in depression, via the inflammatory and dysregulated serotonergic component of the disorder. This review will summarize the data regarding those natural and synthetic cannabinoids that increase pro-inflammatory cytokines. Furthermore, the data on such cytokines associated with KP activation will be further reviewed accordingly. The interaction of the ECS and the KP has been postulated and demonstrated in some studies previously. This review will further contribute to this yet less explored connection and propose the KP to be the missing link between cannabinoid-induced inflammation and depressive symptoms.

Pharmacological Evidence on Augmented Antiallodynia Following Systemic Co-Treatment with GlyT-1 and GlyT-2 Inhibitors in Rat Neuropathic Pain Model.

The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30-60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP.

Shedding Light on the Pharmacological Interactions between µ-Opioid Analgesics and Angiotensin Receptor Modulators: A New Option for Treating Chronic Pain.

The current protocols for neuropathic pain management include µ-opioid receptor (MOR) analgesics alongside other drugs; however, there is debate on the effectiveness of opioids. Nevertheless, dose escalation is required to maintain their analgesia, which, in turn, contributes to a further increase in opioid side effects. Finding novel approaches to effectively control chronic pain, particularly neuropathic pain, is a great challenge clinically. Literature data related to pain transmission reveal that angiotensin and its receptors (the AT1R, AT2R, and MAS receptors) could affect the nociception both in the periphery and CNS. The MOR and angiotensin receptors or drugs interacting with these receptors have been independently investigated in relation to analgesia. However, the interaction between the MOR and angiotensin receptors has not been excessively studied in chronic pain, particularly neuropathy. This review aims to shed light on existing literature information in relation to the analgesic action of AT1R and AT2R or MASR ligands in neuropathic pain conditions. Finally, based on literature data, we can hypothesize that combining MOR agonists with AT1R or AT2R antagonists might improve analgesia.

On the Role of Peripheral Sensory and Gut Mu Opioid Receptors: Peripheral Analgesia and Tolerance.

There is growing evidence on the role of peripheral µ-opioid receptors (MORs) in analgesia and analgesic tolerance. Opioid analgesics are the mainstay in the management of moderate to severe pain, and their efficacy in the alleviation of pain is well recognized. Unfortunately, chronic treatment with opioid analgesics induces central analgesic tolerance, thus limiting their clinical usefulness. Numerous molecular mechanisms, including receptor desensitization, G-protein decoupling, ß-arrestin recruitment, and alterations in the expression of peripheral MORs and microbiota have been postulated to contribute to the development of opioid analgesic tolerance. However, these studies are largely focused on central opioid analgesia and tolerance. Accumulated literature supports that peripheral MORs mediate analgesia, but controversial results on the development of peripheral opioid receptors-mediated analgesic tolerance are reported. In this review, we offer evidence on the consequence of the activation of peripheral MORs in analgesia and analgesic tolerance, as well as approaches that enhance analgesic efficacy and decrease the development of tolerance to opioids at the peripheral sites. We have also addressed the advantages and drawbacks of the activation of peripheral MORs on the sensory neurons and gut (leading to dysbiosis) on the development of central and peripheral analgesic tolerance.

Comparisons of In Vivo and In Vitro Opioid Effects of Newly Synthesized 14-Methoxycodeine-6-O-sulfate and Codeine-6-O-sulfate.

The present work represents the in vitro (potency, affinity, efficacy) and in vivo (antinociception, constipation) opioid pharmacology of the novel compound 14-methoxycodeine-6-O-sulfate (14-OMeC6SU), compared to the reference compounds codeine-6-O-sulfate (C6SU), codeine and morphine. Based on in vitro tests (mouse and rat vas deferens, receptor binding and [35S]GTPγS activation assays), 14-OMeC6SU has µ-opioid receptor-mediated activity, displaying higher affinity, potency and efficacy than the parent compounds. In rats, 14-OMeC6SU showed stronger antinociceptive effect in the tail-flick assay than codeine and was equipotent to morphine, whereas C6SU was less efficacious after subcutaneous (s.c.) administration. Following intracerebroventricular injection, 14-OMeC6SU was more potent than morphine. In the Complete Freund's Adjuvant-induced inflammatory hyperalgesia, 14-OMeC6SU and C6SU in s.c. doses up to 6.1 and 13.2 µmol/kg, respectively, showed peripheral antihyperalgesic effect, because co-administered naloxone methiodide, a peripherally acting opioid receptor antagonist antagonized the measured antihyperalgesia. In addition, s.c. C6SU showed less pronounced inhibitory effect on the gastrointestinal transit than 14-OMeC6SU, codeine and morphine. This study provides first evidence that 14-OMeC6SU is more effective than codeine or C6SU in vitro and in vivo. Furthermore, despite C6SU peripheral antihyperalgesic effects with less gastrointestinal side effects the superiority of 14-OMeC6SU was obvious throughout the present study.

Discovery of Novel µ-Opioid Receptor Inverse Agonist from a Combinatorial Library of Tetrapeptides through Structure-Based Virtual Screening.

Morphine, oxycodone, fentanyl, and other µ-opioid receptors (MOR) agonists have been used for decades in antinociceptive therapies. However, these drugs are associated with numerous side effects, such as euphoria, addiction, respiratory depression, and adverse gastrointestinal reactions, thus, circumventing these drawbacks is of extensive importance. With the aim of identifying novel peptide ligands endowed with MOR inhibitory activity, we developed a virtual screening protocol, including receptor-based pharmacophore screening, docking studies, and molecular dynamics simulations, which was used to filter an in-house built virtual library of tetrapeptide ligands. The three top-scored compounds were synthesized and subjected to biological evaluation, revealing the identity of a hit compound (peptide 1) endowed with appreciable MOR inverse agonist effect and selectivity over δ-opioid receptors. These results confirmed the reliability of our computational approach and provided a promising starting point for the development of new potent MOR modulators.

Kynurenines and the Endocannabinoid System in Schizophrenia: Common Points and Potential Interactions.

Schizophrenia, which affects around 1% of the world's population, has been described as a complex set of symptoms triggered by multiple factors. However, the exact background mechanisms remain to be explored, whereas therapeutic agents with excellent effectivity and safety profiles have yet to be developed. Kynurenines and the endocannabinoid system (ECS) play significant roles in both the development and manifestation of schizophrenia, which have been extensively studied and reviewed previously. Accordingly, kynurenines and the ECS share multiple features and mechanisms in schizophrenia, which have yet to be reviewed. Thus, the present study focuses on the main common points and potential interactions between kynurenines and the ECS in schizophrenia, which include (i) the regulation of glutamatergic/dopaminergic/γ-aminobutyric acidergic neurotransmission, (ii) their presence in astrocytes, and (iii) their role in inflammatory mechanisms. Additionally, promising pharmaceutical approaches involving the kynurenine pathway and the ECS will be reviewed herein.

The Peripheral Versus Central Antinociception of a Novel Opioid Agonist: Acute Inflammatory Pain in Rats.

Opioid analgesics devoid of central side effects are unmet medical need in the treatment of acute pain (e.g. post-operative pain). Recently, we have reported on 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), a novel opioid agonist of high efficacy producing peripheral antinociception in subchronic inflammatory pain in certain doses. The present study focused on the antinociceptive effect of 14-O-MeM6SU compared to morphine in formalin test of an early/acute (Phase I) and late/tonic (Phase II) pain phases. Subcutaneous 14-O-MeM6SU (253-1012 nmol/kg) and morphine (3884-31075 nmol/kg) dose dependently reduced the pain behaviors of both phases. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid antagonist, abolished the antinociceptive effect of 506 nmol/kg 14-O-MeM6SU. On the other hand, the effects of 14-O-MeM6SU (1012 nmol/kg) and morphine (15538 nmol/kg) were only partially affected by NAL-M, indicating the contribution of CNS to antinociception. Locally injected test compounds into formalin treated paws caused antinociception in both phases. Locally effective doses of test compounds were also injected into contralateral paws. Morphine showed effects in both phases, 14-O-MeM6SU in certain doses failed to produce antinociception in either phase. A NAL-M reversible systemic dose of 14-O-MeM6SU and the lowest systemic effective dose of morphine were evaluated for their sedative effects following isoflurane-induced sleeping (righting reflex). In contrast to morphine, 14-O-MeM6SU in certain antinociceptive doses showed no impact on sleeping time. These data highlight that high efficacy opioids of limited CNS penetration in certain doses mitigate somatic and inflammatory pain by targeting MOR at the periphery.

Exploring the first Rimonabant analog-opioid peptide hybrid compound, as bivalent ligand for CB1 and opioid receptors.

Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH2. The bivalent compound (9) has been used for in vitro binding assays, for in vivo antinociception models and in vitro hypothalamic perfusion test, to evaluate the neurotransmitters release.

Interactions between the Kynurenine and the Endocannabinoid System with Special Emphasis on Migraine.

Both the kynurenine and the endocannabinoid systems are involved in several neurological disorders, such as migraine and there are increasing number of reports demonstrating that there are interactions of two systems. Although their cooperation has not yet been implicated in migraine, there are reports suggesting this possibility. Additionally, the individual role of the endocannabinoid and kynurenine system in migraine is reviewed here first, focusing on endocannabinoids, kynurenine metabolites, in particular kynurenic acid. Finally, the function of NMDA and cannabinoid receptors in the trigeminal system-which has a crucial role in the pathomechanisms of migraine-will also be discussed. The interaction of the endocannabinoid and kynurenine system has been demonstrated to be therapeutically relevant in a number of pathological conditions, such as cannabis addiction, psychosis, schizophrenia and epilepsy. Accordingly, the cross-talk of these two systems may imply potential mechanisms related to migraine, and may offer new approaches to manage the treatment of this neurological disorder.

Evaluation of the analgesic effect of 4-anilidopiperidine scaffold containing ureas and carbamates.

Fentanyl is a powerful opiate analgesic typically used for the treatment of severe and chronic pain, but its prescription is strongly limited by the well-documented side-effects. Different approaches have been applied to develop strong analgesic drugs with reduced pharmacologic side-effects. One of the most promising is the design of multitarget drugs. In this paper we report the synthesis, characterization and biological evaluation of twelve new 4-anilidopiperidine (fentanyl analogues). In vivo hot-Plate test, shows a moderate antinociceptive activity for compounds OMDM585 and OMDM586, despite the weak binding affinity on both µ and δ-opioid receptors. A strong inverse agonist activity in the GTP-binding assay was revealed suggesting the involvement of alternative systems in the brain. Fatty acid amide hydrolase inhibition was evaluated, together with binding assays of cannabinoid receptors. We can conclude that compounds OMDM585 and 586 are capable to elicit antinociception due to their multitarget activity on different systems involved in pain modulation.

Receptome: Interactions between three pain-related receptors or the "Triumvirate" of cannabinoid, opioid and TRPV1 receptors.

A growing amount of data demonstrates the interactions between cannabinoid, opioid and the transient receptor potential (TRP) vanilloid type 1 (TRPV1) receptors. These interactions can be bidirectional, inhibitory or excitatory, acute or chronic in their nature, and arise both at the molecular level (structurally and functionally) and in physiological processes, such as pain modulation or perception. The interactions of these three pain-related receptors may also reserve important and new therapeutic applications for the treatment of chronic pain or inflammation. In this review, we summarize the main findings on the interactions between the cannabinoid, opioid and the TRPV1 receptor regarding to pain modulation.

Further Characterization of Hemopressin Peptide Fragments in the Opioid and Cannabinoid Systems.

BACKGROUND: Hemopressin, so-called because of its hypotensive effect, belongs to the derivatives of the hemoglobin α-chain. It was isolated from rat brain membrane homogenate by the use of catalytically inactive forms of endopeptidase 24.15 and neurolysin. Hemopressin has antihyperalgesic features that cannot be prevented by the opioid receptor antagonist, naloxone. METHODS: In the present study, we investigated whether hemopressin (PVNFKFLSH) and its C-terminally truncated fragment hemopressin 1-7 (PVNFKFL) have any influence on opioid-dependent signaling. Peptides have been analyzed using G-protein-stimulating functional and receptor bindings in this experimental setup. RESULTS: These 2 compounds efficiently activated the G-proteins, and naloxone slightly blocked this stimulation. At the same time, they were able to displace radiolabeled [3H]DAMGO, a selective ligand for µ-opioid system, at micromolar concentrations. Displacement caused by the heptapeptide was more modest compared with hemopressin. Experiments performed on cell lines overexpressing µ-opioid receptors verified the opioid activity of both hemopressins. Moreover, the CB1 cannabinoid receptor antagonist, AM251, significantly decreased their G-protein stimulatory effect. CONCLUSIONS: Here, we further confirm that hemopressins can modulate CB1 receptors and can have a slight modulatory effect on the opioid system.

Glycine Transporter 1 Inhibitors: Predictions on Their Possible Mechanisms in the Development of Opioid Analgesic Tolerance.

The development of opioid tolerance in patients on long-term opioid analgesic treatment is an unsolved matter in clinical practice thus far. Dose escalation is required to restore analgesic efficacy, but at the price of side effects. Intensive research is ongoing to elucidate the underlying mechanisms of opioid analgesic tolerance in the hope of maintaining opioid analgesic efficacy. N-Methyl-D-aspartate receptor (NMDAR) antagonists have shown promising effects regarding opioid analgesic tolerance; however, their use is limited by side effects (memory dysfunction). Nevertheless, the GluN2B receptor remains a future target for the discovery of drugs to restore opioid efficacy. Mechanistically, the long-term activation of µ-opioid receptors (MORs) initiates receptor phosphorylation, which triggers ß-arrestin-MAPKs and NOS-GC-PKG pathway activation, which ultimately ends with GluN2B receptor overactivation and glutamate release. The presence of glutamate and glycine as co-agonists is a prerequisite for GluN2B receptor activation. The extrasynaptic localization of the GluN2B receptor means it is influenced by the glycine level, which is regulated by astrocytic glycine transporter 1 (GlyT1). Enhanced astrocytic glycine release by reverse transporter mechanisms as a consequence of high glutamate levels or unconventional MOR activation on astrocytes could further activate the GluN2B receptor. GlyT1 inhibitors might inhibit this condition, thereby reducing opioid tolerance.

Recent Molecular Insights into Agonist-specific Binding to the Mu-Opioid Receptor.

Opioid agonists produce their analgesic effects primarily by acting at the µ-opioid receptor (µOR). µOR agonists with different efficacies exert diverse molecular changes in the µOR which dictate the faith of the receptor's signaling pathway and possibly it's the degree of desensitization. Since the development of the active conformations of the µOR, growing data have been published in relation to ligand-specific changes in µOR activation. In this regard, this review summarizes recent data regarding the most studied opioid agonists in in silico µOR activation, including how these ligands are recognized by the µOR, how their binding signal is transmitted toward the intracellular parts of the µOR, and finally, what type of large-scale movements do these changes trigger in the µOR's domains.

Discovery of Kynurenines Containing Oligopeptides as Potent Opioid Receptor Agonists.

Kynurenine (kyn) and kynurenic acid (kyna) are well-defined metabolites of tryptophan catabolism collectively known as "kynurenines", which exert regulatory functions in host-microbiome signaling, immune cell response, and neuronal excitability. Kynurenine containing peptides endowed with opioid receptor activity have been isolated from natural organisms; thus, in this work, novel opioid peptide analogs incorporating L-kynurenine (L-kyn) and kynurenic acid (kyna) in place of native amino acids have been designed and synthesized with the aim to investigate the biological effect of these modifications. The kyna-containing peptide (KA1) binds selectively the m-opioid receptor with a Ki = 1.08 ± 0.26 (selectivity ratio m/d/k = 1:514:10000), while the L-kyn-containing peptide (K6) shows a mixed binding affinity for m, d, and k-opioid receptors, with efficacy and potency (Emax = 209.7 + 3.4%; LogEC50 = -5.984 + 0.054) higher than those of the reference compound DAMGO. This novel oligopeptide exhibits a strong antinociceptive effect after i.c.v. and s.c. administrations in in vivo tests, according to good stability in human plasma (t1/2 = 47 min).

Long-term systemic administration of kynurenic acid brain region specifically elevates the abundance of functional CB1 receptors in rats.

Kynurenic acid (KYNA) is one of the most significant metabolite of the kynurenine pathway both in terms of functional and potential therapeutic value. It is an N-methyl-D-aspartate (NMDA) receptor antagonist, but it can also activate the G-protein coupled receptor 35 (GPR35), which shares several structural and functional properties with cannabinoid receptors. Previously our group demonstrated that systemic chronic KYNA treatment altered opioid receptor G-protein activity. Opioid receptors also overlap in many features with cannabinoid receptors. Thus, our aim was to examine the direct in vitro and systemic, chronic in vivo effect of KYNA on type 1 cannabinoid receptor (CB1R) binding and G-protein activity. Based on competition and [35S]GTPγS G-protein binding assays in rat brain, KYNA alone did not show significant binding towards the CB1R, nor did it alter CB1R ligand binding and agonist activity in vitro. When rats were chronically treated with KYNA (single daily, i.p., 128 mg/kg for 9 days), the KYNA plasma and cerebrospinal fluid levels significantly increased compared to vehicle treated group. Furthermore, in G-protein binding assays, in the whole brain the amount of G-proteins in basal and in maximum activity coupled to the CB1R also increased due to the treatment. At the same time, the overall stimulatory properties of the receptor remained unaltered in vehicle and KYNA treated samples. Similar observations were made in rat hippocampus, but not in the cortex and brainstem. In saturation binding assays the density of CB1Rs in rat whole brain and hippocampus were also significantly enhanced after the same treatment, without significantly affecting ligand binding affinity. Thus, KYNA indirectly and brain region specifically increases the abundance of functional CB1Rs, without modifying the overall binding and activity of the receptor. Supposedly, this can be a compensatory mechanism on the part of the endocannabinoid system induced by the long-term KYNA exposure.

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.

Preparation of bivalent agonists for targeting the mu opioid and cannabinoid receptors.

In order to obtain novel pharmacological tools and to investigate a multitargeting analgesic strategy, the CB1 and CB2 cannabinoid receptor agonist JWH-018 was conjugated with the opiate analgesic oxycodone or with an enkephalin related tetrapeptide. The opioid and cannabinoid pharmacophores were coupled via spacers of different length and chemical structure. In vitro radioligand binding experiments confirmed that the resulting bivalent compounds bound both to the opioid and to the cannabinoid receptors with moderate to high affinity. The highest affinity bivalent derivatives 11 and 19 exhibited agonist properties in [35S]GTPγS binding assays. These compounds activated MOR and CB (11 mainly CB2, whereas 19 mainly CB1) receptor-mediated signaling, as it was revealed by experiments using receptor specific antagonists. In rats both 11 and 19 exhibited antiallodynic effect similar to the parent drugs in 20 µg dose at spinal level. These results support the strategy of multitargeting G-protein coupled receptors to develop lead compounds with antinociceptive properties.