Compound Nanoparticle 5HMF-Prodrug Formulations for Optimized Anti-Sickling Therapy

BACKROUND: Sickle cell disease (SCD) pathology stems from deoxygenation-induced hemoglobin (HbS) polymerization; allosteric Hb adducts that increase oxyHbS target this mechanistic trigger. 5-hydroxymethylfurfural (5HMF) binds to Hb αVal1, reducing O₂ affinity & sickling and protecting hypoxic SS mice (survival, organ injury). However, 5HMF did not advance beyond PhI/II trials, due to pharmacokinetic/dynamic (PK/PD) limitations. A related molecule (Voxelotor) was approved based on surrogate endpoints (Hb level and hemolytic markers), but without improved vaso-occlusive morbidities. We reformulated 5HMF, a potentially superior agent, to address translation limitations arising from disfavorable PK/PD.

5HMF PK/PD: RBCs from SCD patients were incubated with 5HMF (20% Hct, 37C, HMF: 0-5mM, 0-3 hrs). 5HMF-induced Hb modification was quantified by HPLC (Waters e2695XE, Swift WCX-PEEK); O₂ dissociation curves (ODC) were measured in intact RBCs (HEMOX); pO₂’s at the point of sickling (POS) and point of unsickling (POU) were determined during controlled Hb O₂ unloading/loading, respectively (LORRCA Oxygenscan). We observed a dose dependent increase in 5HMF modified Hb, that was maximal (~75%) at 1 hr and 5mM [5HMF], and that resulted in: (1) 35% increase in HbO₂ affinity (ODC p50 pH 7.4; 31.1 ± 3.1 Torr vs 20.3 ± 1.5 Torr; 0 vs 5mM 5HMF), (2) 30% POS reduction (56.9 ± 10.1 vs 39.8 ± 11.6 Torr; 0 vs 5mM 5HMF) and (3) 45% POR reduction (38.3 ± 9.1 vs 21.1 ± 5.7 Torr; 0 vs 5mM 5HMF) (p < 0.05 for all, RMANOVA).

PRODRUG NPs: We designed two complementary 5HMF (phospholipid and graphitic) prodrugs (Lipid Prodrug; LP and carbon dot (CD)) that were evaluated individually and as an integrated self-assembled compound nanoparticle (CompNP). Phospholipid LP ‘pro-HMF’ synthesis: 5HMF was treated with NaH and THF (RT, N₂), then mixed with 1-Lysophosphatidylcholine (16:0 lysoPC) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-succinimide (NHS) (anhydrous chloroform, RT, ON). Graphitic CD ‘pro-HMF’ synthesis: sucrose in water (0.2 μM, 18 MΩ cm) was heated (270C, 30m), followed by sonication (Q700, 20m: Amp, 1, on: 2s, off: 1s), filtering (0.2 μm) and dialysis (10 kD MWCO, 2d). Simple LNPs (comprised only of LP pro-5HMF) were prepared by combining 5HMF LP (5 mM) with L-α-Phosphatidylcholine (1:1) in chloroform/methanol, then drying (thin film) that was resuspended (PBS), vortexed and sonicated (amp 1, on 2s, off 1s, 10m) and dialyzed (2d, PBS). Compound Nanoparticle (CompNP) self-assembly: CDs in PBS were mixed with the above lipid film, followed by sonication and dialysis (as above) resulting 152±12nm (hydrodynamic size) particles with -34±6mV surface charge. All formulations were extensively characterized (physicochemical features and biodegradation mapping).

NP PK/PD: We found that the prodrugs release 5HMF in RBCs through complimentary mechanisms (acutely from the LP shell by phospholipase A₂ (PLA₂) activity and slowly/sustained from the CD core by enzyme-triggered and peroxide-based degradation). Then, using human SS RBCs, we quantified time- and concentration-dependent in vitro PK/PD parameters (partially presented here): 3h data for the composite NP: Hb-5HMF modification (HPLC, 76.7±8.9% 5HMF modificatied HbS), HbO₂ affinity (p50 15.4±3.8Torr) and antisickling potency (LORRCA deformability/elongation index rose by 0.28±3.8.07AU & point of sickling fell by 21.4±8.2Torr); imaging: ~75% reduced hypoxia-induced sickling (p<0.05, ANOVA, all). Notably, PK (5HMF modified SHb) for LNP & CompNP was sustained well beyond that for free 5HMF and was progressive beyond 24h (likely due to increased PLA₂ and oxidizing milieu in SRBCs). PD (anti-sickling) for the CompNP formulation exhibited the greatest efficacy for (POS) (eg hypoxia tolerance) and POR (simulating O₂ loading during pulmonary circulatory transit); as for our PK data, effect upon POS/POR progressed from 1h to 3h. NB: No produg/NP formulation resulted in hemolysis > 1-2%.

SUMMARY: We developed first in class RBC-targeted, protected, biocompatible, self-assembled 5HMF prodrug nanoparticles for SCD therapy that are designed for eventual transdermal delivery. In vitro proof of concept data demonstrates: (1) improved potency, (2) targeted payload delivery and subsequent extended (>24h) prodrug release within RBCs, and (3) with prolonged pharmacodynamic (antisickling) effect.