Ferenc Zádor

Design, Synthesis and Biological Evaluation of Two Opioid Agonist and Cav2.2 Blocker Multitarget Ligands

N‐type voltage‐dependent Ca2+ channels (CaV2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischaemia and neuropathic pain. CaV2.2 blockers such as the ω‐conotoxin MVIIA (Prialt) are analgesic and have opioid‐sparing effects. With the aim to develop new multitarget analgesic compounds, we designed the first ω‐conotoxin/opioid peptidomimetics based on the enkephalin‐like sequence Tyr‐D‐Ala‐Gly‐Phe (for the opioid portion) and two fragments derived from the loop‐2 pharmacophore of ω‐conotoxin MVIIA. Antinociceptive activity evaluated in vitro and in vivo revealed differential affinity for CaV2.2 and opioid receptors and no significant synergistic activity.

Micromolar concentrations of rimonabant directly inhibits delta opioid receptor specific ligand binding and agonist-induced G-protein activity

WHAT IS KNOWN There is a growing number of evidence showing, that the cannabinoid receptor 1 (CB1) antagonist rimonabant has many non-cannabimimetic actions, such as affecting the opioid system. The direct effect of rimonabant on opioid receptors has been studied so far mainly on μ-opioid receptors. However recently the δ-opioid receptor (DOR) receives much more attention as before, due to its potential therapeutic applications, such as nociception or treatment for psychiatric disorders. OBJECTIVES To investigate the direct effect of rimonabant on DOR specific ligand binding and on the DOR mediated G-protein activation. RESULTS Micromolar concentrations of rimonabant directly inhibited the DOR specific agonist binding in radioligand competition binding experiments using Chinese hamster ovary cells stably transfected with mouse DOR (CHO-mDOR). However the inhibition occurred also in the subnanomolar range during DOR specific antagonist binding in similar experimental conditions. In functional [(35)S]GTPγS binding assays rimonabant significantly decreased the basal receptor activity in CHO-mDOR but also in parental CHO cell membranes. During DOR agonist stimulation, micromolar concentration of rimonabant attenuated the DOR G-protein activation and the potency of the activator ligand in [(35)S]GTPγS binding assays performed in CHO-mDOR, in wild type and also in CB1/CB2 double knock-out mouse forebrain membranes. Yet again this inhibitory action was DOR specific, since it did not occur during other specific GPCR agonist mediated G-protein activation. CONCLUSION Rimonabant directly inhibited DOR function in the micromolar concentrations. The inhibitory actions indicate an antagonistic behavior towards DOR which was established by the followings: (i) rimonabant inhibited DOR antagonist binding more effectively than agonist binding, (ii) the inverse agonistic, agonistic effect of the compound can be excluded, and (iii) additionally according to previous findings the allosteric mechanism can also be foreclosed.

Inhibition of forebrain μ-opioid receptor signaling by low concentrations of rimonabant does not require cannabinoid receptors and directly involves μ-opioid receptors.

Increasing number of publications shows that cannabinoid receptor 1 (CB(1)) specific compounds might act in a CB(1) independent manner, including rimonabant, a potent CB(1) receptor antagonist. Opioids, cannabinoids and their receptors are well known for their overlapping pharmacological properties. We have previously reported a prominent decrease in μ-opioid receptor (MOR) activity when animals were acutely treated with the putative endocannabinoid noladin ether (NE). In this study, we clarified whether the decreased MOR activation caused by NE could be reversed by rimonabant in CB(1) receptor deficient mice. In functional [(35)S]GTPγS binding assays, we have elucidated that 0.1mg/kg of intraperitoneal (i.p.) rimonabant treatment prior to that of NE treatment caused further attenuation on the maximal stimulation of Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO), which is a highly specific MOR agonist. Similar inhibitory effects were observed when rimonabant was injected i.p. alone and when it was directly applied to forebrain membranes. These findings are cannabinoid receptor independent as rimonabant caused inhibition in both CB(1) single knockout and CB(1)/CB(2) double knockout mice. In radioligand competition binding assays we highlighted that rimonabant fails to displace effectively [(3)H]DAMGO from MOR in low concentrations and is highly unspecific on the receptor at high concentrations in CB(1) knockout forebrain and in their wild-type controls. Surprisingly, docking computational studies showed a favorable binding position of rimonabant to the inactive conformational state of MOR, indicating that rimonabant might behave as an antagonist at MOR. These findings were confirmed by radioligand competition binding assays in Chinese hamster ovary cells stably transfected with MOR, where a higher affinity binding site was measured in the displacement of the tritiated opioid receptor antagonist naloxone. However, based on our in vivo data we suggest that other, yet unidentified mechanisms are additionally involved in the observed effects.

Low dosage of rimonabant leads to anxiolytic-like behavior via inhibiting expression levels and G-protein activity of kappa opioid receptors in a cannabinoid receptor independent manner

WHAT IS KNOWN There is an increasing number of studies demonstrating the direct effect of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant on the opioid system. The kappa opioid receptors (KORs) are well known to mediate depression- and anxiety-like behavior. Clinical studies on chronic rimonabant administration have revealed that rimonabant leads to a very similar pathophysiology, suggesting a potential impact of rimonabant on KORs. OBJECTIVES Our objectives were to examine the putative effects of rimonabant on KOR ligand binding, G-protein activity, protein expression and how all these contribute to the development of depression- and anxiety-like behavior. RESULTS In Chinese hamster ovary (CHO) cell membranes transfected with rat KOR (CHO-rKOR) rimonabant inhibited KOR agonist [3H]U69593 binding in the micromolar range in competition binding experiments and specifically reduced KOR basal activity at lower micromolar concentrations in [35S]GTPγS binding assays. Rimonabant significantly inhibited dynorphin (1-11)-induced [35S]GTPγS binding in micromolar range in CHO-rKOR cells, CB1 knockout (CB1 K.O.) and CB1/CB2 double knockout mouse forebrain membranes. A single dose of i.p. 0.1 mg/kg rimonabant significantly reduced dynorphin (1-11)-induced KOR G-protein activity and KOR protein expression levels 24 h following the administration in both wild type and CB1 K.O. mice forebrain. Furthermore, in elevated plus maze mice showed an anxiolytic-like effect upon rimonabant injection that could be reversed by 1 mg/kg KOR antagonist norbinaltorphimine. The anxiolytic-like effects were further confirmed with the light–dark box test. CONCLUSION Rimonabant reduced KOR ligand binding, receptor mediated G-protein activity and protein expression level, which overall leads to altered anxiety-like behavior.

A Novel µ-Opioid Receptor Ligand with High In Vitro and In Vivo Agonist Efficacy

The aims of this study were to synthesize 14-O-Methylmorphine-6-O-sulfate (14-O-MeM6SU) and examine its opioid properties (potency, affinity, efficacy) in receptor ligand binding and isolated tissues (mouse vas deferens, MVD and rat vas deferens, RVD bioassays). The results were then compared to the parent compounds morphine-6-O-sulfate (M6SU) and morphine, as well as the �- opioid receptor (MOR) selective agonist peptide [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). An additional objective was to compare the effect of subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) administered 14-O-MeM6SU, M6SU and morphine in thermal nociception, rat tail-flick (RTF) test. In MVD, the EC50 (nM) value was 4.38 for 14-O-MeM6SU, 102.81 for M6SU, 346.63 for morphine and 238.47 for DAMGO. The effect of 14-O-MeM6SU and DAMGO was antagonized by naloxone (NAL) with Ke value 1-2.00 nM. The Emax values (%) were 99.10, 36.87, 42.51 and 96.99 for 14-O-MeM6SU, M6SU, morphine and DAMGO, respectively. In RVD 14-O-MeM6SU and DAMGO but not M6SU or morphine showed agonist activity. In binding experiments the affinity of 14-OMeM6SU, M6SU, morphine and DAMGO for MOR was 1.12, 11.48, 4.37 and 3.24 nM, respectively. The selectivity of 14-O-MeM6SU was κ/μ= 269 and δ/μ= 9. In G-protein activation experiments, 14-O-MeM6SU and DAMGO showed higher Emax values than M6SU or morphine. S.c. or i.c.v-injected 14-O-MeM6SU, M6SU and morphine produced a dose and time-dependent increase in RTF response latency. 14-O-MeM6SU was the most potent. Our results showed that introduction of 14-O-Me in M6SU increased the binding affinity, agonist potency, and most importantly, the intrinsic efficacy (Emax).

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 &agr;-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 &mgr;-opioid system, at micromolar concentrations. Displacement caused by the heptapeptide was more modest compared with hemopressin. Experiments performed on cell lines overexpressing &mgr;-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.

Inhibition of Opioid Receptor Mediated G-Protein Activity After Chronic Administration of Kynurenic Acid and its Derivative without Direct Binding to Opioid Receptors.

There is an increasing number of evidence showing analgesic properties of the kynurenic acid (KYNA), and also some studies demonstrate that kynurenine might interact with the opioid system. Therefore in this study, for the first time we investigated the direct binding affinity of KYNA and its structural analog KYNA-1 towards mu, kappa and delta opioid receptor in competition binding experiments applying opioid receptor specific radioligands. The binding affinity measurements were performed in Chinese hamster ovary cell lines overexpressing the corresponding opioid receptor (mu and kappa opioid receptor were rat, delta opioid receptor were mouse sequence). Additionally we also examined the chronic effect of these compounds on mu, kappa and delta opioid receptor and also nociceptin peptide receptor mediated G-protein activity in [(35)S]GTPγS binding assays performed in mouse cortex and striatum membranes. Our results showed that KYNA and KYNA-1 had no affinity towards any of the three classic opioid receptors. On the other hand the compounds significantly decreased opioid and nociceptin receptor mediated G-protein activity or in some cases enhanced the potency of the activating ligand. Moreover, the alterations were receptor and brain region specific. Accordingly, we conclude that KYNA and KYNA-1 do not interact directly with the opioid receptors, but more likely alter the receptor functions intracellularly.

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

Abstract 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.

Opioid Receptor Activity and Analgesic Potency of DPDPE Peptide Analogues Containing a Xylene Bridge

d-Pen2,d-Pen5 enkephalin (DPDPE) is one of the most selective synthetic peptide agonists targeting the δ-opioid receptor. Three cyclic analogues of DPDPE containing a xylene bridge in place of disulfide bond have been synthesized and fully characterized as opioid receptors agonists. The in vitro activity was investigated showing a good affinity of 7a-c for μ- and δ-receptors. In vivo biological assays revealed that 7b is the most potent analogue with the ability to maintain high level of analgesia from 15 to 60 min following intracerebroventricular (i.c.v.) administration, whereas DPDPE was slightly active until 45 min. Compound 7b induced long lasting analgesia also after subcutaneous administration, whereas DPDPE was inactive.