Vittoria Borgonetti^a, Paolo Governa^b, Clarissa Caroli^c, Virginia Brighenti^c, Lorenzo Corsi^c, Fabrizio Manetti^b, Nicoletta Galeotti^a, Federica Pellati^c

^a Department of Neuroscience, Psychology, Drug Research and Child Health, University of Florence, Viale G. Pieraccini 6, 50139 Florence, Italy
^b Department of Biotechnology, Chemistry and Pharmacy Department, University of Siena, Via A. Moro 2, 53100, Siena, Italy
^c Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103/287, 41125, Modena, Italy

Neuropathic pain, caused by a lesion or disease of the somatosensory nervous system, is a common chronic pain condition with major impact on quality of life, affecting 7–10% of the world population. Multiple causes of this pathology have been described and its incidence is likely to increase owing to the ageing global population, increased incidence of diabetes mellitus and improved survival from cancer after chemotherapy. Current therapies include antidepressants, antiepileptics, local anesthetics and opioids. These treatments are effective in less than 50% of patients, and they are characterized by many side effects, that limit their long-term use.

Cannabis sativa L. is used in the treatment of chronic pain, thanks to its interaction with the endocannabinoid system. The most investigated cannabinoids are Δ⁹ -tetrahydrocannabinol (Δ⁹ -THC) and cannabidiol (CBD), but the psychotropic properties of Δ⁹-THC, involving its interaction with CB1 receptors, affect patient quality of life, thus making cannabis unsuitable for a long-term medical use.

For these reasons, in this study the possible anti-hyperalgesic effect of non-psychotropic C. sativa oil extracts in animal models of acute and persistent pain was assessed. To identify the possible synergy and interaction between cannabinoids and terpenes, an extract devoid of terpenes and with high a concentration of CBD (K1) and one containing a high level of both cannabinoids and terpenes (K2) were compared.

The detailed chemical characterization the oil extracts was carried out by ultra-high-performance liquid chromatography (UHPLC), coupled with high-resolution mass spectrometry (HRMS), using a targeted metabolomic approach. Among all the identified cannabinoids, the most representative ones were further quantified by HPLC-UV.

The anti-hyperalgesic effect of K2 and K1 after oral administration was assessed by measuring mechanical and thermal allodynia in spared nerve injury (SNI) mice. The mechanism of action of K2 was investigated using spinal cords samples in a cell-based in vitro model of neuroinflammation. Oral administration of K2 at 25 mg kg ⁻¹ ameliorated mechanical and thermal allodynia in SNI mice, with a rapid and a long-lasting effect, without inducing an alteration of locomotor activity, with an efficacy higher than K1. K2 reduced p-ERK, p-p38 and p-JNK1 protein levels compared to the untreated mice. Moreover, the extract downregulated protein expression of neuroinflammatory factors (HDAC-1, p-65, NOS2).

In addition, hemp essential oil (EO) was also fully characterized by GC-MS and GC-FID and its bioactivity was assessed in vivo in the SNI showing significant anti-hyperalgesic activity.

Finally, to deeper investigate the possible synergism of CBD and β-caryophyllene (BCP), which are the two main compounds among cannabinoids and terpenes, respectively, an in silico structure-based approach was performed on CB2 receptors. Together, CBD and BCP were able to resemble the experimental binding mode of the reference ligand and the interactions with the binding site residues better than CBD or BCP alone, which could explain the experimental data obtained. In vitro binding and functional assays are currently ongoing to validate the in silico results.

Acknowledgements

The project was financed by the European Union – Next Generation EU “Targeting microglia CB2 Receptors with novel multisite ligands: a multidisciplinary and translational study for the identification of an innovative multiple sclerosis therapy” (2022BNSNS2, CUP E53D23012360006).

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