Quinine-Hemin Compound As an Anti-Inflammatory Therapy in Sickle Cell Disease

Free hemin released during intravascular hemolysis induces inflammatory responses and plays a pivotal role in the pathogenesis of various hemolytic disorders, including sickle cell disease (SCD). Several strategies including the use of hemopexin have been developed to eliminate or inactivate the pro-inflammatory effects of free hemin. Compared to biologic drugs, small molecule drugs are more affordable, which is critical for the SCD patient cohort. Several hemin binding small molecule drugs, including quinine derivatives, have been developed and approved by FDA as malaria therapies, but it is unclear whether they can alleviate the detrimental effects of free heme and be used as a therapeutic for SCD. These approved drugs have been extensively studied for safety, pharmacokinetics and targets, which will substantially reduce the effort required for anti-hemolysis drug development. Because in a recent publication (Pal et al, Blood, 2021), quinine demonstrated immunologic bioactivity against B cells after binding with free heme, we selected quinine as the first candidate.

Using human primary monocytes, we found that quinine can inhibit hemin-mediated inflammatory cytokine production. Specifically, we measured the levels of IL-1β, IL-6, and TNF-α produced by human monocytes treated with medium, LPS alone, LPS plus hemin (25µM) or quinine alone (25µM), and LPS plus hemin (25µM) mixed with 2.5-25µM quinine for 4 hours. We found increased IL-1β (~3.6 fold) and TNF-α (~1.5 fold) production in LPS plus hemin treatment compared to LPS alone treatment but no change on IL-6 production (quinine alone treatment had no effect). However, adding quinine to hemin led to a dose-dependent inhibition of all three cytokines, such that TNF-α was reversed to the level to the LPS alone group while IL-1β and IL-6 levels were inhibited below the level of the LPS alone group (IL-1β:15% of LPS alone, IL-6: 36% of LPS alone), indicating a novel anti-inflammatory activity of the quinine hemin compound (QHC). To test the potential of quinine binding free heme in vivo and exhibiting anti-inflammatory activities in hemolytic diseases, we treated SCD mice and control mice with quinine (I.P., 2 weeks) using a dose (5mg/kg BW) lower than normally used (>20mg/kg BW). We found quinine treatment (I.P., 2 weeks) decreased plasma levels of IL-1β, IFN-β, IL-6, and TNF-α, and reduced liver tissue damage in SCD mice, while it had no effect on control mice. These results demonstrated the anti-inflammatory activity of quinine in hemolytic conditions, and the mechanism needed further studies to elucidate.

Our data suggest that QHC can inhibit the production of multiple inflammatory cytokines. We decided to focus on the mechanism of QHC inhibiting IL-1β production because it is specifically controlled by the inflammasome pathway. Using ATP, an NLRP3 inflammasome agonist, treatment on human monocytes as the model, we found that QHC (2.5µM), but not hemin alone or quinine alone, reduced >80% IL-1β production and ~50% cell death induced by ATP through inhibiting Caspase 1 and GASDMD activation. Similar results were obtained using other NLRP3 agonists, NLRC4 agonist, AIM2 agonist, and non-canonical inflammasome agonist. These results indicate that QHC is a broad-spectrum inhibitor of inflammasome activation. In addition to quinine, we tested the effect of combination hemin with chloroquine, amodiaquine, and dihydroartemisinin on IL-1β, and no effect was identified at the concentration of 2.5 µM. The results indicates that specific molecular space structure of QHC is required for the activity. In vivo, QHC (1.25mg/kg BW, I.P.), but not hemin or quinine alone, inhibited ~70% of IL-1β production and ~50% of neutrophil infiltration in peritoneal lavage in a classic alum crystal I.P. injection inflammasome activation mouse model. alum crystal I.P. injection can cause ~70% lethality in SCD mice. Pre-treating SCD mice with QHC (1.25mg/kg BW, I.P.) protected 100% of the mice from alum-induced lethality, while the same dose of hemin alone or quinine alone showed no effects. Our data support that QHC inhibits inflammasome activation both in vitro and in vivo.

In summary, our data support the use of hemin-binding small molecules as potential therapeutics for SCD and other inflammasome activated diseases.