PP-14, a Novel Structurally-Enhanced Antisickling Allosteric Hemoglobin Effector, Increases Oxygen Affinity and Disrupts Hemoglobin S Polymer Formation
Authors: Safo Martin K. , Pagare Piyusha P , Ghatge Mohini , Casu Carla , Ghiaccio Valentina , Anabaraonye Nancy , Xu Xiaomeng , Rivella Stefano , Hines Patrick C. , Liu Ke , Zhang Yan , Venitz Jürgen , Abdulmalik Osheiza 
Published: February 2020

We have previously designed and investigated novel allosteric effectors of hemoglobin (AEHs), as potential targeted treatment of sickle cell disease (SCD). In general, AEHs transiently covalently bind to hemoglobin (Hb), increase its affinity for O2, increasing the fraction of oxygenated sickle Hb (HbS), thus reducing HbS polymerization and countering red blood cell (RBC) sickling. In the current study, we designed a novel class of AEH molecules, incorporating a secondary mechanism of action (MOA), which is independent of Hb O2-affinity by interacting with the F-helix of deoxygenated HbS to directly destabilize its polymerization. Here, we report current results from our in-vitro and in-vivo studies with a representative AEH compound (PP-14).

First, we assessed the anti-sickling properties in-vitro by incubating 0.5, 1, and 2 mM of PP-14 with whole blood suspensions from a subject with homozygous SCD (SS, hematocrit: 20%) under hypoxic conditions, with subsequent RBC sickling assessment by microscopy. Next, we subjected the samples to anoxia (100% N2 gas) to demonstrate the O2-affinity-independent antisickling mechanism. Subsequently, we tested residual samples for the degree of Hb modification (i.e., HbS-AEH adduct formation) and O2-affinity (p50) shifts. In a second experiment to further assess the secondary MOA, we subjected SS blood samples treated with various concentrations of PP-14 to hypoxia in the Hemox analyzer, which permitted us to obtain aliquot samples at defined pO2 values to establish pO2-dependent sickling. Additionally, we conducted in silico and in-vitro ADME studies to evaluate possible metabolic inhibition of a panel of CYP enzymes. Finally, we conducted a preliminary in-vivo PK/PD study in wild-type mice administered single doses of PP-14 via the oral (P.O.: 100-200 mg/kg) and intraperitoneal (I.P.: 75 mg/kg) routes. Serial blood samples were collected for up to 52 h after P.O., and up to 30 h after I.P. administration, and samples were assayed to quantify PP-14 concentrations. Residual blood samples were assayed for in-vivo Hb-AEH adduct formation, and the corresponding change in O2-affinity (Δp50, %).

Our in-vitro studies demonstrated concentration-dependent inhibition of cell sickling of 25.5±11%, 44.4±3.8% and 90.8±1%, at 0.5, 1 and 2 mM of PP-14, respectively. HbS was modified correspondingly (38.9±9%, 55.7±4.9 %, and 92.4±9.8%), and was correlated linearly with the left-shift in OEC (Δp50 values of 11.3±5.1%, 29.0±13.2%, and 67.5±8.2%). Importantly, the antisickling effect was sustained under anoxic conditions (100% N2), strongly supporting the notion of a secondary, O2-affinity-independent MOA. Furthermore, we observed a dose-dependent delay in sickling, with initiation of sickling recorded at a pO2 level of 40 mmHg in absence of PP-14; and at 30 and 20 mmHg at 0.5, and 1 mM PP-14 concentrations, respectively. Complete inhibition of sickling was observed at 2 mM PP-14 through the lowest recorded pO2 level of 1.5 mmHg, a unique, effect not previously observed in any analogous AEH. In-vitro partitioning studies demonstrated that &gt;90% of PP-14 partitioned into the RBC compartment when whole blood was incubated with 100-300 µM concentrations. Metabolic studies using pooled human liver microsomes (HLM) and isozyme-specific probe substrates suggested that up to 100 µM PP-14 did not inhibit CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 or CYP2B6.

Wild-type mice after I.P. administration of PP-14 (75 mg/kg, n=5) showed peak concentrations in blood at 7 hrs (416.3±81.2 µM), with corresponding PD effects (Δp50 of 41.6±13.5%; modified Hb levels of 43.6±8.0%). Orally-treated mice had peak drug concentrations after 10-24 hrs, (~150 µM at 200 mg/kg, n= 2), with corresponding PD effects (Δp50 of 36.5±7.0%; modified Hb levels of 28.8±4.9%), which declined by 52 hrs.

Overall, our data confirm that PP-14 is novel antisickling AEH with a secondary, O2-independent MOA in addition to the primary O2-dependent effect, as demonstrated by the inhibition of sickling under anoxic conditions. Additionally, PP-14 showed: excellent partitioning into the RBC compartment; acceptable in-silico ADME properties and in-vivo oral bioavailability; PD effects; and low liability for metabolic drug-drug interactions.

Further studies to investigate formal detailed pharmacokinetic properties, and biological activity after single- or repeat-doses in a SCD mouse model are ongoing.

Disclosures

Safo: Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Pagare:Virginia Commonwealth University: Patents & Royalties. Ghatge:Virginia Commonwealth University: Patents & Royalties. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy. Hines:Functional Fluidics: Equity Ownership. Liu:Functional Fluidics: Employment. Zhang:Virginia Commonwealth University: Patents & Royalties. Venitz:Virginia Commonwealth University: Patents & Royalties. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent.