Targeting p38 Mitogen Activated Protein Kinase to Ameliorate Pain in SCD

Pain is a major comorbidity of sickle cell disease (SCD). Central sensitization is associated with chronic sickle cell pain. Earlier studies showed that p38 mitogen activated protein kinase (MAPK) is activated in the spinal cords of sickle mice with nociceptor sensitization of 2˚ neurons (Cataldo et al., Pain 2015). Novel approaches targeting p38 MAPK phosphorylation or its upstream targets may alleviate chronic pain. Cannabinoids represent a novel approach for ameliorating pain, including the endogenous cannabinoid-like lipid mediator palmitoylethanolamide (PEA). PEA has been shown to reduce neuropathic pain and depression scores, while improving sleep concomitant with reduced c-reactive protein (CRP) in a clinical evaluation of diabetic peripheral neuropathic pain and inflammation (Pickering et al., Inflammopharmacology 2022). We therefore examined if, [i] p38 MAPK is activated in dorsal root ganglion (DRG) which transmits action potentials to spinal neurons; [ii] if PEA administration reduces spinal neuroinflammation, oxidative stress and neuronal p38 MAPK phosphorylation; and [iii] inhibition of p38 MAPK phosphorylation ameliorates chronic pain in SCD.

Female Berkeley (BERK) sickle (HbSS) expressing >99% human sickle hemoglobin and/or control (HbAA) expressing normal human HbA, mice (3.5-5.0 months) were treated for 14-days with PEA (i.p., 20 mg/kg/d) or vehicle (veh; i.p., 7.5% DMSO, 7.5% Tween 20, in sterile PBS). At endpoint, spinal cords and plasma were collected. HbSS mice have constitutively higher spinal malondialdehyde (MDA) levels, a marker of oxidative stress, compared to HbAA mice (~50%, P<.05), and PEA treatment significantly reduced HbSS spinal MDA levels (~53%, P<.05, vs veh); pro-apoptotic caspase-3 (~91%, P<.01) and pro-inflammatory interleukin (IL)-1β (~42%, P<.05), which are constitutively elevated in HbSS compared to HbAA mice (~45%, P<.05). Concomitant with reduced spinal inflammation, PEA reduced global marker of inflammation, serum amyloid P (~42%, P<.01) in the circulation, an analog for human CRP, which is significantly higher in HbSS compared to HbAA mice (~700%, P<.01). Thus, PEA ameliorates spinal inflammation and oxidative stress, and inhibits apoptosis. DRG neurons in vitro isolated from sickle mice were treated with tumor necrosis factor α (TNF α; 1 ng/mL) and hemin (40 mM) to create a sickle microenvironment replete with inflammation and oxidative stress, which significantly increased p38 MAPK phosphorylation (~494%, P<.001). PEA (30 mM) treatment of the neurons significantly inhibited TNF α and hemin-induced MAPK phosphorylation and its nuclear translocation (~65%, P<.001). Thus, it is likely that both peripheral and central mechanisms mediated by p38 MAPK signaling contribute to the initiation and maintenance of pain in SCD which are inhibited by PEA.

Neflamapimod (Nef) is a novel inhibitor of p38 MAPK phosphorylation with significant anti-inflammatory effects, mediated by IL-1β and TNF α. Nef is orally bioavailable and has been suggested to improve synaptic transmission. We evaluated the effect of orally administered Nef (6 and 12 mg/kg 2x daily; veh 0.1% pluronic acid in sterile H₂O) in female sickle mice (9.0 months) with hyperalgesia. Treatment with Nef 12 mg/kg/d for 7-days reduced mechanical (~42% & ~34%, both P<.01, vs veh & baseline, respectively) and cold hyperalgesia (~46% & ~34%, both P<.001, vs veh & baseline, respectively). A decrease was also observed in mechanical (~29%, P<.05, vs veh) and cold hyperalgesia (~24%, P<.05, vs veh) after 7 days with 6 mg/kg Nef treatment. These data suggest p38 MAPK signaling contributes to chronic pain in SCD.

Together, our observations demonstrate the therapeutic potential of PEA and Nef in targeting p38 MAPK as novel interventions to ameliorate sickle cell pain and central sensitization. PEA is a safe nutraceutical which has shown effectiveness in many pain conditions including osteoarthritis. Nef has shown efficacy in improving cognitive function in Phase II clinical trials in dementia patients. Pain and cognitive impairment co-exist in SCD. Therefore, Nef has the potential to reduce pain and improve cognitive function in SCD.