Superfine powdered activated carbon (SPAC) is sub-micron sized activated carbon with superior adsorption kinetics compared to regular powdered activated carbon (PAC) due to the short intraparticle diffusion times. SPAC has recently been demonstrated as an excellent adsorbent; however, subsequent removal of spent SPAC in suspended reactors may be challenging due to its fine size. Thus, SPAC may benefit from immobilization into a macroscopic scaffolding if its adsorptive properties can be preserved. Composition of non-woven electrospun polystyrene (PS) fibers consisting of SPAC is a novel approach to immobilize SPAC; however, the potential losses of adsorption capacity due to polymer-SPAC interactions is still mostly unknown. In this study, SPAC-laden electrospun PS with intentionally tuned SPAC sizes were fabricated for the first time. The centrifugal separa-tion of SPAC generated two different particle sizes with median diameters of 0.30 +/- 0.15 mu m (SPAC-Fine) and 0.62 +/- 0.25 mu m (SPAC-Coarse). Both SPAC-Fine and SPAC-Coarse were added to the spinning solution and compared against the PS-only control fibers. Phenanthrene was used as a probe molecule to investigate the accessibility of adsorption sites on PS/SPAC composite fibers. This study showed that most SPAC adsorption sites are still accessible when incorporated into electrospun polymer fibers, regardless of whether the carbon particle size is larger or smaller than the fiber diameter. It can be speculated that the disposition of fine particles on the external surface of PS fibers as well as partial incorporation of large SPAC particles in the fiber allow diffusion of probe molecules onto the ultimate sorption sites of SPAC particles.

Hepcidin peptidomimetics that are orally stable and systemically active will mark a paradigm change in management of blood disorders that exhibit aberrant iron homeostasis (e.g. hereditary hemochromatosis) and in conditions that can be influenced by modulating stressed iron homeostasis (e.g. polycythemia vera). Hepcidin modulates the iron exporter membrane protein ferroportin and is the master regulator of iron homeostasis in the body. Orally bioavailable "Minihepcidins" have been previously shown to be efficacious in lowering serum iron in mice when dosed peroral (PO) (Preza GC et. al., Journal of Clinical Investigation 2011). Here we describe hepcidin mimetic peptides that are metabolically stable in the gastrointestinal tract, systemically absorbed when delivered orally, and pharmacodynamically active in reducing serum iron parameters in pre-clinical models. Further, we also demonstrate improvement in disease parameters in a mouse model for hereditary hemochromatosis. The oral peptides, PN20076 and PN20089, have EC50 of 16.5 nM and 1.39 nM respectively in cell based ferroportin internalization assay (Table 1). In comparison EC50 was 67.8 nM for Hepcidin and 6.12 nM for PTG-300. (PTG-300 is an injectable hepcidin mimetic currently in Phase 2 clinical studies for polycythemia vera and hereditary hemochromatosis.) Oral stability of the peptides was evaluated in a panel of assays, including in vitro matrices simulating the gastric and intestinal conditions, and ex vivo matrices of serum/plasma from different species. Table 1 shows data for peptides PN20018, PN20076 and PN20089. PN20076 demonstrated extended stability in gastric and intestinal conditions, and degradation half-life of >24 hr in mouse plasma and 14.8 hr in rat serum. Based on their stability and potency data from the above battery of screening assays, the peptides were selected for in vivo evaluation in healthy mice to characterize their pharmacodynamic (PD) and pharmacokinetic (PK) properties. PN20076 and PN20089 showed equivalent PD response of reduction in serum iron concentration in wild type mice. After two successive PO doses of PN20076 or PN20089 approximately 24 hr apart, serum iron concentration was reduced from ~30 µM to ~10 µM (group averages), i.e. ~66% reduction, at 4.5 hr post-second dose for both peptides (Fig. 1). At 4.5 hr post-dose, the serum concentration of PN20076 was ~262 nM. PN20076 was further evaluated for its effect in lowering iron overload in a mouse model for hemochromatosis (HFE2-/- with homozygous deletion of hemojuvelin, a positive regulator of hepcidin expression). This mouse model is marked by hyper-absorption of dietary iron, higher transferrin saturation and deposition of excessive iron in liver, all manifestations of aberrant iron homeostasis caused by the genetic disruptions of the hepcidin-iron pathway. Liver iron accumulation was significantly prevented in groups treated with PN20076 once daily (QD) by PO administration for over two weeks, as compared to vehicle treated controls (Fig. 2). The reduction in non-heme iron concentration in liver homogenates (measured using a colorimetric iron assay) was statistically significant in the female group treated with PN20076. We have described orally stable and systemically active hepcidin mimetic peptides and demonstrated oral activity in preventing liver iron overload in hemochromatosis mice. The effective reduction of iron absorption from the diet and the steady state lowering of transferrin-saturation can potentially prevent tissue iron toxicity in hereditary hemochromatosis. Similarly, the sustained reduction of systemic iron levels with an oral hepcidin mimetic to control stressed iron homeostasis should reduce excessive erythrocytosis, a hallmark of polycythemia vera and other congenital and acquired erythropoietic disorders. Disclosures Bourne: Protagonist Therapeutics: Current Employment, Other: shareholder. Zhang:Protagonist Therapeutics: Current Employment, Other: shareholder. Frederick:Protagonist Therapeutics: Current Employment, Other: shareholder. Tran:Protagonist Therapeutics: Current Employment, Other: shareholder. Vengalam:Protagonist Therapeutics: Current Employment, Current equity holder in private company. McMahon:Protagonist Therapeutics: Current Employment, Other: shareholder. Huie:Protagonist Therapeutics: Current Employment, Other: shareholder. Ledet:Protagonist Therapeutics: Current Employment, Other: shareholder. Zhao:Protagonist Therapeutics: Current Employment, Other: shareholder. Tovera:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Lee:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Yang:Protagonist Therapeutics: Current Employment, Other: shareholder. Dion:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Yuan:Protagonist Therapeutics: Current Employment, Other: shareholder. Zemede:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Nguyen:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Masjedizadeh:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Cheng:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Mattheakis:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Liu:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Smythe:Protagonist Therapeutics: Current Employment, Other: shareholder.

Hepcidin-Ferroportin axis dictates optimal absorption of dietary iron as well as systemic iron levels. This is crucial for providing sufficient iron needed for cellular functions while also preventing iron toxicity. PTG-300 (currently in a Phase 2 clinical study for beta-thalassemia) is a peptide mimetic of natural hepcidin that targets the major iron transporter, ferroportin, and causes its internalization & subsequent degradation. The pharmacodynamic effects of PTG-300 are the reductions in serum iron and transferrin-saturation (TSAT) due to reduced ferroportin expression on cells that store or recycle iron. We chose to demonstrate in two mouse models with iron dysregulation, that our hepcidin mimetics improve disease parameters by correcting dysregulated iron homeostasis. Beta-thalassemia is characterized by an imbalance in alpha-beta globin ratio in erythrocytes due to underlying beta-globin gene mutations. The excess alpha-globin, along with associated heme and iron, form "hemichrome" aggregates that integrate into the membranes of RBCs. The labile iron in these hemichromes generate ROS and are toxic to the cells, causing premature hemolysis of circulating RBCs and reduction in their lifespan. In a mouse model for beta-thalassemia, Hbbth3/+, we investigated the efficacy of a hepcidin mimetic in reducing hemichrome aggregation by limiting iron in the erythroid progenitors, and thereby reducing iron toxicity in RBCs. Subcutaneous injections of 1 mg/kg PN-8772 (analog of PTG-300 which has similar in vitro and in vivo potency) were administered every other day (Q2D) for a period of 4 weeks. At the end of the study, hemichrome aggregates were extracted from RBC membranes, and then analyzed on a TAU gel to quantify the cytoskeleton α-globin band intensities (Casu et al, Blood 2016). Hemichrome aggregates were reduced in groups treated with PN-8772 as compared to untreated controls, with concurrent improvements in hemoglobin and reductions in reticulocytes. Treatment with oral chelator Deferasirox (200 mg/kg; daily) did not show reduction in hemichrome aggregation, while it significantly lowered liver iron-overload. RBCs in Hbbth3/+ mice express aberrant morphologies due to the underlying hemichrome toxicity, similar to the phenotypes expressed in human beta-thalassemia. Chronic treatment with PN-8772 (as described above) also resulted in a significant reduction in aberrant morphologies that are indicative of hemolysis, viz. spherocytes & schistocytes. In a separate study, flow cytometry was used to monitor the survival of RBCs in Hbbth3/+ mice. At the end of 4 weeks of PTG-300 treatment (1 mg/kg, Q2D) the RBCs were marked by an in-life biotinylation method (Schmidt et al, Blood 2013) and subsequently followed over 49 days with continued treatment. There was a significant increase in survival of RBCs as compared to untreated controls. In summary, we demonstrate that by limiting iron in the developing erythroblasts and iron toxicity in RBCs, PTG-300 therapy has the potential to improve the quality of the RBCs and their oxygen carrying capacity, thereby ameliorating anemia. In beta-thalassemia, the clinical presentation includes secondary iron overload in various organs because of hyperabsorption of dietary iron, exacerbated by frequent blood transfusions that are required for management of anemia. Similarly, in hereditary hemochromatosis (HH) there is hyperabsorption of dietary iron leading to primary iron overload. We used a hemochromatosis mouse model (HFE) to demonstrate the effectiveness of PTG-300 therapy in limiting systemic iron toxicity by regulating TSAT and in preventing hyper-iron absorption. The model is characterized by homozygous deletion of HFE with severely low hepcidin levels and consequently very high TSAT (~100%). In this model, a single dose of PTG-300 at 2.5mg/kg reduced TSAT by ~60% at 10-hour post-dose, as compared to untreated controls. Sustained TSAT reduction by chronic treatment will therefore mitigate toxic effects of labile iron. Two weeks of chronic treatment with PTG-300 (2.5 mg/kg, Q2D) effectively prevented iron deposition in the liver. Overall our data suggests that PTG-300 has the potential to be an effective treatment in hemoglobinopathies, like beta-thalassemia, and Hereditary Hemochromatosis, by reducing systemic labile iron toxicity by limiting TSAT, preventing organ iron deposition & improving anemia (in case of thalassemia). Disclosures Taranath: Protagonist Therapeutics: Employment. Bourne:Protagonist Therapeutics: Employment. Zhao:Protagonist Therapeutics: Employment. Frederick:Protagonist Therapeutics: Employment. King:Protagonist Therapeutics: Employment. Liu:Protagonist Therapeutics: Employment.

Background:The &agr;4&bgr;7 integrin is a clinically validated target in inflammatory bowel disease (IBD). Vedolizumab (Entyvio®), a humanized monoclonal antibody that specifically binds to the &agr;4&bgr;7 integrin, is FDA-approved for the treatment of moderate-to-severe ulcerative colitis and Crohn's disease. Vedolizumab binds to &agr;4&bgr;7 on circulating memory/effector T cells in the blood and blocks their homing to intestinal tissues expressing the ligand MAdCAM-1. The aim of this study is to characterize PTG-100, a novel oral &agr;4&bgr;7 antagonist peptide that is largely restricted to the gut tissues, and is pharmacologically active in murine colitis models and in normal cynomolgus monkeys. Methods:Pharmacokinetic (PK) studies of PTG-100 were conducted in mice, rats, and cynomolgus monkeys, with peptide concentrations measured by mass spectrometry. Pharmacodynamic (PD) studies were conducted in murine colitis models and in healthy cynomolgus monkeys. Cell trafficking in blood and gut lymphoid tissues was measured by FACS or immunohistochemistry (IHC). Results:PTG-100 is a potent antagonist of &agr;4&bgr;7 (IC50 = 1 nM), but inactive against &agr;4&bgr;1, &agr;L&bgr;2 or &agr;E&bgr;7 as measured in a variety of biochemical and cellular assays. Oral dosing of PTG-100 in normal or dextran sodium sulfate (DSS)-treated mice and rats showed dose-dependent exposure in the small intestine, colon, mesenteric lymph nodes (MLN) and Peyer's Patches (PP), but much lower exposure if any in blood and urine. Oral dosing of a fluorescent dye conjugate of PTG-100 and imaging by fluorescence microscopy or IHC showed the peptide accumulates in the lamina propria of tissues from the small intestine. Daily dosing with PTG-100 in murine DSS colitis models showed a dose-dependent reduction of CD4+CD44high CD45RBlow &bgr;7+ T cells in the MLN and PP, and a concomitant increase in the spleen and blood as measured by FACS. There was also a strong reduction of &bgr;7+ cell infiltration into lamina propria lesions of the distal colon as measured by IHC. PTG-100 also caused a dose-dependent reduction in body weight loss and mucosal injury as assessed by endoscopy. Daily oral dosing of PTG-100 in normal cynomolgus monkeys resulted in high blood receptor occupancy of memory T cells, and a dose-dependent increase in the percentage of &agr;4&bgr;7 memory CD4+ T cells in the blood. There were no adverse clinical or microscopic changes with PTG-100 administration in 6-week GLP toxicology studies in rat and monkey up to 90 and 75 mg·kg−1·d−1, respectively. Safety pharmacology and mutagenesis studies demonstrated no adverse findings. Conclusions:PTG-100 is a first-in-class oral &agr;4&bgr;7-selective antagonist being developed for the treatment of patients with IBD. PTG-100 reaches high concentrations in gut tissues and alters the trafficking of gut-homing T cells in mice and cynomolgus monkeys. The lack of toxicity in the full battery of safety and toxicology studies to date coupled with low exposure in blood suggest that PTG-100 will be suitable for human trials.

Background:The heterodimeric IL-23 receptor is comprised of the IL-12R&bgr;1 subunit in complex with IL-23R subunit. The ligand IL-23 is also a heterodimer of the unique p19 subunit coupled with the common p40 subunit shared with IL-12. Binding of IL-23 ligand to the IL-23R complex leads to phosphorylation of STAT3, and IL-23-dependent expression of pro-inflammatory cytokines. Clinical trials in Crohn's Disease or psoriasis with ustekinumab and briakinumab (which target the common p40 subunit) and tildrakizumab, guselkumab, MEDI2070, and BI-655066 (which target the unique p19 subunit of IL-23) highlight the potential of IL-23 signaling blockade in treatment of human inflammatory diseases. The aim of this study is to develop orally stable IL-23R antagonist peptides that act locally in the intestinal tissue for treatment of IBD. Methods:Potent, selective and orally stable peptide antagonists of IL-23R were identified through a combination of phage display technology and medicinal chemistry. To evaluate oral stability, the peptides were incubated in a variety of ex vivo intestinal/colonic washes or simulated gastric/intestinal fluids, and half-lives determined by mass spectrometry. Pharmacokinetic (PK), pharmacodynamic (PD) and colitis studies were conducted in rats. Results:Using a combination of phage display technology and medicinal chemistry, we identified functional inhibitory peptides of IL-23R that are stable in assays that mimic the harsh redox and proteolytic conditions of the GI environment. These peptides potently neutralize IL-23-mediated STAT3 signaling in the transformed human B lymphoblast DB cell line, and block IL-23 stimulated IFNg release from human primary NK cells. They are also active against the rat and cynomolgus monkey IL-23R. The peptides do not block the interaction between IL-6 and IL-6R or antagonize the IL-12 signaling pathway. In PK studies, oral dosing of the peptides results in high exposure in GI tissues, but very low exposure in the blood. In a rat TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced acute colitis model, oral dosing of the peptides caused a significant reduction in neutrophil infiltration as measured by myeloperoxidase (MPO) activity, and a reduction in inflammation and other disease parameters as assessed by histopathology. These in vivo activities of the peptides were comparable to that of an anti-IL-23p19 mAb. Conclusions:We have discovered a suite of potent, selective, and orally efficacious IL-23R peptide antagonists that are promising GI restricted therapeutics for the treatment of IBD. Peptides are effective and comparable to an anti-IL-23p19 monoclonal antibody in attenuating colitis in a TNBS-induced rat colitis model. PK studies show these peptides have significant exposure in intestinal tissues, but not blood and urine indicating the exposure of these peptides is gut-restricted, and supports the potential of locally blocking IL-23 signaling while minimizing immunogenicity and the risk of opportunistic infections associated with systemically delivered immunosuppressants and biologics.

The α4β7 integrin is a clinically validated target for inflammatory bowel disease (IBD). The anti-α4β7 antibody vedolizumab is approved by the FDA for treating moderate-to-severe ulcerative colitis and Crohn's disease. Vedolizumab binds to α4β7 on circulating memory/effector T cells in the blood and blocks their homing to intestinal tissues expressing the ligand MAdCAM-1. The aim of this study is to develop orally stable α4β7 antagonist peptides that act locally in the intestinal tissue. These peptides have minimal systemic exposure, yet are effective in blocking T cell homing and are efficacious in murine models of IBD. Potent, selective and orally stable peptide antagonists of α4β7 integrin were identified through Protagonist’s peptide and peptidomimetic technology platform. To evaluate oral stability, the peptides were incubated in a variety of ex vivo intestinal/colonic washes or simulated gastric/intestinal fluids, and half-lives determined by mass spectrometry. Pharmacokinetic (PK), pharmacodynamic (PD) and chronic colitis studies were conducted in mice. The peptides are potent against α4β7, but not α4β1 and αLβ2 as measured in biochemical and cell adhesion assays. In α4β7 specific cell adhesion assays, the peptides block adhesion of the human RPMI 8866 cell line (B cell lymphoblastoid) or mouse TK-1 (T cell lymphoblast) to immobilized MAdCAM-1 (IC50 < 20 nM). In the α4β1 or αLβ2 specific cell adhesion assay using human Jurkat cells, they are inactive up to the highest tested concentration, 100 μM. To facilitate oral delivery, we chemically engineered the peptides to be resistant to chemical and proteolytic degradation in a variety of gastric and intestinal fluids, while maintaining their potency and selectivity. PK studies in normal or dextran sodium sulfate (DSS) treated mice and rats showed that oral dosing results in exposure in the small intestine, colon and mesenteric lymph nodes (MLN), but no significant measurable levels in the blood and urine. A PD assay was used to assess the effect of oral dosing on trafficking of endogenous memory T cells in the mouse. Mice treated with DSS were orally dosed daily with peptides for 9 or 13 days, and harvested tissues were analyzed by FACS. FACS analysis of tissues from animals dosed with peptides showed that there is a reduction of CD4+ CD44high CD45RBlow β7+ T cells in the MLN and Peyer’s Patches, but not in the spleen or blood. There was also marked reduction of clinical disease symptoms. We also evaluated these peptides in a T cell adoptive transfer chronic colitis model. Daily oral dosing with peptides reduced the severity of disease as measured by colon weight length ratio and histology. Potent, selective and orally stable peptide antagonists of α4β7 integrin were shown in oral PK studies to have significant exposure in intestinal tissues, but not blood and urine. Despite low blood exposure, these peptides block T cell homing to gut associated lymphoid tissue and attenuate disease in murine models of IBD. These results support the therapeutic potential of locally blocking T cell homing while minimizing immunogenicity and the risk of opportunistic infections associated with systemically delivered immunosuppressants and biologics.