Background: Few models exist that can control for placebo and expectancy effects commonly observed in clinical trials measuring ‘Cannabis’ pharmacodynamics. We used the Foramen Rotundum Inflammatory Constriction Trigeminal Infraorbital Nerve injury (FRICT-ION) model to measure the effect of “full-spectrum” whole plant extracted hemp oil on chronic neuropathic pain sensitivity in mice. Methods: Male BALBc mice were submitted to the FRICT-ION chronic neuropathic pain model with oral insertion through an incision in the buccal/cheek crease of 3 mm of chromic gut suture (4-0). The suture, wedged along the V2 trigeminal nerve branch, creates a continuous irritation that develops into secondary mechanical hypersensitivity on the snout. Von Frey filament stimuli on the mouse whisker pad was used to assess the mechanical pain threshold from 0–6 h following dosing among animals (n = 6) exposed to 5 μL of whole plant extracted hemp oil combined with a peanut butter vehicle (0.138 mg/kg), the vehicle alone (n = 3) 7 weeks post-surgery, or a naïve control condition (n = 3). Results: Mechanical allodynia was alleviated within 1 h (d = 2.50, p < 0.001) with a peak reversal effect at 4 h (d = 7.21, p < 0.001) and remained significant throughout the 6 h observation window. There was no threshold change on contralateral whisker pad after hemp oil administration, demonstrating the localization of anesthetic response to affected areas. Conclusion: Future research should focus on how whole plant extracted hemp oil affects multi-sensory and cognitive-attentional systems that process pain.

Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of K_D = 1.794 × 10^–8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.

The intrathecal drug delivery system (IDDS) is successfully utilized for the treatment of chronic pain conditions; however, they are associated with complications related to human error and system failure. A case report is presented of a patient with opioid withdrawal (OW) secondary to assumed catheter microfraclure. Interrogation of the IDDS allowed for the collection of pre- and post-treatment/stabilization cerebrospinal fluid (CSF), which is used to investigate the possible physiological determinants of OW. A 46-year-old female with a history of low back pain after traumatic low back injury status post-IDDS placement for failed back surgery syndrome presented with signs and symptoms concerning for OW. After every other possible explanation was ruled out, it was hypothesized that there may be IDDS catheter microfracture(s), and catheter replacement led to symptom resolution. There were no significant differences in cytokine levels tested in pre-CSF versus post-CSF samples. Whole-cell patch-clamp electrophysiology analysis of human-induced pluripolert stem cell-derived nociceplors after treatment with pre- and post-CSF samples demonstrated modulation of action potential waveform. In patients presenting with acute OW attribution IDDS malfunction, catheter microfracture must be in the differential, and non-conventional interrogation of the IDDS catheter should be considered. The possible differences in pre-CSF and post-CSF may be more complicated than previously postulated, as there were no significant differences in cytokine profiles; however, treatment of in vitro neurons with pre- and post-CSF resulted in differential neuronal excitability, which may account for some of the symptoms of OW.