RATIONAL DESIGN OF ALLOSTERIC MODULATORS OF HEMOGLOBIN AS DUAL ACTING ANTISICKLING AGENTS

Intracellular polymerization of deoxygenated sickle hemoglobin (Hb S) remains the principal cause of the pathophysiology associated with sickle cell disease (SCD). Naturally occurring and synthetic allosteric effectors of hemoglobin (AEH) have been investigated as potential therapeutic agents for the treatment of SCD. Several aromatic aldehydes, including vanillin, have been studied previously as AEHs for their antisickling activities. Specifically, these compounds form Schiff- base adduct with Hb to stabilize the oxygenated (R) state, increase Hb affinity for O2 and concomitantly prevent the hypoxia-induced primary pathophysiology of Hb S polymerization and RBC sickling, in turn, ameliorating several of the cascading secondary adverse effects. These compounds, however, undergo significant metabolism leading to suboptimal pharmacokinetic properties, e.g. short duration of pharmacologic action and low bioavailability. These drawbacks have severely hampered the use of aromatic aldehydes as AEHs to treat SCD. To counter these challenges, we designed and synthesized 14 novel compounds (PP- compounds) based on previously studied pyridyl derivative of vanillin. These modifications were expected to increase binding interactions with the protein and thus stabilize the Schiff-base adduct, as well as lead to perturbation of the surface-located F-helix that would stereospecifically destabilize polymer contacts. We investigated the in vitro pharmacokinetic/pharmacodynamic properties of these newly synthesized compounds to ascertain sustained binding and modification of normal human Hb. Subsequently, we conducted in vitro screening assays to test for inhibition of sickling, modification of Hb to the high-affinity form, as well as for a direct left-shift in oxygen equilibrium curves (OEC). Three selected compounds, PP6, PP10, and PP14, that demonstrated not only high antisickling activity but also showed sustained pharmacologic action were chosen for preliminary in vivo studies. Our results showed maximal levels of Hb modification, which were sustained for the entire 24 h experimental period. In contrast, TD-7 after reaching maximum effect at 1 h gradually decreased in potency and at 24 h has lost 45% of its activity, consistent with the low bioavailability of this compound. These findings suggested that our modifications appeared to successfully limit drug metabolism in red blood cells. Most of these compounds showed almost complete inhibition of sickling at 2 mM concentration; with significant modification of Hb to a higher affinity Hb as well as an increase in O2 affinity of Hb. Interestingly, while TD-7 demonstrated a clear linear correlation between its ability to increase Hb-O2 affinity and antisickling activity, PP2, PP3, PP6, PP9, PP10, and PP14, showed a weak correlation between these parameters. In fact, these compounds demonstrated high antisickling effect despite only marginally increasing Hb affinity for O2. This observation indicated that these compounds possibly exhibit the dual mechanism of antisickling activity. We hypothesize that their secondary mechanism of action is due to interactions with the surface located αF-helix that would lead to direct or stereospecific inhibition of polymer formation. To establish the mode of interaction with Hb, we further conducted x-ray crystallography studies and co-crystallized PP2, PP6, PP9 and PP11 with CO-liganded Hb. Our studies showed that these compounds bind in symmetry-related fashion at the α-cleft of Hb to form Schiff-base adducts with the N-terminal Val1 and showed direct interactions with the F-helix which overall enhanced interactions with Hb. PP6, PP10, and PP14 demonstrated significant in vivo modification of intracellular Hb in mice after IP administration, with increasing levels from 1 h to the 6 h experimental period. They also showed corresponding changes in O2 affinity observed at 3 h and 6 h, compared to 0 h pre-treatment samples in vivo. Thus, our results establish these compounds as a novel, promising group of potent anti-sickling agents, demonstrate their proposed mechanism of action and provide proof-of-concept justifications for our structure-based approach to developing potent therapeutics for SCD

Improving the Solubility and Oral Bioavailability of a Novel Aromatic Aldehyde Antisickling Agent (PP10) for the Treatment of Sickle Cell Disease

Background: Aromatic aldehydes, with their ability to increase the oxygen affinity of sickle hemoglobin, have become important therapeutic agents for sickle cell disease (SCD). One such compound, voxelotor, was recently approved for SCD treatment. Methyl 6-((2-formyl-3-hydroxyphenoxy)methyl) picolinate (PP10) is another promising aromatic aldehyde, recently reported by our group. Like voxelotor, PP10 exhibits O2-dependent antisickling activity, but, unlike voxelotor, PP10 shows unique O2-independent antisickling effect. PP10, however, has limited solubility. This study therefore aimed to develop oral and parenteral formulations to improve PP10 solubility and bioavailability. Methods: Oral drug tablets with 2-hydroxypropyl beta cyclodextrin (HP-β-CD), polyvinylpyrrolidone, or Eudragit L100-55 PP10-binary system, and an intravenous (IV) formulation with d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) or HP-β-CD, were developed. The pharmacokinetic behavior of the formulations was studied in Sprague-Dawley rats. PP10, a methylester, and its acid metabolite were also studied in vitro with sickle whole blood to determine their effect on Hb modification, Hb oxygen affinity, and sickle red blood cell inhibition. Results: Aqueous solubility of PP10 was enhanced ~5 times with the HP-β-CD binary system, while the TPGS aqueous micelle formulation was superior, with a drug concentration of 0.502 ± 0.01 mg/mL and a particle size of 26 ± 3 nm. The oral tablets showed relative and absolute bioavailabilities of 173.4% and 106.34%, respectively. The acid form of PP10 appeared to dominate in vivo, although both PP10 forms demonstrated pharmacologic effect. Conclusion: Oral and IV formulations of PP10 were successfully developed using HP-β-CD binary system and TPGS aqueous micelles, respectively, resulting in significantly improved solubility and bioavailability

Design, Synthesis, and Biological Evaluation of Ester and Ether Derivatives of Antisickling Agent 5‑HMF for the Treatment of Sickle Cell Disease

Candidate drugs to counter intracellular polymerization of deoxygenated sickle hemoglobin (Hb S) continue to represent a promising approach to mitigating the primary cause of the pathophysiology associated with sickle cell disease (SCD). One such compound is the naturally occurring antisickling agent, 5-hydroxymethyl-2-furfural (5-HMF), which has been studied in the clinic for the treatment of SCD. As part of our efforts to develop novel efficacious drugs with improved pharmacologic properties, we structurally modified 5-HMF into 12 ether and ester derivatives. The choice of 5-HMF as a pharmacophore was influenced by a combination of its demonstrated attractive hemoglobin modifying and antisickling properties, well-known safety profiles, and its reported nontoxic major metabolites. The derivatives were investigated for their time- and/or dose-dependent effects on important antisickling parameters, such as modification of hemoglobin, corresponding changes in oxygen affinity, and inhibition of red blood cell sickling. The novel test compounds bound and modified Hb and concomitantly increased the protein affinity for oxygen. Five of the derivatives exhibited 1.5- to 4.0-fold higher antisickling effects than 5-HMF. The binding mode of the compounds with Hb was confirmed by X-ray crystallography and, in part, helps explain their observed biochemical properties. Our findings, in addition to the potential therapeutic application, provide valuable insights and potential guidance for further modifications of these (and similar) compounds to enhance their pharmacologic properties