Developing the required biomarkers that define ferroportin target engagement and impact on downstream signaling for clinical translation

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Abstract Developing the required biomarkers that define ferroportin target engagement and impact on downstream signaling for clinical translation Iron is essential for life, but is also highly toxic. Consequently, iron acquisition, transport, storage and utilization are tightly regulated. Hepcidin is the master regulator of iron; it controls the uptake of iron from diet and the release from iron stores through its interaction with the iron transporter ferroportin. Dysfunctional hepcidin production can cause serious health issues, including β-thalassemia and hereditary hemochromatosis. In both diseases, low hepcidin levels increase intestinal iron absorption and increase release of recycled iron from the reticuloendothelial system, which causes depletion of macrophage iron, relatively lower levels of serum ferritin, increased liver iron concentration, and free iron release into the circulation, causing organ damage. Without treatment, iron continues to accumulate, and a considerable proportion of patients eventually reach toxic iron-overload levels. Patients with iron overload diseases are currently treated with combinations of phlebotomy, blood transfusions and/or iron chelators, depending on the disease. Phlebotomy and blood transfusions are inconvenient and require hospital visits, while chelation therapy is associated with considerable toxicity. Thus, there is a significant need for new treatments that are safer and better tolerated. Because of hepcidin's complicated 4-disulfide structure, insolubility, aggregation potential and rapid clearance, it is unsuitable as a treatment and hepcidin mimetics have been proposed as potential therapeutics. To overcome the physicochemical limitations of hepcidin, Protagonist engineered more potent, stable, soluble and efficacious alternative scaffolds using a purposefully built structure-based drug design environment (Vectrix™). During the SBIR Phase I proposal, the hepcidin mimetic PTG-300 was developed after optimizing potency, stability, solubility and other physicochemical properties. The overall objective of this Phase II SBIR proposal is to develop methods for characterizing in vivo target engagement, including pharmacokinetic and pharmacodynamic methods to characterize the binding of PTG-300 to ferroportin and how binding affects downstream biomarkers and efficacy. These biomarkers will be used to aid dose selections and to provide early- stage clinical proof-of-concept. The specific objectives are to: 1) develop methods to characterize the extent of target engagement (TE) of ferroportin, 2) develop pharmacodynamic (PD) biomarkers that describe the effect of ferroportin engagement on downstream signaling, 3) correlate the TE and effect on PD biomarkers with efficacy in a preclinical model of thalassemia, 4) provide further mechanistic data on the clinical utility of PTG-300. These are important steps towards our ultimate objective of demonstrating clinical benefit in late-stage clinical trials. The biomarkers developed in this study will permit the assessment of mechanism-specific and disease- related parameters to guide clinical design.