Synergistic combinations of the dual enkephalinase inhibitor PL265 given orally with various analgesic compounds acting on different targets, in a murine model of cancer-induced bone pain

Background: The first line pharmacological treatment of cancer pain is morphine and surrogates but a significant pain relief and a reduction of the side-effects of these compounds makes it necessary to combine them with other drugs acting on different targets. The aim of this study was to measure the antinociceptive effect on cancer-induced bone pain resulting from the association of the endogenous opioids enkephalin and non-opioid analgesic drugs. For this purpose, PL265 a new orally active single dual inhibitor of the two degrading enkephalins enzymes, neprilysin (NEP) and aminopeptidase N (APN) was used. It strictly increased the levels of enkephalin at their sites of releases. The selected non-opioid compounds are: gabapentin, A-317491 ( P2X(3) receptor antagonist), ACEA (CBI receptor antagonist), AM1241 (CB2 receptor antagonist), JWH-133 (CB2 receptor antagonist), URB937 (FAAH inhibitor), and NAV26 (Nav1.7 channel blocker). Methods: Experiments. Experiments were performed in 5-6 weeks old (26-33 g weight) C57BL/6 mice. Cell culture and cell inoculation. B16-F10 melanoma cells were cultured and when preconfluent, treated and detached. Finally related cells were resuspended to obtain a concentration of 2 x 10(6) cells/100 mu L. Then 10(5) cells were injected into the right tibial medullar cavity. Control mice were treated by killed cells by freezing. Behavioural studies. Thermal withdrawal latencies were measured on a unilatered hot plate (UHP) maintained at 49 +/- 0.2 degrees C. Mechanical threshold values were obtained by performing the von Frey test using the up and down method. To evaluate the nature (additive or synergistic) of the interactions between PL265 and different drugs, an isobolographic analysis following the method described by Tallarida was performed. Results: The results demonstrate the ability of PL265, a DENKI that prevents the degradation of endogenous ENK5, to counteract cancer-induced bone thermal hyperalgesia in mice, by exclusively stimulating peripheral opioid receptors as demonstrated by used of an opioid antagonist unable to enter the brain. The development of such DENKIs, endowed with druggable pharmacokinetic characteristics, such as good absorption by oral route, can be considered as an important step in the development of much needed novel antihyperalgesic drugs. Furthermore, all the tested combinations resulted in synergistic antihyperalgesic effects. As shown here, the greatest synergistic antinociceptive effect (doses could be lowered by 70%) was produced by the combination of PL265 with the P2X(3) receptor antagonist (A-317491), cannabinoid CB1 receptor agonist (exogenous, ACEA and endogenous URB937-protected-AEA) and Na(v)1.7 blocker (NAV26) whose mechanism of action involves the direct activation of the enkephalinergic system. Conclusions: These multi-target-based antinociceptive strategies using combinations of non-opioid drugs with dual inhibitors of enkephalin degrading enzymes may bring therapeutic advantages in terms of efficacy and safety by allowing the reduction of doses of one of the compounds or of both, which is of the utmost interest in the chronic treatment of cancer pain. Implications: This article presents synergistic antinociceptive effect produced by the combination of PL265 with non-opioid analgesic drugs acting via unrelated mechanisms. These multi-target-based antinociceptive strategies may bring therapeutic advantages by allowing the reduction of doses, which is of great interest in the chronic treatment of cancer pain. (C) 2016 Scandinavian Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.