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2360 Compound Nanoparticle 5HMF-Prodrug Formulations for Optimized Anti-Sickling Therapy

Program: Oral and Poster Abstracts
Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Sickle Cell Disease, Translational Research, Hemoglobinopathies, drug development, Diseases, Therapies
Sunday, December 11, 2022, 6:00 PM-8:00 PM

Stephen C Rogers, PhD1*, Parikshit Moitra, PhD2*, Mary Brummet1*, Zohreh Safari, PhD1*, Qihong Wang, MD1*, Joy Okhuevbie, BS1*, Nancy Anabaraonye3*, Osheiza Y. Abdulmalik, DVM4*, Martin K. Safo, PhD5*, Nicole Glynn-Cunningham, RN6*, Jennie Y. Law, MD7, Maria R. Baer, MD8, Dipanjan Pan, PhD9* and Allan Doctor, MD1

1Center for Blood Oxygen Transport and Hemostasis, University of Maryland School of Medicine, Baltimore, MD
2Department of Bioengineering, Pennsylvania State University School of Engineering, University Park, PA
3Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA
4Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA
5Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA
6Department of Hematology, University of Maryland Medical Center, Baltimore, MD
7Department of Medicine, University of Maryland School of Medicine, Baltimore, MD
8University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD
9School of Engineering; Huck Institutes for the Life Sciences, Pennsylvania State University, University Park, PA

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.

Disclosures: Baer: Kite: Research Funding; Ascentage: Research Funding; Kura Oncology: Research Funding; AbbVie: Research Funding; Forma: Research Funding; Takeda: Research Funding.

*signifies non-member of ASH