Attenuation of Opioid Tolerance in Mice by Systemic Inhibition of the Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling Pathway.

Opioids remain the mainstay of clinical analgesia but after prolonged administration they induce therapeutic desensitization (tolerance) that significantly restricts their clinical usefulness. Therefore, there is a pressing need for the identification of new therapeutic strategies to improve efficacy of opioid-based treatments. While the mammalian target of rapamycin complex 1 (mTORC1), a kinase which controls protein synthesis, is well established as a regulator of chronic pain sensitivity, its role in the regulation of opioid efficacy has also been recently promoted. It was found that the activation of μ-opioid receptor by morphine in naïve rodents triggers the mTORC1 activity within the nociceptive pathway and promotes morphine-induced protein translation changes associated with morphine tolerance and hyperalgesia. However, these observations were recorded following direct mTORC1 inhibition with rapamycin, a therapy restricted due to adverse effects. For this reason, we extended these observations and focused on upstream regulation of mTORC1 activity via targeting 5' adenosine monophosphate-activated protein kinase (AMPK) with metformin. This drug is widely given to treat type-2 diabetes and was shown to negatively regulate translation via inhibition of mTORC1.

In adult male C57BL/6J mice (n=6) opioid tolerance was induced by morphine (20 mg/kg, i.p.) given twice daily at 12 h intervals for 9 consecutive days. The influence of mTORC1 inhibition was assessed by repeated injections of the rapamycin analogue CCI-779 (25 mg/kg, i.p.) or the AMPK activator metformin (200 mg/kg, i.p.) once daily, 24 h before morning morphine injection on each testing day. Pain threshold was assessed by tail-flick daily 30-60 min after morphine. To determine the effect of a single injection of CCI-779 or metformin on restoring analgesic effect of morphine, separate groups of morphine tolerant mice received a single injection of CCI-779 (25 mg/kg, i.p.) or metformin (200 mg/kg, i.p.), 24 h before subsequent morphine dose. Western blotting was used to determine changes in mTORC1 activity after treatment with CCI-779 and metformin.

Our study showed for the first time that chronic metformin blocked the development and maintenance of morphine tolerance, while a single metformin injection fully restored the analgesic effect of morphine in naïve mice (pain threshold after chronic morphine on day 9: 4.1±0.2s vs. in the presence of chronic metformin: 7.7±0.5s or single dose of metformin: 6.9±0.9s). In parallel studies using the direct mTORC1 inhibitor, CCI-779 showed that these effects were attributed to the inhibition of mTORC1 (pain threshold after chronic morphine on day 9: 4.9±0.2s vs. in the presence of chronic CCI-779: 8.1±0.4s or single dose of CCI-779: 7.8±0.3s). This mechanism was confirmed by immunoblotting showing inhibition of mTORC1 activity in the dorsal spinal cord after metformin and CCI-779 observed as a decrease in the phosphorylation levels of two mTORC1 downstream targets, P-p70 S6 kinase (68.4±16.4% decrease after metformin) and P-S6 ribosomal protein (61.6±10.1% decrease after CCI-779).

Since chronic pain and tolerance to antinociceptive effects of morphine share some common pathological mechanisms, our study may suggest that mTORC1 represents a novel and tractable target for the improvement of opioid analgesic efficacy in chronic pain. An important aspect of our observations is related to the use of metformin, a widely clinically available and relatively safe anti-diabetic drug, in contrast to other mTORC1 inhibitors that display numerous side effects (e.g. rapamycin), thus leading to an immediate novel avenue for the improvement of opioid therapy in humans.

ACKNOWLEDGEMENTS: This work was funded by the BPS Pump-Priming Award.