In hereditary hemochromatosis, hepcidin deficiency is a consequence of destructive mutations in the genes encoding hepcidin (24
) or one of its regulators, HFE (3
) (most common), transferrin receptor 2 (25
), or hemojuvelin (26
). Hepcidin deficiency results in excessive intestinal iron absorption and maldistribution of tissue iron. If severe and untreated, the disease can progress to cirrhosis, hepatocellular carcinoma, and endocrine and cardiac problems. In the US, about 0.4% of people of mixed European descent have hereditary hemochromatosis mutations that put them at risk for iron overload (27
Iron overload also occurs in β-thalassemia, a disease that affects millions of patients whose origins are in the Mediterranean basin and Asia, regions historically affected by endemic malaria (28
). In β-thalassemia, hepcidin is suppressed by increased erythropoietic activity through mechanisms that are still incompletely defined. In the absence of transfusions (β-thalassemia intermedia), hepcidin levels are very low (4
), and patients develop iron overload similar to that of severe hereditary hemochromatosis and may succumb to iron overload if untreated (29
). In β-thalassemia major, transfusions rather than dietary iron absorption are the predominant cause of iron overload. Here, hepcidin levels are higher, because ineffective erythropoiesis is suppressed by transfusions and also because the additional iron load stimulates hepcidin production. However, in the intervals between transfusions, hepcidin concentrations progressively decrease toward the end of the interval (4
), and this recurrent lowering of hepcidin may also contribute to iron overload in β-thalassemia major.
Milder hepcidin insufficiency is seen in chronic liver disease, including viral hepatitis (6
). The cause of hepcidin suppression in these diseases is not yet clear, but the resulting chronic iron loading in the liver worsens the prognosis (32
Therapeutic augmentation of hepcidin levels would be expected to curb hyperabsorption of dietary iron in these patients. The proof of this concept was achieved in mouse model of HFE hereditary hemochromatosis, in which the introduction of a hepcidin transgene into Hfe–/–
mice prevented the development of iron overload (33
). Hepcidin also causes redistribution of iron when iron overload is already established. Hepcidin induction in HFE-null mice carrying a Tet-inducible hepcidin construct did not acutely reverse the iron overload but did shift the excess iron from hepatocytes and other parenchymal cells to macrophages (34
). Macrophages are relatively resistant to the toxic effects of iron (35
), as demonstrated by the relatively benign course of “ferroportin disease,” in which iron accumulates in macrophages. Most affected patients have no clinical manifestations, despite often severe iron overload (36
). Thus, hepcidin-mediated redistribution of iron from parenchyma to macrophages in iron-overloaded patients could potentially limit iron toxicity in the heart, the pancreas, and the liver.
In addition to the beneficial effect of hepcidin on iron balance and distribution, recent studies suggest that hepcidin may also improve disordered erythropoiesis in β-thalassemia. Although the mechanism is still not understood, transgenic expression of hepcidin in a mouse model of β-thalassemia intermedia increased hemoglobin and decreased extramedullary erythropoiesis (37
Using full-length hepcidin for the treatment of iron overload conditions would be expected to be expensive, not only because the synthesis and refolding of hepcidin with 4 disulfide bonds allows for a large number of alternative folds but also because of the relatively high dose required for its biological effect. Even assuming that hepcidin could be produced at a cost comparable to that of recombinant insulin, a typical dose of hepcidin would likely be many fold higher than that of insulin. In human patients with type 1 diabetes mellitus (and mouse models), a typical dose of insulin is 0.2–0.7 U/kg/d or 9–32 μg/kg/d (1 U is the biological equivalent of 45.5 μg pure crystalline insulin). A typical dose of hepcidin, on the other hand, is 50 μg/mouse/d or 2 mg/kg/d, which is nearly 100-times higher than the typical dose of insulin. Given the current limitations of peptide synthetic technology, designing more potent and less expensive hepcidin analogs would be advantageous.
In this study, we developed small peptides, minihepcidins, that act as hepcidin agonists. Their rational design was facilitated by the identification of the region on hepcidin and ferroportin molecules that is critical for their binding. Together with our previous structure-function studies (16
), we showed that hydrophobic contacts dominate the ligand-receptor interaction and that several N-terminal amino acids of hepcidin, up to residue 9, are critical for its activity. We therefore used the first 9 residues as the scaffold for minihepcidin design. We also found that the ability to participate in thiol-thiol interactions is essential for minihepcidin activity. Blocking the only cysteine (C7) with a protective group abrogated the peptide activity unless the protective group was added in such a way that disulfide exchange was possible. Furthermore, in contrast to its Cys-containing counterpart, minihepcidin bearing the Cys to Ser substitution did not cause serum iron decrease in mice after intraperitoneal injection.
To improve agonist activity, we developed the minihepcidins with structural modifications that addressed some undesirable physicochemical properties of the minihepcidin scaffold, such as thiol instability, hydrophobicity, poor gastrointestinal absorption, susceptibility to proteolysis, and instability in the bloodstream. We succeeded in designing minihepcidins of 7– or 9–amino acids in length that were active in mice in vivo. Intraperitoneal injection of proteolysis-resistant retro-inverso minihepcidins caused hypoferremia similar to that caused by native hepcidin. In a proof-of-principle experiment, chronic administration of minihepcidins significantly decreased iron loading in a mouse model of hereditary hemochromatosis. Hepcidin-1 knockout mice, which received intraperitoneal injections of a retro-inverso minihepcidin daily for 2 weeks, had significantly lower liver iron content that hepcidin-1 knockout mice injected with solvent.
Importantly, minihepcidin conjugated to fatty or bile acids caused hypoferremia after oral administration by gavage also. Given the number and rich variety of peptides involved in biological processes, relatively few have been FDA approved as therapeutics, and their efficient delivery by the oral route is still an unmet goal. Development of oral hepcidin analogs would represent a major advance in peptide pharmacology.
The mainstay of treatment for patients with iron overload is phlebotomy if they are not anemic and iron chelation if they have anemia. Although these measures are effective at reducing excess iron, they are frequently not well tolerated by patients, and compliance with iron-depleting therapy is suboptimal. If hepcidin therapy proves to be effective and relatively free of side effects, it could represent a major improvement over existing therapies, either alone or in combination with current approaches to allow modifications that would make the treatment less burdensome and better accepted by patients.