Targeting Elastase with Alpha1 Anti-Trypsin Reduces Hyperalgesia in Mice with Sickle Cell Disease

Sickle cell disease (SCD) is associated with increased activity of proteases including tryptase and elastase, which can contribute to a wide variety of pathologies including endothelial activation and injury to the neural system. In turn, these effects of proteases contribute to pain, a major comorbidity of SCD (Vincent et al., Blood 2013; Vicuña et al., Nature Med 2015). The enzyme activity of proteases released from granulocytes including neutrophils is regulated by serine protease inhibitors (Serpins). Transcriptomic analysis revealed downregulation of Serpins in the dorsal root ganglion (DRG) of sickle mice (Paul et al., Sci Data 2017). Increased neutrophil activation has been observed in patients with SCD and in mouse models of SCD (Zhang et al., Blood. 2016). Therefore, we hypothesized that increased neutrophil elastase activity contributes to pain in SCD and that a Serpin, a₁ anti-trypsin (A1AT) would inhibit elastase activity and reduce pain in SCD. We used transgenic homozygous HbSS-BERK (sickle) and HbAA-BERK (control) mice expressing human sickle hemoglobin and normal human hemoglobin A, respectively, and Prolastin C (Grifols) an A1AT, a Food and Drug Administration (FDA) approved drug, to examine our hypothesis. We observed a significant increase in elastase activity in the plasma, lungs and DRG of sickle mice compared to control mice (p<0.002, 0.05 & 0.05, respectively). Treatment with 80 mg/Kg/day for 3 days with intraperitoneal (ip) dose of A1AT led to a significant decrease in elastase activity in the plasma, lungs and DRG of sickle mice compared to vehicle treatment (p<0.02, 0.001 & 0.05, respectively). A1AT had no significant effect on elastase activity in any of these tissues in control mice, perhaps due to constitutively low activity of elastase in control mice. DRG neurons transmit nociceptive action potentials to the dorsal horn of spinal cord where signals are further processed by the activation of neuromodulators and signaling pathways. One of the key pathways is p38 mitogen activated protein kinase (MAPK), which is activated in chronic pain and has been found to be significantly activated in the spinal cords of BERK sickle mice compared to control mice. A1AT treatment resulted in a decrease in the phosphorylation of p38 MAPK in the dorsal horn of the spinal cord of sickle mice, suggesting that inhibition of elastase attenuates the central mechanisms of chronic pain in sickle mice. Next, we examined the effect of A1AT on hyperalgesia in male sickle mice using 3 different doses of 40, 80 and 200 mg/kg injected ip on day 1 of a 3-day cycle for 3 - 5 cycles. A significant decrease for heat hyperalgesia with 40 mg/kg A1AT was observed on the 3^rd day of Cycles 1, 2 and 3 (p < 0.01). The 80 mg/kg dose also reduced heat hyperalgesia on the 3^rd day of Cycles 1 and 2 (p < 0.005) but at 24 hours after the injection for Cycle 3 (p < 0.05). This reduction remained significant through the end of Cycle 5. A significant difference for cold hyperalgesia with 40 mg/kg (p < 0.005) was observed after the first injection for Cycle 1. This reduction remained significant through the end of Cycle 3 (p < 0.01). Similar, but more sustained effect was noted with 80 and 200 mg/Kg dose. Thus, an optimum dose of 80 mg/Kg A1AT reduced thermal hyperalgesia significantly, which was sustained over a period of 15 days without leading to tolerance. However, none of the doses of A1AT had a significant effect on mechanical or deep tissue hyperalgesia in male sickle. Different nociceptors and channels on C-fibers mediate thermal sensitivity. It is likely that A1AT inhibition of proteases targets thermal but not mechanoreceptors. Nonetheless, it is noteworthy that cold hyperalgesia, a characteristic feature of sickle pain, is ameliorated with A1AT. In conclusion, our data demonstrate a novel role for A1AT, an FDA approved agent, to reduce thermal sensitivity in SCD. Since A1AT deficiency is known to adversely influence lung function, A1AT may even provide benefit in ameliorating lung injury observed in SCD. We speculate that proteases may be treatable targets and provide a therapeutic effect on sickle pathobiology in addition to ameliorating pain. Since several drugs containing A1AT including Prolastin C used by us are FDA approved for clinical use, our observations have the potential for developing these agents for the treatment of pain in SCD following clinical trials.