Investigation On Receptor Mechanisms Underlying Powerful Antinociception And Low Addiction Of Novel Opioid Receptor Agonist 030418

At present, opioid drugs, such as morphine, are still most effective drugs for the treatment of server pain. Opioid drugs show remarkable analgesic activity, but have serious adverse reactions in central nervous system, especially respiratory depression, tolerance and addiction, which greatly limit their clinical use. Therefore, people have been working to develop a ideal opioid drug, which has high-efficacious antinociception and low-potential addiction. During the course of studying structure-activity relationship of opioid drugs, these drug targets in vivo—opioid receptor systems also have been further understood.Most searchers focused on modifying of the chemical structure of morphine, and series of semi-synthetic and completely synthetic opioid drugs have emerged. Among them, thienorphine, a novel opioid receptor partial agonist, has been synthesized in our institute. Compared with buprenorphine, previous studies have found that thienorphine has a higher potency, better oral bioavailability, and more potent antimorphine effect; the relative clinical research has been investigated. Therefore thienorphine has a pharmacological profile indicating use as a potential treatment for opiate abuse. Nevertheless, thienorphine has been demonstrated to be a low-intrinsic activity opioid receptor agonist, which might show poor analgesic effect. To improve the antinociceptive effect of thienorphine, 030418 (N-methyl-7α-[(R)-1-hydroxy-1-methyl- 3-(thien-3-yl)-propyl]-6,14-endo-ethanotetrahydronororipavine), which was a derivative of thienorphine and possessed both bridge ring and thiophene ring, was synthesized. Preliminary animal experiments showed that 030418 exhibited powerful analgesic effects (maximal ED = 6μg/kg, s.c.), long duration of action (t1/2 = 8 h ) and limited dependence liability in the most of animal dependant models, which is different from pharmacological properties of morphine, fentanyl, buprenorphine or thienorphine. Many reports have indicated that analgesia and dependence of opioid drugs are mediated by opioid recepoters, however, due to different selectivity and agonistic activity of drugs on opioid recepoters, there are remarkable differences in pharmacological properties of opioid drugs. Now receptor mechanisms underlying pharmacological effects of 030418 have not been clarified, thus the research works are carried out as follow:Objective:The current study systematically investigated interaction characteristics of between 030418 and opioid receptors. On the basis of works above, the study also identified possible pharmacological mechanisms underlying powerful antinociception and low dependence of opioid receptor full agonist 030418 and heterodimerization of opioid receptors. Eventually, these studies help to further analyse structure-activity relationship of long-acting opioid drugs and the correlation of opioid receptors, and provide theoretical and experimental evidences into design of high-efficient and low-addictive"the ideal analgesic".Methods:Firstly, binding affinity and efficacy of 030418 were determined using receptor binding and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) assays in CHO-μ, CHO-κ, CHO-δ, and CHO-ORL1 stable transfected cell membranes. Secondly, analgesic effects and tolerance of 030418 were evaluated in the mouse tail-flick test and the mouse 55 ?C hot-plate test, and physical dependence of 030418 was examined in the naloxone-precipitated withdrawal sign in mouse. Using in vivo pharmacological blockade of opioid receptor antagonists, effects of different subtypes of opioid receptor on analgesia, tolerance and physical dependence of 030418 were further investigated. Thirdly, in the isolated guinea-pig ileum experiment, the dissociation kinetic of 030418 from the opioid receptor was investigated by prolonged washing. In CHO-μcells, desensitization and overshoot of adenosine 3',5'-cyclic phosphate (cAMP) level induced by long-term treatment of 030418 were measured by time-resolved fluorescence resonance energy transfer immunoassay method. Finally, immuno- fluorescence and co-immunoprecipitation experiments using HA, Myc or Flag-tagged receptors were performed to examine colocaliztion and heterodimerization ofκ-opioid receptor and ORL1 receptor in primary culturing rat neurons, Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. With recombinant plasmid and transfection mediated with liposome,κ-opioid receptor and ORL1 receptor are stably co-expressed in CHO cells.Results:1. 030418 displayed high binding affinity toμ,κ,δ-opioid receptors and ORL1 receptor, and the Ki values were 0.91 nM, 0.60 nM, 0.58 nM and 1.6 nM, respectively. In [35S]GTPγS binding assay, the subtype selective high-efficacy agonists DAMGO, (±)U50,488, SNC80, and N/OFQ were selected as the control forμ,κ,δ-opioid receptor and ORL1 receptor, respectively. 030418 produced maximal stimulation of [35S]GTPγS binding onμ- (89%),κ- (86%),δ- (67%) opioid receptors and also the ORL1 receptor (91%), and the EC50 values were 0.10 nM, 0.15 nM, 0.38 nM and 15.0 nM, respectively. The results showed that 030418 had nonselective affinities of opioid receptors, and had potent activities on opioid receptors, in order,μ≈κ≈ORL1 >δ.2. In the mouse tail-flick and hot-plate tests, the antinociceptive ED50 of 030418 was respectively calculated to be 2.39μg/kg (s.c.) and 2.90μg/kg (s.c.), similar with dihydroetorphine. The maximally effective dose of 030418 had the longest duration of antinociceptive effect in comparison with that of dihydroetorphine and morphine. Compared with morphine, chronic treatment of 030418 could result in dramatic development of tolerance, but mice repeatedly injected of increasing doses of 030418 could not produce the obvious naloxone-precipitated jumping symptoms. Animal experiments further demonstrated that 030418 had powerful antinocicetive effects, long-lasting action, and limited degree of physical dependence.3. In the mouse hot-plate model, nonselective opioid receptor antagonist naloxone (1.0 mg/kg, s.c.) could completely block 030418-induced antinociception, and theμ-opioid receptor antagonist, naloxonazine (25μg/mouse, i.c.v.),β-FNA (10μg/mouse, i.c.v.), orκ-opioid receptor antagonist, nor-BNI (3.7μg/mouse, i.c.v.), attenuated 030418-induced antinociception. By contrast, the ORL1 receptor antagonist, J-113397 (4.0μg/mouse, i.c.v.), could enhance the antinociceptive effect of 030418. In the analgesic tolerance model, pretreatment with antagonist J-113397 could partly attenuate the development of morphine and dihydroetorphine tolerance, but could not effectively prevent 030418 tolerance. In the mouse physical dependence model, selective antagonist nor-BNI or J-113397 could not reverse limited physical dependence of 030418. The results indicated that activation ofμ-, andκ-opioid receptors appears to be involved in the antinociceptive effect of 030418, and activation of ORL1 receptor could inhibit antinociception induced by 030418. Fast development of tolerance and limited physical dependence of 030418 might involve inκ-opioid receptor and ORL1 receptor.4. In the isolated guinea pig ileum preparation, stimulation induced by 1μM acetylcholine chloride produces ileum muscle contraction, which can be inhibited by action of 030418 (0.1 mM). The existing action of 030418 could, with more difficulty, be removed by prolonged washing than equivalent effects of morphine (5 mM) and dihydroetorphine (0.2 mM). The finding revealed that 030418 had high binding affinity to opioid receptors, and had slow receptor dissociation kinetics.5 The desensitization and overshoot of cAMP signaling ofμopioid receptor were examined following chronic treatment of 030418. Compared with the control group (83% of inhibition), 24 h chronic treatment of 10μM DAMGO or 10μM 030418 could produce significant decrease of DAMGO-induced inhibition of increased cAMP level of caused by 10μM forskolin in CHO-μcells, and inhibition% values were 31% and 8%, respectively. Additionally, after CHO-μcells were treated by 10μM morphine or 10μM DAMGO for 24 h and precipitated by naloxone (10μM), intracellular cAMP level was respectively increased to 339% and 291% compared with that of vehicle control. However, chronic treatment of 030418 failed to cause significant cAMP overshoot in CHO-μcells. The resultes indicated that the effects of 030418 were different from that of DAMGO and morphine in cAMP signaling ofμopioid receptor.6. Fluorescence of bothκ-opioid receptor and ORL1 receptor were seen overlapping in primary culturing hippocampal and cortical neurons. Similarly in co-expressing CHO or HEK293 cells, HA-KOR and Myc-ORL1 were almost exclusively confined to the membranes, revealing extensive colocalization. When Flag-KOR and Myc-ORL1 were co-expressing in CHO cells, heterodimerization was identified by the ability to co-immunoprecipitate ORL1-receptors withκ-opioid receptor and vice versa. These findings suggested that two subtypes of opioid receptors, KOR and ORL1-receptors, might exist the direct interaction to form the heterodimerization.7. Recombinant pcDNA3.1/hygro(+)-hKOR was transfected into CHO-hORL1 cells. In the selective cultured medium containing G418 and HygroB, 35 monoclones of co-transfected cells were obtained. Through screening by RT-PCR and ligand binding assay, the CHO cell model, in whichκopioid receptor and ORL1 receptor were highly co-expressed, was established. Between CHO-KOR/ORL1 cells and CHO-KOR cells, the Ki value of selective ligands (±)U50,488, N/OFQ and 030418 for the inhibition of [3H]diprenorphine binding to the KOR did not change. The results provided experimental base for futher study interaction of KOR and ORL1. Conclusion:030418 is a novel opioid receptor agonist. The results in vivo and in vitro show that 030418 has nonselective and high binding affinity to all subtypes of opioid receptors, and 030418 can fully activate theμ,κ-opioid receptor, as well as ORL1 receptors, and 030418 has slow receptor dissociation kinetics. Pharmacological properties of 030418 are closely correlated with high activity of methyl group at N17 position and high hydrophobicity of C7-thiophene group in 030418 chemical structure. Further studies reveal that the powerful antinociceptive effects of 030418 result from activation ofμ-, andκ-opioid receptors; slow receptor dissociation kinetics may account for the long duration of agonist effect of 030418; the rapid tolerance of 030418 may be involved inμ-opioid receptor desensitization induced by chronic treatment of 030418 on cAMP signaling pathway; mechanisms of low physical dependence of chronic treatment of 030418 are partially associated with no significant naloxone-precipitated overshoot of cAMP level in CHO-μcell. In addition, we provide further evidence for the direct interaction of two similar"antiμ-opioid receptor"subtypes of opioid receptors, KOR and ORL1-receptors, to co-localize in the cellular level and form the heterodimerization. But the relationship between interaction of KOR and ORL1 receptor and low dependence liability of 030418 needs further study.