Mechanism-Based Treatable Targets for Organ Damage and Pain in Sickle Cell Disease

Sickle cell disease (SCD), characterized by oxidative stress and inflammation, affects most organs. Antioxidants and anti-inflammatory drugs have not been successful in preventing/treating organ damage or pain. Using a “Whole Person” approach, we examined novel mechanisms in different organs and the nervous system using humanized BERK sickle (HbSS) and BERK control mice (HbAA). To validate the treatable potential of the targets we used a novel transdermal curcumin (TDC) gel with high bioavailability, ability to cross blood brain barrier and devoid of toxic side effects. TDC gel (Vasceptor®, Vascarta Inc.), containing 0.1 M of curcuminoids, or vehicle, were applied (0.1 mL) topically by gentle rubbing to the abdomen on alternating days for 3 wks followed by analysis of organs. For the in vitro study, we diluted TDC or vehicle gel in complete culture medium. We examined the mechanisms by which TDC exerts an anti-inflammatory and antioxidant activity in organs, peripheral- and central-nervous system (PNS & CNS). We isolated neuronal cells from the dorsal root ganglion (DRG) and hippocampal region of the brain of HbSS and HbAA mice. To mimic the SCD microenvironment, we incubated the neuronal cells with TNF-α 1 ng/mL + hemin 40 µM (T+H) in the presence/absence of 100 µM TDC, or 100 µM TDC alone or vehicle. We observed a significant increase in reactive oxygen species (ROS) in DRG neurons treated with T+H for 4 hours vs. vehicle (P<.001) and that TDC co-incubated with T+H inhibited ROS vs. T+H treatment alone (P<.01). No significant changes occurred in ROS in TDC alone vs. vehicle. We next stained neurite outgrowth from DRG neurons using anti-β3-tubulin antibody. After 4 and 20 hours of incubation, vehicle-treated DRG neurons showed robust neurite growth. In a sickle microenvironment with T+H, neurites appeared disorganized and tortuous, with swollen terminals resembling “retraction bulbs” and thinning with discontinuous β3-tubulin expression at 20-hours. T+H reduced neurite intersections compared to vehicle at 4- (P<.0001) and 20-hours (P<.0001), and co-incubation with TDC prevented neurite loss at 4- (P<.001) and 20-hours (P<.001). T+H-induced ROS and axonal injury in DRG neurons were accompanied by a significant increase in phosphorylation of p38 (P<.0001 vs. vehicle), which was inhibited by co-incubation with TDC and a p38 inhibitor neflamamipod. Similarly, in hippocampal neurons, T+H induced p38 phosphorylation (p<0.01 vs. vehicle) and increased postsynaptic density protein 95 (PSD95) puncta (P<.01 vs. vehicle), indicating more dendritic spine sprouting, a process associated with neuronal hyperexcitability. Co-incubation with TDC significantly reduced PSD95 puncta density (P<.05 vs. T+H). HbSS mice treated with TDC for 3 weeks mitigated oxidative stress in spinal cords analyzed by malondialdehyde content (p<.03 vs. vehicle). Cell-free heme and oxidative stress can lead to microglial activation, thus promoting inflammation in the CNS. HbSS mice brains showed an increase in Iba-1 specific microglial activation compared to HbAA, which was mitigated by 3 wks of TDC treatment. These data suggest that SCD microenvironment leads to neuronal injury in the PNS and CNS via a p38 mediated mechanism, which may underlie central sensitization and neuropathic pain in SCD. This is mitigated by TDC. Analysis of peripheral organs showed that HbSS mice have decreased mitochondrial respiratory complex I activity in heart (P<.002); complex II-III activity in liver (P<.003); and complex IV activity in liver (P<.03), kidney (P<.025), and heart (P<.007), vs. HbAA. TDC treatment in HbSS mice led to a significant improvement in mitochondrial respiratory activity of complex I in heart (P<.004) and complex IV activity in liver (P<.04). ATP is critical to maintain the high metabolic requirement in SCD and its decrease may contribute to organ pathology. Together, our data show that multiple mechanisms converge at increased oxidative stress and inflammation, which challenges treatment strategies that target a single mechanism. TDC has the potential to target multiple causal mechanisms and mitigate the multi-system comorbidities in SCD. The ease of topical application, affordability and a high safety profile suggest that TDC may be a novel treatment option for SCD patients, including children, globally.

This presentation reflects the views of the authors and should not be construed to represent FDA's views or policies.

Gupta:Novartis: Research Funding; Zilker LLC: Research Funding.