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Synergistic attenuation of cancer-related pain and implications on adverse effects by the use of methadone and Phα1β in C57BL/6J mice
* 1 , 2 , 2
1  Programa de pós graduação em ciências da saúde, Santa Casa de Belo Horizonte Ensino e Pesquisa, Belo Horizonte, Minas Gerais, Brazil
2  Programa de pós graduação em ciências da saúde, Santa Casa de Belo Horizonte Ensino e Pesquisa, Belo Horizonte, Minas Gerais, Brazil

Published: 14 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Introduction and Goals: Cancer pain produces severe distress and disrupt the life quality of patients and very often is not effectively treated. Opioids are practically the only analgesics capable of controlling cancer pain but this therapy leads to distinct side effects that limit its use. Methadone is a valuable opioid analgesic, which can be administered in case of cancer pain and can reverse other opioids tolerance like morphine. However, methadone has some side effects as other opioids. Phα1β toxin from the spider Phoneutria nigriventer has an antinociceptive action in several models of pain in rodents and it is known that it induces analgesic effect in a model of cancer pain in mice. This toxin is a dual blocker of TRPA1 channels and voltage-gated calcium channels and exhibits greater selectivity for N-type channels. One strategy to improve the therapeutic utility of opioids is to co-administer with other analgesic agents, such as Phα1β toxin, looking for overall dose reduction and also reducing side effects to improving the quality of analgesia. This work aims to analyze by an Isobolographic analysis whether antinociceptive interaction of Methadone and Phα1β is subadditive, additive, or synergic. Methodology: B16F10 cells inoculation was performed on the right paw on C57BL/6J for tumor induction. The PWT (Von-Frey filaments) was measured before (baseline), at day 7 and at day 14 before and after drug treatment. N= 6-9 per group. Dose-response curves of drugs alone or in combination were performed using a fixed proportion design. Data interpretation was performed using isobolographic analysis to determine the interaction index of the combination. To evaluate the possible side effects from this combination, the Open Field test, Rotarod test and quantification of the gastrointestinal transit were performed. To check whether the combination is capable of reversing morphine tolerance induced by several morphine doses, the protocol with tail-flick apparatus was used. All the procedures were authorized by CEPEEA, the Ethics Committee in animals’ experimentation from Santa Casa of Belo Horizonte Education and Research (Protocol 002/2018). Results: Fourteen days after B16-F10 right hind paw inoculation, marked hyperalgesia was induced measured by Von Frey filaments. This hyperalgesia was reversed by the i.t. treatment with Phα1β at 100 pmol/site and also by the methadone s.c injection at 1mg/kg, other doses were tested and the final dose used to combine these two drugs were made as described by Tallarida in a protocol of fixed proportions by two components. The antinociceptive effect of Phα1β and Methadone was dose-dependent, with ED50 values of 1.076 pmol/site for Phα1β and 86.849 pmol/site for Methadone. The combination of Phα1β + Methadone has an ED50 which is lower than the theoretical additive ED50 (p<0.05), indicating synergism. The required dose to reach synergism is 20 pmol/site for Phα1β (IC95%: 7-58pmol/site) and 15.410 pmol/site for Methadone (IC95%: 5.480-41.680pmol/site). Since we have observed strong potentiation in the analgesic effect, we assess the animal behavior in an open field, as well as motor impairment checking fall latency and intestinal motility after the administration of drugs alone or in combination at ED50 doses. No changes in animal behavior were observed in an open field (traveled distance, movement number and movement time duration) after drug administration, p>0,05; the same occurs in latency to fall, no difference was seen between the groups p>0,05. Combined drugs reduced 27% of gastrointestinal transit compared with the control group (p=0,02), whereas the two drugs alone did not significantly differ with the control group (p<0,05). Methadone is currently used to reverse morphine tolerance, methadone given alone at its ED50 dose was able to reverse morphine-induced tolerance in animals with significant difference (p<0,05) to control (PBS). Phα1β also reversed morphine tolerance as described earlier. At lower doses, Phα1β + Methadone ED50 value reversed morphine tolerance without significant difference (p>0,05) to isolated compounds but statistically different to the control group (p<0,05). Conclusions: Our data show that synergism occurred when s.c methadone was administered simultaneously with i.t Phα1β suggesting potentiation on the analgesic effect of these drugs when both are added together. Even with strong potentiation in the analgesic effect, no relevant side effects associated with this combination were observed. In addition to producing an antinociceptive effect, the combination of these compounds was able to reverse morphine-induced tolerance.

Keywords: Cancer pain, melanoma, Phα1β toxin, Methadone, synergism, isobolographic analysis