Calpain-1 Contributes to Pain and Organ Damage in Sickle Cell Disease

Abstract

Sickle cell disease (SCD) is complex with disabling and life-threatening consequences including pain and organ damage. We hypothesized that calpain-1, a calcium-dependent protease may contribute to both organ damage and pain in SCD because deletion of calpain-1 in HbSS Townes mice ameliorated hyperalgesia (Nwankwo et al., Haematologica 2016), and BDA-410, a selective calpain-1 inhibitor, significantly improved age-associated organ damage in a mouse model of aging (Nabeshima et al., Sci Reports 2014). We performed a randomized double-blind placebo-controlled trial in homozygous female HbSS BERK (sickle) mice to examine the effect of BDA-410 on pain and organ pathology. Mice were treated daily intraperitoneally with vehicle (50 μl DMSO in 950 μl sesame oil) and BDA-410 at 30 mg/kg or 100 mg/kg for 2 weeks. Mechanical-, thermal- (heat and cold), and deep hyperalgesia were assessed at baseline (before treatment), during treatment (1-hour post-injection, as well as on days 4, 8, 12), and after discontinuation of treatment (days 16, 20, 24, and 28). Using 2-way repeated measures ANOVA with Tukey's correction, we observed a significant decrease in mechanical, heat, and cold hyperalgesia in sickle mice treated with BDA-410. Compared to baseline, we observed a significant decrease in, [a] mechanical hyperalgesia, on day 8 with 30 mg/kg (p=0.0312), and days 8, 12, 16, 20, and 24 with 100 mg/kg dose (p=0.0012; p<0.0001; p<0.001; p=0.0006 and p=0.0007, respectively); [b] heat hyperalgesia, on days 8, 12, 16, and 20 with 30 mg/kg (p=0.044, 0.0027, 0.0172 and 0.0326, respectively), and on day 12 with 100 mg/kg (p=0.0138); and [c] cold hyperalgesia on days 8, 12, 16, 20, 24, and 28 with both 30 mg/kg (p= 0.0254, 0.0155, 0.001, 0.003, 0.035 and 0.008, respectively) and 100 mg/kg (p= 0.0054, 0.0035, 0.005, 0.009, 0.011 and 0.003, respectively). No significant difference in deep hyperalgesia was observed with either treatment. No significant effect of vehicle was found on any measures. Although BDA-410 treatment was discontinued after 2 weeks, analgesic effect was maintained for a significantly longer time, suggesting amelioration of the pathobiology of pain and/or inhibition of neural transmission of pain mechanisms. Therefore, BDA-410 is effective in reducing chronic pain in sickle mice without causing tolerance. In addition to pain, extensive Purkinje cell damage in the cerebellum and liver infarcts have been demonstrated in BERK sickle mice (Manci et al., Blood 2006). We observed reduced liver infarction in number and area as well as a significant decrease in Purkinje cell damage in sickle mice treated with 30 mg/kg and 100 mg/kg BDA-410 vs. vehicle (p= 0.0007 & 0.0233, respectively). Thus, BDA-410 is effective at significantly reducing Purkinje cell damage in the brain, which is associated with motor dysfunction as well as pain. We therefore examined gait parameters using Mouse Walker equipment which captures natural walking gait based on optical total internal reflection (TIR). Foot contacts disrupt the effect causing frustrated total internal reflection (fTIR) resulting in illuminated points of contact detected by a high-speed camera (Lumenera Lt425C) and a high-performance recording software (StreamPix 7, Norpix). Gait-related parameters were extracted using MouseWalker software. Sickle mice showed significantly lower walking speed (p=0.0062), increased stance duration and stance instability (p= 0.001 and 0.030, respectively) compared to control mice. Gait parameters correlated positively for mechanical hyperalgesia with stance duration (r=0.72, p=0.004) and negatively for grip force with stance instability and stance duration (r = -0.70, p=0.0075 and r = -0.84, p=0.0002). Since decreased grip force demonstrates increased hyperalgesia, deep hyperalgesia also correlates positively with stance instability and stance duration. Thus, Purkinje cell damage is associated with alterations in gait and pain in sickle mice. Since BDA-410 inhibited Purkinje cell damage and reduced pain, it demonstrates the potential of targeting calpain-1 as a treatable target to develop novel therapeutics for treating pain and organ damage in SCD. These data also suggest that gait measures may serve as biomarkers for Purkinje cell damage and pain. We speculate that gait measures may have utility as a diagnostic and prognostic tool in the progression of SCD. BDA-410 and funding in part were provided by 1910 Genetics. Disclosures Pagare: 1910 Genetics: Employment. Nwankwo:1910 Genetics: Equity Ownership. Gupta:1910 Genetics: Research Funding.