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    • 专利摘要:
    MODIFIED MINI-HEPCIDINE PEPTIDES AND METHODS OF USE THEREOF.Peptides are disclosed here which exhibit hepcidin activity and methods for preparing and using them.
    公开号:BR112014013697A2
    申请号:R112014013697-1
    申请日:2012-12-06
    公开日:2020-11-03
    发明作者:Tomas Ganz；Elizabeta Nemeth；Piotr Ruchala
    申请人:The Regents Of The University Of California；
    IPC主号:
    专利说明:

    [1] [1] This application claims the benefit of US Patent Application Serial No. 61 / 568,724, filed on December 9, 2011, which is incorporated herein by reference in its entirety.
    [2] [2] This application is related to US Patent Application Serial No. 13 / 131,792, which is an entry in National Phase 371 of PCT / US2009 / 066711, filed on December 4, 2009, and the provisional US application Serial number 61 / 120.277, deposited on December 5, 2008, all of which are incorporated herein by reference in their entirety. GOVERNMENT SUPPORT NOTICE
    [3] [3] This invention was made with Government support NIH / NIDDK R01 DK090554 grant number, granted by the National Institutes of Health. The Government has certain rights in this invention. REFERENCE TO A SEQUENCE LISTING VIA EFS-WEB
    [4] [4] The contents of the ASCII text file of the sequence listing named “034044_097WO1_ST25”, which is 2.53 kb in size, was created on 1 November 2012 and electronically submitted via EFS-Web, attached to the application, is incorporated herein by reference in its entirety.
    [5] [5] BACKGROUND OF THE INVENTION
    [6] [6] 1. FIELD OF THE INVENTION.
    [7] [7] The present invention relates generally to peptides that show hepcidin activity.
    [8] [8] Hepcidin, a peptide hormone produced by the liver, is a regulator of iron homeostasis in humans and other mammals. Hepcidin acts by binding to its receptor, the ferroportin iron export channel, and generating its internalization and degradation.
    [9] [9] Abnormal hepcidin activity is associated with iron overload diseases, which include hereditary hemochromatosis and iron-loading anemias and myelodysplasia. Hereditary hemochromatosis (HH) is a genetic iron overload disease that is caused mainly by hepcidin deficiency, or very rarely by hepcidin resistance. This allows excessive absorption of iron from the diet and development of iron overload. Clinical manifestations of HH can include liver disease (liver cirrhosis, hepatocellular carcinoma), diabetes, and heart failure.
    [10] [10] Iron-loading anemias are hereditary anemias with ineffective erythropoiesis, such as β-thalassemia, which are accompanied by severe iron overload. Complications of iron overload are the main cause of morbidity and mortality in these patients. Hepcidin deficiency is the main cause of iron overload in non-transfused patients, and contributes to iron overload in transfused patients. The current treatment for iron overload in these patients is iron chelator which is very heavy, sometimes ineffective and accompanied by frequent side effects. SUMMARY OF THE INVENTION
    [11] [11] The present invention relates generally to peptides that show hepcidin activity and methods of using them.
    [12] [12] The present invention provides peptides, which can be isolated and / or purified, comprising, consisting essentially or consisting of the following structural formula IA or IB: where A1 is Asp, D-Asp, Glu, D-Glu, pyroglutamate, D- pyroglutamate, Gln, D-Gln, Asn, D-Asn, or an unnatural amino acid commonly used as a substitute for it, such as bhAsp, Ida, Ida (NHPal), and N-MeAsp, preferably Ida and N-MeAsp ; A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile, D-Ile, Ala, D-Ala or an unnatural amino acid commonly used as a substitute for it, such as Tle, Inp, Chg , bhThr, and N-MeThr; A3 is His, D-His, Asn, D-Asn, Arg, D-Arg, or an unnatural amino acid commonly used as a substitute for it, such as L-His (π-Me), D-His (π-Me ), L-His (τ-Me), or D-His (τ-Me); A4 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Trp, D-Trp, Tyr, D-Tyr, or an unnatural amino acid commonly used as a substitute for it, such as Phg, bhPhe,
    [13] [13] In some embodiments, the peptides form a cyclic structure through a disulfide bridge. In some embodiments, the peptides exhibit hepcidin activity. In some embodiments, the peptides bind ferroportin, preferably human ferroportin.
    [14] [14] In some embodiments, the present invention provides compositions and medicaments that comprise at least one peptide, which can be isolated, synthesized and / or purified, comprising, consisting essentially of or consisting of Structural Formula IA or IB as set forth herein. In some embodiments, the present invention provides a method of making medicaments to treat diseases of iron metabolism, such as iron overload diseases, which comprise at least one peptide, which can be isolated and / or purified, comprising , consisting essentially of or consisting of Structural Formula IA or IB as set forth herein.
    [15] [15] In some embodiments, the dose is administered as a weekly dose, for example, from 1-10,000 µg / kg / dose. In some embodiments, the daily dose is about 1-1,000, preferably about 10-500 µg / kg / day. Dosages may vary according to the type of formulation of the peptidyl drug administered, as well as the route of administration. A person skilled in the art will be able to adjust the dosage by changing the route of administration or formulation, so that the dosage administered should result in a similar pharmacokinetics or biological profile as could result from the preferred dosage ranges described herein. In some embodiments, the composition to be administered is formulated for oral, pulmonary or mucosal administration.
    [16] [16] Some modalities include any dosage with any route of administration which results in an effective pharmacokinetic and pharmacodynamic profile, reducing serum iron values by 10-80%. Some preferred doses include those that result from a desired reduction in serum iron. Administration of the peptidyl or protein formulations of the present invention includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, a doctor who instructs a patient to self-administer a drug and / or provides a patient with a prescription for a drug is considered to be administering the drug to the patient.
    [17] [17] In some embodiments, the present invention provides methods of attaching a ferroportin or inducing internalization and degradation of ferroportin, which comprises contacting ferroportin with at least one peptide or a composition as disclosed herein.
    [18] [18] In some embodiments, the present invention provides kits that comprise at least one peptide or composition as disclosed herein packaged together with a reagent, device, instructional material, or a combination thereof.
    [19] [19] In some embodiments, the present invention provides complexes comprising at least one peptide as disclosed herein attached to a ferroportin, preferably a human ferroportin, or an antibody, such as an antibody that specifically binds to a peptide as disclosed herein, Hep25 , or a combination thereof.
    [20] [20] In some embodiments, the present invention provides the use of at least one peptide, which can be isolated and / or purified, comprising, consisting essentially or consisting of Structural Formula IA or IB, as defined herein, or a composition comprising , consisting essentially of, or consisting of said at least one peptide to manufacture a drug to treat a disease of iron metabolism and / or decrease the amount of iron, in a subject in need of the same, in which the drug is prepared for be administered in an effective daily dose, as a single daily dose, or in divided daily doses. In some embodiments, the dose is about 1-1,000, preferably about 10-500 µg / kg / day. In some modalities, the drug is formulated for subcutaneous injection or oral, pulmonary or mucosal administration.
    [21] [21] Both the previous general description and the following detailed description are exemplary and explanatory only and are intended to provide a further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated and form part of this specification, illustrate various embodiments of the invention and, together with the description serve to explain the principles of the invention. DESCRIPTION OF THE DRAWINGS
    [22] [22] The present invention is best understood with reference to the drawings in which:
    [23] [23] Figure 1 is a graph showing the relative hepcidin activity of alanine substitutions in Hep25.
    [24] [24] Figure 2A is a graph showing the relative hepcidin activities of F4 substitutions in Hep25.
    [25] [25] Figure 2B is a graph showing the relative hepcidin activities of F9 substitutions in Hep25.
    [26] [26] Figure 3A is a graph showing the activities of hepcidin Hep1-9 and Hep1-10 C7A in relation to Hep25 (A).
    [27] [27] Figure 3B is a graph showing the activities of hepcidin Hep1-7 and Hep1-8 in relation to Hep 1-9 or Hep25.
    [28] [28] Figure 3C is a graph showing the activities of hepcidin Hep4-7, Hep3-7, Hep3-8 and Hep3-9 in relation to Hep25.
    [29] [29] Figure 4 is a graph showing the hepcidin activities of modified C7 peptides in relation to Hep25 and Hep 1-9.
    [30] [30] Figure 5 is a graph showing the in vivo effect (as measured by serum iron levels in mice) of mini-hepcidins Hep 1-9, PR6 and PR12 compared to Hep25 or control (PBS). The peptides were injected intraperitoneally, 50 µg of peptide per mouse.
    [31] [31] Figure 6 is a graph showing the in vivo effect (as measured by serum iron levels in mice) of intraperitoneally injected mini-hepcidin PR27 (20 and 200 nmoles). The amount of Hep25 injected was 20 nmoles.
    [32] [32] Figure 7 is a graph showing the in vivo effect (as measured by serum iron levels in mice) of intraperitoneally injected mini-hepcidin riHep7∆DT (20 and 200 nmoles). The amount of Hep25 injected was 20 nmoles.
    [33] [33] Figure 8 is a graph showing the in vivo effect (as measured by serum iron levels in mice) of mini-hepcidins PR27 and PR28 that were first mixed with liposomes and injected intraperitoneally (20 nmoles). The amount of Hep25 injected was 20 nmoles.
    [34] [34] Figure 9 is a graph showing the in vivo effect
    [35] [35] Figures 10A-10C show mini-hepcidin PR65 and its activity in wild type mice. Figure 10A shows the structural formula of PR65. Ida = iminodiacetic acid, Dpa = diphenylalanine, bhPro = beta-homo proline, bhPhe = beta-homo phenylalanine. Figure 10B shows serum iron in C57BL / 6 wild-type mice, 4 hours after intraperitoneal injection of solvent, native hepcidin or PR65 (n = 4-8 in each group). ** p = 0.01, * p = 0.005. Figure 10C shows serum iron in C57BL / 6 wild type mouse 4 hours after intraperitoneal or subcutaneous injection of 20 nmoles of PR65 (n = 4 in each group). * p = 0.007, ** p = 0.04. In Figures 10B and 10C the bars represent the mean values and standard deviations of error bars.
    [36] [36] Figures 11A and 11B show the hypoferremic effect of PR65 in iron-loaded hepcidin knockout mice. Figure 11A shows that PR65 induced a dose-dependent reduction in serum iron within 24 hours after a subcutaneous injection. Mean values and standard deviations are shown, n = 3-5 mice per point. #p = 0.005, & p = 0.004, * p <0.001. Figure 11B shows the time course of hypoferremia induced by a subcutaneous injection of 100 nmoles of PR65. Mean and standard deviations are shown, n = 4-6 mice per point. #p = 0.008, * p <0.001.
    [37] [37] Figure 12 shows changes in iron distribution in PR65-treated hepcidin knockout mice. Tissue iron was visualized by increased Perls increased 0-48 hours after subcutaneous injection of PR65 (100 nmoles). Representative images are shown. The horizontal bars indicate 400 µm (10X) and 100 µm (40X).
    [38] [38] Figures 13A-13E show that PR65 prevented iron loading in iron-depleted hepcidin knockout mice. All mice were placed on an iron-deficient diet (4 ppm of iron) between the ages of 5-6 weeks up to 12 weeks. The “baseline” group (n = 7) was examined at 12 weeks of age (white bars). The rest of the mice were fed an iron loading diet (300 ppm) for an additional 2 weeks, while receiving daily subcutaneous injections of solvent (gray bars, n = 6) or PR65, at 20, 50 or 100 nmoles per day ( black bars, n = 4 per dose). The mice were analyzed 24 hours after the last injection. Compared to the solvent, PR65 injections resulted in: Figure 13A - iron retention in the spleen; Figure 13B - a dose-dependent decrease in serum iron; Figure 13C - a dose-dependent decrease in Hb levels; Figure 13D - a decrease in iron in the heart at higher doses; and Figure 13E - decreased iron in the liver. Iron content in the liver in mice injected with PR65 did not differ significantly from the base group of mice, indicating that little or no new iron was absorbed or deposited in the liver during a 2-week treatment. The graphs show mean and standard deviation. Student's t test was used to compare the mean of each condition to that of the solvent treatment (p-value on bars).
    [39] [39] Figure 14 shows the cellular distribution of iron after 2 weeks of PR65 injections to prevent iron overload. Representative images are shown. The horizontal bars indicate 400 µm (10X) and 100 µm (40X). Iron accumulation was observed in the red pulp of the spleen of mice treated with PR65, but not with mice treated with solvent. Likewise, the accumulation of iron in duodenal enterocytes was observed only in mice treated with PR65. Compared to iron staining in the heart of mice injected with solvent, there was less accumulation of iron in the heart of animals injected with 50 and 100 nmoles of PR65, according to the quantitative method in Figure 4. Loading of iron into the liver in mice treated with 20 and 50 nmoles of PR65 was similar to the baseline group and much less than the iron loading in the solvent treated group. At the highest dose of PR65, liver iron was lower than at the beginning of the study, indicating that the mice were able to mobilize liver iron despite the high activity of mini-hepcidin.
    [40] [40] Figures 15A-15E show that the two-week treatment with PR65 of iron-loaded hepcidin knockout mice caused the most modest redistribution of iron. Hepcidin knockout mice were maintained on a 300 ppm iron diet for their entire life span.
    [41] [41] Figure 16 shows the cellular distribution of iron after 2 weeks of PR65 injections to treat established iron overload. The tissue sections correspond to the animals analyzed in Figures 15A-15E, with representative images shown. The horizontal bars indicate 400 µm (10X) and 100 µm (40X). Improved Perls staining confirmed that spleen macrophages and duodenal enterocytes retained iron in mice treated with PR65, but not in those treated with solvents. In comparison with the solvent-treated controls, less intense iron staining was observed in the liver of mice treated with PR65. No consistent difference between solvent-treated and PR65 mice was observed in the heart sections (not shown).
    [42] [42] Figure 17 shows some of the structures of the molecules cited in Tables 1, 3 and 4. DETAILED DESCRIPTION OF THE INVENTION
    [43] [43] The present invention provides peptides that are useful in the study and treatment of diseases of iron metabolism.
    [44] [44] As used herein, an “iron metabolism disease” includes diseases where abnormal iron metabolism directly causes the disease, or where blood iron levels are unregulated causing the disease, or when iron deregulation is a consequence of another disease, or where diseases can be treated by modulating iron levels, and the like. More specifically, an iron metabolism disorder according to that disclosure includes iron overload diseases, iron deficiency disorders, iron biodistribution disorders, other iron metabolism disorders and other diseases potentially related to iron metabolism, etc. Iron metabolism disorders include hemochromatosis, HFE mutation hemochromatosis,
    [45] [45] In some cases, diseases and disorders included in the definition of “iron metabolism disease” are not typically identified as being related to iron. For example, hepcidin is highly expressed in the murine pancreas suggesting that diabetes (Type I or Type II), insulin resistance, glucose intolerance and other disorders can be ameliorated by treating underlying disorders of iron metabolism. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26, which is incorporated herein by reference. As such, these diseases fall within the scope of the broad definition. Those skilled in the art are readily able to determine whether a given disease is an "iron metabolism or disease" according to the present invention, using methods known in the art, including the tests of WO 2004092405, which is incorporated herein by reference, and of assays that monitor hepcidin, hemojuvelin, or iron and expression levels, which are known in the art, such as those described in US Patent No. 7,534,764, which is incorporated herein by reference.
    [46] [46] In preferred embodiments of the present invention, iron metabolism diseases are iron overload diseases, which include hereditary hemochromatosis, iron loading anemias, alcoholic liver diseases and chronic hepatitis C
    [47] [47] As used herein, the terms "protein", "polypeptide" and "peptide" are used interchangeably to refer to two or more amino acids linked together. Except for the abbreviations for unusual or unnatural amino acids indicated in Table 2, below, the three letter and one letter abbreviations, as used in the art, are used here to represent the amino acid residues. Except when preceded by "D-", the amino acid is an L-amino acid. Groups or chains of amino acid abbreviations are used to represent the peptides. Unless otherwise indicated, peptides are indicated with the N-terminus on the left and the sequence is written from the N-terminus to the C-terminus.
    [48] [48] The peptides of the present invention can be made using methods known in the art, including chemical synthesis (solid phase, solution phase, or a combination of both), biosynthesis or in vitro synthesis using recombinant DNA methods, see , for example, Kelly & Winkler (1990) Genetic Engineering Principles and
    [49] [49] In some embodiments, the peptides of the present invention are substantially purified. As used herein, a "substantially purified" compound
    [50] [50] As used herein, an "isolated" compound refers to a compound that is isolated from its native environment. For example, an isolated peptide is one that lacks its native amino acids, which correspond to the full-length polypeptide, flanking the N-terminal, C-terminal, or both. For example, isolated Hep1-9 refers to an isolated peptide comprising amino acid residues 1-9 of Hep25, which may have non-native amino acids at their N-terminal, C-terminal end, or both, but have no a cysteine amino acid residue after its 9th amino acid residue at the C-terminal. As stated here, references to the amino acid positions correspond to the amino acid residues of Hep25. For example, reference to amino acid position 9, corresponds to the 9th amino acid residue of Hep25.
    [51] [51] The peptides of the present invention bind ferroportin, preferably human ferroportin. The preferred peptides of the present invention specifically bind to human ferroportin. As used herein, "specifically bind" refers to the preferred interaction of a specific binding agent with a given binder over the other agents in a sample. For example, a specific binding agent that specifically binds to a given linker, binds to the determined linker, under suitable conditions, of an amount, or a level that is observable over that of any non-specific interaction with other components present in the sample . The suitable conditions are those that allow the interaction between a specific binding agent and a given ligand.
    [52] [52] The peptides of the present invention, which simulate the activity of Hep25 hepcidin, the bioactive human form of 25-amino acids, are referred to herein as "mini-hepcidins".
    [53] [53] In some embodiments, the peptides of the present invention have in vitro activity as tested for their ability to cause ferroportin to internalize and degrade in a cell line expressing ferroportin as taught in Nemeth et al. (2006) Blood 107: 328-33. In vitro activity can be measured by the dose-dependent loss of fluorescence of cells modified to exhibit ferroportin fused to the green fluorescent protein, as in Nemeth et al. (2006) Blood 107: 328-33.
    [54] [54] Other methods known in the art can be used to calculate hepcidin activity and in vitro peptide activity according to the present invention.
    [55] [55] Previous studies indicate that the N-terminal segment of Hep25 is important for its hepcidin activity and is likely to form the contact interface with ferroportin. However, the importance of each of the N-terminal amino acids for hepcidin activity was unknown. Therefore, alanine scan mutagenesis was performed on residues 1-6 of Hep25 to determine the contribution of each N-terminal amino acid to hepcidin activity. As shown in Figure 1, the T2A substitution did not substantially impact hepcidin activity.
    [56] [56] To determine whether highly conserved and apparently structurally important phenylalanine F4 is important for hepcidin activity, the amino acid F4 of Hep25 has been systematically replaced with other amino acids. As shown in Figure 2A, making the side chain more polar (F4Y) led to a substantial loss of hepcidin activity as did the substitution with D-phenylalanine (f), or charged amino acids (D, K and Y). However, hepcidin activity was maintained when residue F4 was replaced with non-aromatic cyclohexylalanine, thus indicating that a bulky hydrophobic residue is sufficient for the activity.
    [57] [57] To determine whether the highly conserved and apparently structurally important F9 phenylalanine is important for hepcidin activity, the amino acid F9 of Hep25 has been replaced by other amino acids. As shown in Figure 2B, hepcidin activity not only decreased when F9 was replaced with alanine, but also when it was replaced with non-aromatic cyclohexylalanine, thus indicating that an aromatic residue may be important for the activity.
    [58] [58] Mutational studies indicate that C326, the cysteine residue at position 326 of human ferroportin, is the critical residue involved in binding to hepcidin. Thus, several N-terminal fragments of Hep25 containing a thiol, for example, Hep4-7, Hep3-7, Hep3-8, Hep3-9, Hep1-7, Hep1-8, Hep1-9, and Hep1-10 C7A, were chemically synthesized, refolded and their activities in relation to Hep25 were analyzed using flow cytometry quantification of ferroportin-GFP degradation, estimation of iron efflux based on cellular ferritin measurements, and radioisotope iron efflux studies. The sequences and EC50 of these N-terminal fragments are shown in Table 1.
    [59] [59] Surprisingly and unexpectedly, as shown in Figure 3, Hep1-9 and Hep1-10 C7A were found to be very active in the ferroportin-GFP internalization flow cytometry assay. On a mass basis, Hep1-9 and Hep1-10 C7A were only about four times less potent and on a molar basis, about 10 times less potent than Hep25. Thus, Hep1-9 and Hep1-10 C7A were used as a basis for the construction of other peptides that have hepcidin activity.
    [60] [60] To determine the importance of cysteine thiol in Hep 1-9 hepcidin activity, Hep 1-9 C7 residue has been replaced with amino acids that have a similar shape, but disulfide bonds cannot be formed to generate Hep9-C7S (serine replacement) and Hep9C7-tBut
    [61] [61] Other peptides based on Hep1-9 and Hep1-10 C7A were constructed to be cyclized F4 and F9 disulfide residues, have unnatural amino acid substitutions, are retroinverted, modified, or have a positive charge. The C-terminal amino acid was in the amidated form. The resulting changes and hepcidin activities are shown in Table 1.
    [62] [62] As shown in Table 1, with the exception of PR40 and PR41, mini-hepcidins that exhibit EC50 of about 1000 nM or less, contain at least 6 contiguous amino acid residues, which correspond to residues 3-8 of Hep25 ( see Hep3-8). Thus, in some embodiments, the preferred mini-hepcidins have at least 6 contiguous amino acid residues, which correspond to 6 contiguous Hep1-9 amino acid residues, preferably 3-8 residues. Amino acid residues can be unnatural or unusual amino acids, L- or D-amino acid residues, modified residues, or a combination thereof.
    [63] [63] In some embodiments, the mini-hepcidins of the present invention have at least one amino acid substitution, modification, or addition. Examples of amino acid substitutions include substituting an L-amino acid residue for its corresponding D-amino acid residue, substituting Cys for homocysteine, Pen, (D) Pen, Inp, or the like, replacing Phe with bhPhe, Dpa, bhDpa, Beep, 1Nal and the like. The names and structures of the substitution residues are exemplified in Table 2. Other suitable substitutions are exemplified in Table 1. Examples of modification include the modification of one or more amino acid residues in such a way that the peptide forms a cyclic structure, retroinversion, and modifying a residue being able to form a disulfide bond. Examples of an addition include the addition of at least one amino acid residue or at least one compound, at N-terminal, C-terminal, or both, as exemplified in Table 1.
    [64] [64] As shown in Table 1, most mini-hepcidins exhibiting EC50 of about 100 nm or less, contain at least one Dpa or bhDpa amino acid substitution. Thus, in some embodiments, the mini-hepcidins of the present invention have at least one Dpa or bhDpa amino acid substitution.
    [65] [65] Taking into account the alanine substitution data of Figure 1, in some embodiments, the mini-hepcidins of the present invention may have an Ala at amino acid positions in addition to amino acid positions 4 and 9, provided that a thiol is available to form a disulfide bond at the amino acid position 7. See Hep9F4A and Hep9C-SStBut in Table 1.
    [66] [66] Taking into account the amino acid substitution data of position 4 of Figure 2 and Table 1, the minihepcidins of the present invention can have an amino acid substitution in position 4, which do not result in a substantial change in their charge or polarity when compared to that of Hep25, Hep1-9 or Hep1-10 C7A.
    [67] [67] The original mini-hepcidins as mentioned herein have the following Structural Formula I where A1 is Asp, Glu, pyroglutamate, Gln, Asn, or an unnatural amino acid commonly used as a substitute for it;
    [68] [68] In some modalities, the A1 is Asp; A2 is Thr; A3 is His; A4 is Phe; A5 is Pro; A6 is Ile; A7 is Ala; A8 is Ile; A9 is Phe; and A10 is Cys in the form of an amide; where A1 or A1 to A2 are optionally absent.
    [69] [69] In some embodiments, A1 is Asp, A2 is Thr, A3 is His, A4 is Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural amino acid thiol, A8 is Ile, A9 is Phe in amide form, and A10 is absent.
    [70] [70] In some embodiments, A1 and A2 are absent, A3 is His, A4 is Phe, A5 is Pro, A6 is Ile, A7 is Cys or a non-natural amino acid thiol, A8 is Ile in the form of amide, and A9 and A10 are absent.
    [71] [71] In some embodiments, A1 and A2 are absent, A3 is His, A4 is Phe, A5 is Pro, A6 is Ile, A7 is Cys or an unnatural thiol amino acid in the form of amide, and A8 to A10 are absent.
    [72] [72] In some embodiments, the unnatural A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, or a combination thereof is the corresponding D-amino acid. For example, for A1, the unnatural amino acid can be D-
    [73] [73] In some embodiments, the unnatural amino acid of: A1 is D-Asp, D-Glu, D-pyroglutamate, D-Gln, D-Asn, bhAsp, Ida, or N-MeAsp; A2 is D-Thr, D-Ser, D-Val, Tle, Inp, Chg, bhThr, or N-MeThr; A3 is D-His, D-Asn, D-Arg, Dpa, (D) Dpa, or 2-aminoindan; A4 is D-Phe, D-Leu, D-Ile, D-Trp, Phg, bhPhe, Dpa, Bip, 1Nal, bhDpa, Amc, PheF5, hPhe, Igl, or cyclohexylalanine; A5 is D-Pro, D-Ser, Oic, bhPro, trans-4-PhPro, cis-4-PhPro, cis-5-PhPro, Idc; A6, D-Ile, D-Leu, Phg, Chg, Amc, bheIle, Ach, and N-MeIle; A7 is D-Cys, D-Ser, D-Ala, Cys (S-tBut), homoCys, Pen, (D) Pen, Dap (AcBr), and Inp; A8 is D-Ile, D-Leu, D-Thr, D-Val, D-Arg, Chg, Dpa, bhIle, Ach, or N-MeIle; A9 is D-Phe, D-Leu, D-Ile, PheF5, N-MePhe, benzalimide, bhPhe, Dpa, Bip, 1Nal, bhDpa, cyclohexylalanine; and A10 is D-Cys, D-Ser, D-Ala.
    [74] [74] In some embodiments, amino acid substitution (and addition, if indicated) for: A1 is Ala, D-Ala, Cys, D-Cys, Phe, D-Phe, Asp or D-Asp linked to Cys or D -Cys, Phe or D-Phe linked to a PEG molecule linked to kenodeoxycholate, ursodeoxycholate, or palmitoil, or Dpa or (D) Dpa linked to palmitoil; A2 is Ala, D-Ala, Cys, D-Cys, Pro, D-Pro, Gly, D-Gly; A3 is Ala, D-Ala, Cys, D-Cys, Dpa, Asp or D-Asp linked to Dpa or (D) Dpa; A4 is Ala, D-Ala, Pro, or D-Pro; A5 is Ala, D-Ala, Pro, D-Pro, Arg, D-Arg; A6 is Ala, D-Ala, Phe, D-Phe, Arg, D-Arg, Cys, D-Cys; A7 is His, or D-His; A8 is Cys or D-Cys; and A9 is Phe or D-Phe linked to RA, Asp, D-Asp, Asp or D-Asp linked to RB, bhPhe linked to RC, or cysteamide, where RA is -CONH2-CH2-CH2-S, -D -Pro connected to Pro-Lys or Pro-Arg, - bhPro connected to Pro connected to Pro-Lys or Pro-Arg-, D-Pro connected to bhPro-Lys or bhPro-Arg, where RB is -PEG11- GYIPEAPRDGQAYVRKDGEWVLLSTFL, - (PEG11) - (GPHyp) 10, and where RC is -D-Pro linked to Pro-Lys or Pro-Arg, -D-Pro linked to bhPro-Lys or bhPro-Arg.
    [75] [75] In some embodiments, minihepcidine is a 10-mer sequence where A7 is Ala and A10 is Cys.
    [76] [76] In some embodiments, minihepcidines form a cyclic structure through a disulfide bond.
    [77] [77] In some embodiments, mini-hepcidin is a retroinverted peptide such that A1 is the C-terminal and A10 is the N-terminal and the amino acid residues are D-
    [78] [78] In some embodiments, mini-hepcidin has an amino acid substitution at position 4, position 9, or both. In some embodiments, the amino acid substituent is Phg, Phe, D-Phe, bhPhe, Dpa, Bip, 1Nal, Dpa, bhDpa, Amc, or Cysteamide.
    [79] [79] In some embodiments, the mini-hepcidin has an amino acid substitution at position 7. In some embodiments, the amino acid substituent is Cys (S-tBut), Ala, D-Ala, Ser, D-Ser, homoCys, Pen, (D) Pen, His, D-His, or Inp.
    [80] [80] Examples of some original mini-hepcidins are given in Table 1.
    [81] [81] Hep25 was synthesized at the UCLA Peptide Synthesis Core Facility using solid phase 9 fluorenylmethyloxycarbonyl (fmoc) chemistry. Specifically, the peptides were synthesized on a peptide synthesizer
    [82] [82] The other peptides set out in Table 1 were synthesized by the solid phase method using the automated Symphony® peptide synthesizer (Protein Technologies Inc., Tucson, AZ) or CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC), applying 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry (Fields & Noble (1990) Int J Pept Protein Res. 35: 161-214) and commercially available amino acid derivatives and reagents (EMD Biosciences, San Diego, CA and Chem-Impex International, Inc., Wood Dale, IL). The peptides were cleaved from the resin using the modification reagent K (94% TFA (v / v); phenol, 2% (w / v), water, 2% (v / v); TIS, 2% (v / v), 2 hours) and precipitated by adding ice-cooled diethyl ether. Subsequently, the peptides were purified by preparative reverse phase high-performance liquid chromatography (RP-HPLC) at> 95% homogeneity and their purity assessed by matrix assisted laser desorption ionization spectrometry (MALDI-MS, UCLA Mass Spectrometry Facility, Los Angeles, CA), as well as analytical RP-HPLC using a Varian ProStar 210 HPLC system equipped with a ProStar 325 dual wavelength UV-Vis detector with the expected wavelengths of 220 nm and 280 nm ( Varian Inc., Palo Alto, CA).
    [83] [83] Other methods known in the art can be used to synthesize or obtain the peptides according to the present invention. All peptides were synthesized as carboxylamides (-CONH2) which creates a neutrally charged end more similar to a peptide bond than the negatively charged -COOH end. However, peptides having the negatively charged -COOH end are contemplated herein. ACTIVITY TESTS
    [84] [84] FLOW CYTOMETRY. The peptide activity of the present invention was measured by flow cytometry as previously described. See Nemeth et al. (2006) Blood 107: 328-333, which is incorporated herein by reference.
    [85] [85] FERRITINE TEST. Cells treated with peptides having hepcidin activity will retain iron and contain higher amounts of ferritin. Thus, the following ferritin assay can be used to identify the mini-hepcidins according to the present invention. Briefly, HEK293-Fpn cells are incubated with 20 µΜ FAC with or without 10 µΜ ponasterone.
    [86] [86] IN VIVO TESTS. Test of serum iron. The decrease in serum iron after peptide administration is the main measure of hepcidin activity. Thus, as provided herein, the hepcidin activity of selected peptides of the present invention was assessed in vivo, by measuring iron in the serum of test subjects.
    [87] [87] As illustrated in Figure 5, by intraperitoneal (i.p.) administration of 50 µg of PR12 per mouse in PBS caused a significant decrease in serum iron after 4 hours when compared to i.p. of PBS. The decrease in serum iron was similar to that caused by i.p. 50 mg of Hep25. Injection (i.p.) of Hep9 did not result in a decrease in iron in the serum.
    [88] [88] As illustrated in Figure 6, i.p.
    [89] [89] As illustrated in Figure 7, i.p.
    [90] [90] As shown in Figure 8, i.p.
    [91] [91] As shown in Figure 9, oral administration of PR27 200 nmoles by gavage in a Cremophor EL solution caused a decrease in serum iron in mice, compared to oral administration of PBS in the same formulation. Cremophor EL increases the solubility of chemicals, and is often used as an excipient or additive in drugs. Cremophor EL solution was prepared by mixing Cremophor EL (Sigma), ethanol and PBS in a 12.5: 12.5: 75 ratio. 250 µl of the solution was administered by gavage to the mice.
    [92] [92] Thus, the present invention can be used to decrease serum iron in subjects. A preferred mini-hepcidin according to the present invention is a retroinverted peptide comprising a PEG molecule, such as PEG11, attached to its N-terminal amino acid. In some embodiments, the PEG molecule is linked to a palmitoyl group or diaminopropionic acid linked to one or more palmitoyl groups.
    [93] [93] In addition to analyzing the effect on serum iron content, other in vivo assays known in the art can be conducted to identify mini-hepcidins according to the present invention and / or determine the therapeutically effective amount of a particular peptide or mini-hepcidin according to the present invention. Examples of such tests include the following:
    [94] [94] Tissue iron test. In addition to, or instead of, iron assay in the above serum, the iron distribution in the tissue can be determined by improved Perl staining of liver and spleen sections obtained from treated mice. Briefly, the tissue sections are fixed in 4% paraformaldehyde / PBS, incubated in Perl solution (1: 1.2% HCl and 2% potassium ferrocyanide) and diaminobenzidine in 0.015% hydrogen peroxide. Non-heme tissue iron can be quantified using the micromethod of Rebouche et al. (2004) J Biochem Biophys Methods. 58 (3): 239-51; Pak et al. (2006) Blood 108 (12): 3730-5. Pieces of 100 mg of liver and spleen are homogenized and acid is added to release non-heme bound iron, which is detected by colorimetric reaction using ferrozine and compared to controls. Treatment with mini-hepcidins would be expected to cause redistribution of iron from other spleen tissues.
    [95] [95] Hematological tests. Hematological assays can be used to identify minihepcidins according to the present invention and / or to determine the therapeutically effective amount of a particular peptide or minihepcidin according to the present invention. Briefly, the blood of treated subjects is collected in tubes containing heparin. Hemoglobin, RBC, MCV, EPO, white cell parameters, reticulocyte count and reticulocyte Hb content are determined using methods known in the art and compared to controls. Treatment with mini-hepcidins would be expected to cause a decrease in MCV and decrease the Hb content of reticulocytes. Administration of mini-hepcidins in excessive amounts would be expected to decrease Hgb.
    [96] [96] IRON EXPORT TESTS. Iron export assays (55Fe) known in the art using primary hepatocytes and macrophages can be used to identify the mini-hepcidins according to the present invention and / or to determine the therapeutically effective amount of a particular peptide or mini-hepcidin according to the present invention. Peptides with hepcidin activity will decrease or reduce the release of 55Fe from cells. Briefly, the cells are incubated with 55Fe-NTA or 55Fe-Tf for 36 hours. After elimination of unincorporated 55Fe, cells are treated with a given peptide or a control. In the case of ferroportin mutants, transfection is performed before the addition of 55Fe and expression is allowed to continue during the 36 hour iron loading period. Aliquots of the medium are collected after 1, 4, 8, 24, 36, 48 and 72 hours and the radioactivity is determined by a scintillation counter. Cell-associated radioactivity can be measured by centrifuging the cells using silicone oil to decrease the non-specific binding of radiolabelled iron to the cells using methods known in the art.
    [97] [97] To determine whether a given peptide modifies the internalization and degradation of endogenous ferroportin, the levels of protein and cellular distribution of ferroportin in hepatocytes and macrophages treated with the peptide can be tested using Western blotting, immunohistochemistry and ferroportin antibodies known in technical. MODIFIED MINI-HEPCIDINES
    [98] [98] Additional minihepcidins according to the present invention are shown in Table 3 and Table 4 as follows:
    [101] [101] In Tables 3 and 4, PR47, PR48, PR49, PR50, PR51, PR52, PR76, PR77, PR78, and PR79 are retroinverted mini-hepcidins and are shown, from left to right, from their C end -terminal to the N-terminal end, to exemplify the alignment between amino acid residues and residues 1-10 of Hep25. Thus, the conventional mention of these retroinverted mini-hepcidins from their N-terminals to their C-terminals are as follows (the D-amino acids are underlined):
    [102] [102] As shown in Table 4, the route of administration may play a role in the activity of the determined mini-hepcidin (compare, for example, PR65 and PR66). Thus, the indication of no activity for some of the minihepcidins in Tables 3 and 4 should not be interpreted as an indication that a certain minihepcidin has no activity in any route of administration and / or dosage. In fact, as shown in Table 3, very few of these mini-hepcidins exhibit significant activity in vitro at considerably lower dosages than the original mini-hepcidins.
    [103] [103] These additional mini-hepcidins are modifications of the mini-hepcidins as established in PCT / US2009 / 066711 (hereinafter referred to as “original mini-hepcidins” and with structural formula I). As used herein, mini-hepcidins are modifications of the original mini-hepcidins which are referred to herein as "modified" mini-hepcidins. As used herein, "mini-hepcidins" refers to both the original mini-hepcidins and modified mini-hepcidins. Mini-hepcidins modified according to the present invention have the following structural formula IA or IB: A1 is Asp, D-Asp, Glu, D-Glu, pyroglutamate, D-pyroglutamate, Gln, D-Gln, Asn, D-Asn , or an unnatural amino acid commonly used as a substitute for them, such as bhAsp, Ida, Ida (NHPal), and N-MeAsp, preferably Ida and N-MeAsp; A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile, D-Ile,
    [104] [104] Five of the modified mini-hepcidins in Table 4 contain tryptophan in order to facilitate measurements of their concentrations. Thus, in some embodiments, the modified minihepcidins of the present invention contain tryptophan. In some embodiments, tryptophan residues are eliminated or replaced with another amino acid. Other modified mini-hepcidins were made by modifying the original mini-hepcidins, replacing the two isoleucines flanking the cysteine with arginines or arginine derivatives. Unexpectedly, it was found that the replacement of these isoleucines with arginines resulted in mini-hepcidins with increased activity. The unexpectedly higher activity is believed to be the result of the presence of arginine in the 6th position of the amino acid and / or the 8th position of the amino acid. Thus, in some embodiments, the amino acid residue at position A6 and / or A8 of formula I is arginine. Additional modifications of such mini-hepcidins also resulted in unexpectedly higher activities. The mini-hepcidins modified in accordance with the present invention show an unexpectedly higher activity compared to the modified mini-hepcidins exemplified in USSN 13 / 131,792.
    [105] [105] In some embodiments, the N-terminal amino acid has a free amine. In some embodiments, the N-terminal amine is blocked with a group that removes the charge, preferably an acetyl group or a formal group.
    [106] [106] In some embodiments, the side chain of amino acid A1 can be modified, as indicated for the N-terminal amine. For example, the free carbonyl group at amino acid A1 can be modified, for example, blocked by an acyl group, such as palmitoyl (see PR71).
    [107] [107] In some embodiments, A4 is a bulky hydrophobic amino acid such as Phe, Tyr, Trp, Leu, or Ile or any unnatural amino acid commonly used as a substitute for it containing preferably four or more carbon atoms in its side chain , a cyclic structure, such as Phg, Bip, 1Nal, 2Nal, Amc, PheF5, Igl (L-2-indanylglycine) or Cha (L-cyclohexylalanine), preferably Dpa. In some embodiments, A4 contains the beta-homo form of the hydrophobic roughage amino acids above, for example, bhPhe, or bhDpa. Other changes in the side chain include aromatic substituents, such as those disclosed in Wang et al. (2002) Tetrahedron 58: 3101-3110 and Wang et al. (2002) Tetrahedron 58: 7365-7374. In some embodiments, the A4 residue is a D-amino acid.
    [108] [108] In some embodiments, the amino acid at position A9 is a bulky hydrophobic amino acid such as Phe, Tyr, Trp, Leu, or Ile or any unnatural amino acid commonly used as a substitute for those containing four or more carbon atoms in its side chain, preferably a cyclic structure, such as Phg, Dpa, Bip, 1Nal, 2Nal, Amc, PheF5, Igl or Cha, as a cyclic or aromatic group containing one or more aromatic or substituent rings. In some embodiments, residue A9 is Dpa or Trp.
    [109] [109] In some embodiments, the mini-hepcidins of the present invention are modified or formulated in order to maintain and / or increase their bioavailability in vivo.
    [110] [110] The modified minihepcidins were demonstrated in a mouse model of hemochromatosis whose daily administration of the modified minihepcidins, for example, PR65, prevented iron overload. Therefore, modified mini-hepcidins according to the present invention, alone or in combination with one or more original mini-hepcidins, can be administered to individuals in order to treat, for example, inhibit and / or reduce, the overload of iron in individuals, like humans. Therefore, original and modified mini-hepcidins according to the present invention can be used in medications and treatments to treat iron overload disorders, for example, beta-thalassemia and hereditary hemochromatosis, by inhibiting and / or reducing iron overload. in subjects. In some embodiments, at least a modified mini-hepcidin and / or at least an initial mini-hepcidin is administered to subjects before, during, after, or a combination of them, the symptoms of iron overload are observed and / or be diagnosed as having an iron overload disorder.
    [111] [111] Thus, in some embodiments, one or more modified mini-hepcidins, alone or in combination with one or more original mini-hepcidins, is provided in the form of a composition comprising a vehicle suitable for its intended purpose. The compositions can also include one or more additional ingredients suitable for the intended purposes. For example, for assays, compositions can include liposomes, niclosamide, solubilizing agent SL220 (NOF, Japan), cremophor EL (Cysma), ethanol, and DMSO. To treat an iron overload disease, the compositions may comprise different absorption promoters and protease inhibitors, solid microparticles or nanoparticles for peptide encapsulation (such as chitosan and hydrogels), the conjugation of macromolecules, lipidization and other chemical modification.
    [112] [112] The present invention also provides kits comprising one or more modified mini-hepcidins, alone or in combination with one or more original mini-hepcidins, and / or compositions of the present invention packaged together with reagents, devices, instructional material , or a combination thereof. For example, kits may include reagents used for testing, drugs and compositions for the diagnosis, treatment, or monitoring of disorders of iron metabolism, devices for obtaining samples to be tested, devices for mixing reagents and testing, and the like.
    [113] [113] Since the peptides of the present invention exhibit hepcidin activity, that is, they act as ferroportin degradation agonists, one or more modified mini-hepcidins, alone or in combination with one or more original mini-hepcidins, can be used to treat iron overload diseases. For example, one or more modified mini-hepcidins, alone or in combination with one or more original mini-hepcidins, can be administered to a subject to improve symptoms and / or pathologies associated with iron overload in iron loading anemias ( especially β-thalassemias) where phlebotomy is contraindicated and iron chelators are the mainstay of treatment, but are often poorly tolerated.
    [114] [114] Thus, one or more modified mini-hepcidins, alone or in combination with one or more original mini-hepcidins can be administered to an individual, preferably a mammal as a human. In some embodiments, administration to the subject is, before, during and / or after the subject shows an increase in iron levels and / or abnormally high levels of iron. In some modalities, the subject to be treated is one who is at risk of having high levels of iron and / or has a genetic predisposition to have an iron overload disease. In some embodiments, the peptides are administered in the form of a pharmaceutical composition. In some embodiments, the peptides are administered in a therapeutically effective amount.
    [115] [115] A therapeutically effective amount can be readily determined by conventional methods known in the art. The doses to be administered can be determined by a person skilled in the art depending on the clinical severity of the disease, the subject's age and weight, or the subject's exposure to iron. Preferred effective amounts of mini-hepcidins range from about 0.01 to about 10 mg / kg of body weight, about 0.01 to about 3 mg / kg of body weight, about 0.01 to about 2 mg / kg, about 0.01 to about 1 mg / kg, or about 0.01 to about 0.5 mg / kg of body weight for parenteral formulations. The effective amounts for oral administration can be up to 10 times greater. In addition, treating a subject with a peptide or composition of the present invention can include a single treatment or, preferably, can include a series of treatments.
    [116] [116] The pharmaceutical compositions of the invention can be prepared in an appropriate unit dosage form for the desired mode of administration. The compositions of the present invention can be administered for therapy by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). A variety of routes of administration can be used according to the present invention, including orally, topically, transdermally, nasal, pulmonary, transpercutaneously (in which the skin has been broken, mechanically or by energy), rectally, buccally , vaginal, through an implanted reservoir, or parenteral. Parenteral includes subcutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques, as well as injectable materials (including polymers) for localized therapy. In some modalities, the route of administration is subcutaneous. In some embodiments, the composition is in a sealed sterile glass bottle. In some embodiments, the composition contains a preservative. The pharmaceutical compositions can be formulated as a bulk powder, tablets, liquids, gels, lyophilized, and the like, and can be further processed for administration. See, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 20th ed. (2000) Lippincott Williams & Wilkins. Baltimore, MD, which is incorporated herein by reference.
    [117] [117] It will be appreciated that the preferred route will vary with the condition and age of the beneficiary, the nature of the condition to be treated, and the chosen peptide and composition.
    [118] [118] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of at least one peptide as disclosed herein and a pharmaceutically acceptable carrier or diluent, which can be inert. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, bulking agent, coatings, antibacterial and antifungal agents, preservatives, absorption retarding and isotonic agents, and the like, compatible with administration pharmaceutical and known in the art. Except to the extent that any conventional medium or agent is incompatible with the active compound, its use in the compositions is contemplated.
    [119] [119] Supplementary compounds can also be incorporated into the compositions. Supplementary compounds include niclosamide, liposomes, solubilizing agent SL220 (NOF, Japan), Cremophor EL (Cysma), ethanol, and DMSO.
    [120] [120] The toxicity and therapeutic efficacy of the peptides and compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the lethal dose for 50% of the population) and ED50 ( the therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Peptides that exhibit large therapeutic indexes are preferred. Although peptides that exhibit toxic side effects can be used, care must be taken to design a delivery system that targets such peptides to the affected tissue site in order to minimize the potential damage to uninfected cells and thereby reduce secundary effects.
    [121] [121] The data obtained from cell culture assays and animal studies can be used in formulating a dosage range for use in humans. The dosage of the peptides of the present invention is preferably within a range of concentrations that includes the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration used. For any peptide used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a range of circulating plasma concentrations that includes the IC 50 (i.e., the concentration of the test compound that achieves a semi-maximum inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography or mass spectroscopy.
    [122] [122] PEPTIDE SUMMARY. Mini-hepcidins modified according to the present invention were synthesized using standard solid phase Fmoc chemistry and were purified by reverse phase HPLC. For PR65, from A1 to A9 of structural formula IA (that is, from N to C terminal), the main sequence was all L-amino acids as follows: iminodiacetic acid, threonine, histidine, diphenylalanine, beta-homo proline , arginine, cysteine, arginine and beta-homo phenylalanine. The C-terminal carboxyamide was derivatized with polyethylene glycol (PEG) ligand and the palmitic acid groups (Figure 10A). Human hepatocyte was purchased from Peptides International (Louisville, KY).
    [123] [123] STUDIES IN ANIMALS. All studies were approved by the UCLA Office of Animal Research Oversight.
    [124] [124] The therapeutic effects of PR65 have been studied in hepcidin-1 knockout mice (Hamp1 - / -) (Lesbordes-Brion JC, et al. (2006) Blood 108 (4): 1402-1405) and backcrossed to C57BL background / 6 (N4, 99% of the identity marker gene), using marker-assisted accelerated backcrossing (Charles River Laboratories, Wilmington, MA). PR65 was administered subcutaneously in 100 µl of SL220 solubilizer. Short-term studies were conducted for up to 48 hours to determine the effectiveness of the single injection. Long-term studies ("prevention" and "treatment") were performed for 2 weeks using daily injections and iron and hematological parameters measured 24 hours after the last injection.
    [125] [125] To test the ability of PR65 to prevent, inhibit, or reduce iron load ("prevention" studies), male Hamp1 - / - mice were depleted on iron, putting them on a low iron diet (4 ppm iron) for 2 months, starting at the age of 5-6 weeks. The regimen was developed to match the liver iron content of C57B6 wild-type mice, about 2-3 µmoles / g of wet liver (Ramos E, et al. (2011) Hepatology 53 (4): 1333-1341) . One group of mice was analyzed immediately after iron depletion (baseline group), and the remaining animals were switched to an iron loading diet (standard feed, about 300 ppm Fe) and the daily subcutaneous injection received of solvent or PR65 (20, 50 or 100 nmoles) for 2 weeks. All rat diets were obtained from Harlan-Teklad (Madison, WI).
    [126] [126] To test the effect of PR65 in iron-loaded Hamp1 - / - mice ("treatment" studies), male mice were kept on a normal diet throughout their lifespan. Beginning at 12-14 weeks of age, 50 nmoles of PR65 or solvent was injected daily subcutaneously for 2 weeks.
    [127] [127] IRON MEASUREMENT AND HEMATOLOGICAL PARAMETERS.
    [128] [128] STATISTICAL ANALYSIS. The statistical significance of the differences between the means of the groups was assessed using T-Student and Sigmaplot 11.0 (Systat Software, San Jose, CA).
    [129] [129] PR65 (Figure 10A) was selected for prevention and treatment studies in hepcidin null mice based on pilot studies in the C57BL / 6 wild type. PR65 was shown to be among the most potent mini-hepcidins and its molar bioactivity after intraperitoneal injection was comparable to that of native hepcidin (Figure 10B). In addition, PR65 maintained full activity with subcutaneous administration compared to intraperitoneal administration (Figure 10C) and its cost of synthesis was favorable compared to other mini-hepcidins. Based on the qualitative assessment of more than 80 mini-hepcidins, the high bioactivity of PR65 compared to the prototype peptide, containing the nine human hepcidin N-terminal amino acids (SEQ ID NO: 9), is probably due to the increase in aromaticity, solubility, proteolysis resistance, as well as lower renal clearance, due to the increased binding to plasma proteins mediated by the palmitoil group.
    [130] [130] To establish optimal dosing parameters for a long-term minihepcidin treatment regimen, dose-response experiments (Figure 11A) and time course (Figure 11B) in iron-overloaded hepcidin knockout mice, Hamp1 -/-, were done. After 24 hours, subcutaneous injection of 20 and 50 nmoles of PR65 caused 15% and 10%) (p = 0.005, p = 0.004) reductions in serum iron, while doses of 100 and 200 nmoles resulted in a reduction of 85 % and 95% (p <0.001 for both). Due to the fact that 100 nmoles of PR65 produced hypoferremia close to the maximum, this dose was selected for a time course experiment to determine the time and duration of its maximum effect. The maximum reduction (88%) in Fe in serum occurred 12 hours after subcutaneous injection (p <0.001), and Fe in serum remained severely suppressed (82%) in 24 hours, but returned to solvent control levels 48 hours after injection.
    [131] [131] PR65 activity (100 nmoles) was also assessed for 48 hours, through its effect on tissue iron retention. Interestingly, the accumulation of iron in the spleen was not observed for 48 hours after PR65 injection (Figure 12). This is probably because the spleen in hepcidin knockout mice is completely depleted of iron and takes more than 2 days to accumulate enough iron so it is conclusively detectable by increasing Perls staining. Iron content in the liver, which was already high in these mice, did not change visibly during the course of the experiment. From 1-4 hours after injection, sections of the duodenum showed distinct iron staining around villous capillary networks indicating continued high ferroportin activity and iron transfer did not occur to plasma. From 12-24 hours after the PR65 injection, iron accumulated within enterocytes consistent with the expected mini-hepcidin-induced loss of ferroportin and decreased iron transfer to plasma. As the effect of mini-hepcidin dissolved 48 hours after the injection, iron was no longer retained in enterocytes.
    [132] [132] Thus, in some modalities, subjects are treated with a certain dose, for example, about 100 µg / kg, of a mini-hepcidin daily, and after about a week, the dose is halved if the The subject's serum iron concentration is below about 10 µΜ or doubled if the serum iron is above about 30 µΜ. By the beginning of the third week of treatment, the dose can be increased or decreased by about 25-50% to maintain serum iron levels between 10-30 µΜ. In some embodiments, after about 1 week of administration of one or more mini-hepcidins, the levels of iron, and / or ferroportin, and / or the levels of mini-hepcidin in the subject can be monitored by methods known in the art or as described herein, and then, based on the levels, the subject can be treated accordingly, for example, administered one or more subsequent doses of one or more mini-hepcidins which may be higher or lower than the starting dose . The mini-hepcidins of subsequent doses may be the same or different from the mini-hepcidins of the first dose.
    [133] [133] The ability of PR65 to prevent iron overload in hepcidin in subjects was examined using mice as models. KO mice on hepcidin were placed on an iron-deficient diet for 8 weeks to reduce their iron stores to a level comparable to that of WT mice. After iron depletion, a group of mice was analyzed to establish baseline iron and hematological parameters and the rest of the mice were placed on an iron loading diet (300 ppm Fe) for 2 weeks, while simultaneously receiving subcutaneous injections. daily of solvent only (control) or PR65 (20, 50 or 100 nmoles) in solvent. It is postulated that, compared to solvent treatment, PR65 would cause iron retention in the spleen, decrease serum iron and prevent iron from being loaded into the liver. Because cardiac iron overload is a poor prognostic marker in patients loaded with iron, cardiac iron was also measured. Hemoglobin concentrations were monitored to detect potential iron restriction effects of excess hepcidin in erythropoiesis.
    [134] [134] Hepcidin agonist activity of mini-hepcidins was confirmed in all groups treated by increased iron retention in macrophages manifested as an increase in spleen iron content. In comparison with the almost undetectable non-heme iron content in the control spleens injected with solvents, the three doses of mini-hepcidins caused a 15-30 fold increase in the iron content in the spleen (p = 0.01 for all) (Figure 13 A). Serum iron did not change in mice that received 20 nmoles of PR65 daily (p = 0.26), but decreased by 69% and
    [135] [135] Perls dyes from organ sections of mice treated with minihepcidins compared to baseline iron-depleted mice indicated that iron stores in the liver did not increase from baseline groups of 20 and 50 nmoles, and were even more so lower than baseline at a dose of 100 nmoles (Figure 14). In contrast, the liver sections of mice injected with solvents showed very high levels of iron. A similar pattern of differences between the solvent and PR65 groups was observed in the heart, with a total absence of iron staining in the heart of mice that received 100 nmoles of peptide. Significant accumulation of iron in the red pulp of the spleen was observed in all groups of mini-hepcidin, but not in mice that received the solvent, or in mice with baseline iron depletion. Duodenal sections at baseline showed no iron staining, while PR65-treated mice showed iron retention in duodenal enterocytes, again confirming that PR65 blocked the iron efflux of enterocytes.
    [136] [136] Thus, in some embodiments, the present invention provides methods of preventing iron overload in subjects with abnormally low or none levels of hepcidin which comprises the chronic administration of one or more mini-hepcidins according to the present invention.
    [137] [137] To assess the potential of mini-hepcidins as a stand-alone treatment for iron overload in subjects, 12-week-old iron-overloaded hepcidin knockout mice were injected with 50 nmoles of PR65 daily for 14 days. This dose was chosen as the maximum tolerated dose, since mice that received 100 nmoles in the previous experiment became moderately anemic. Peptide activity was confirmed by a 15-fold increase in the iron content of the spleen (p <0.001) (Figure 15A). In contrast to mice that were depleted of iron prior to PR65 administration, in mice with serum iron levels overloaded with iron, they were not reduced 24 hours after the last dose compared to solvent-treated mice (p = 0.682) ( Figure 15B). However, the decrease of 2 g / dl in hemoglobin (p = 0.012) (Figure 15C), suggests that serum iron could have been transiently reduced during treatment. In less than 24 hours the hypoferremic effect of each 50 nmole dose may have been due to severe iron overload in hepcidin knockout mice at this age. Iron accumulated in the hepatocyte can stimulate ferroportin synthesis and iron efflux from plasma hepatocytes, alleviating the inhibition of ferroportin translation by iron regulatory proteins (IRP) interacting with a 5 'iron (5' IRE) regulatory element in the ferroportin mRNA and possibly other mechanisms.
    [138] [138] Improved Pearls staining demonstrated iron retention in the spleen and duodenum in mice treated with PR65 and a change in the pattern of iron distribution in the liver (Figure 16). No statistically significant difference was noticeable in the heart and pancreas sections of the mice treated with solvents and PR65. In total, staining and quantitative analysis indicate that treatment with 2-week mini-hecepdin can not only inhibit the absorption of iron from the diet, but also redistribute a modest amount of iron from the liver to the spleen.
    [139] [139] Thus, in some embodiments, human subjects being treated for an iron overload disease are treated with one or more mini-hepcidins for a period of at least the minimum period necessary to detect that the treatment prevented accumulation of iron in the liver by available imaging technologies (eg FerriScan), for example, about three months. In some subjects, treatment may be continued for several years, or for the life of the subject.
    [140] [140] As shown in Figure 13, PR65 acted predominantly in reducing iron absorption, but also redistributed iron to spleen macrophages when administered prophylactically. Thus, in some embodiments, one or more mini-hepcidins can be administered to a subject as a prophylactic treatment against the subject's iron overload. For example, where a subject having iron in the liver who is within a normal range, but has a predisposition for iron overload disease (eg, genetically predisposed to adsorption of excess iron), or is at risk of developing an abnormally high level of iron, the subject can be administered with one or more mini-hepcidins to prevent and / or reduce the likelihood that the subject will develop an iron overload disease and / or abnormally high levels of iron.
    [141] [141] According to Figure 13, the distribution of iron was calculated based on the following assumptions and equations: Organ masses estimated based on the average weight of 25 g, volume of blood (5.5%) = 1.4 ml, liver mass (5%) = 1.3 g, spleen mass (0.3%) = 0.08 g,% Fe in Hb = 0.34% based on the molecular mass of Hb = 64,000 Dalton and 4 iron atoms with a total atomic mass of 224 Dalton, total iron in Hb = (concentration of Hb) x (volume of blood) x (% Fe in Hb) and Total iron in an organ = molar concentration of iron x mass of organ x 56 g / mol. The values for the solvent group and the PR65 group are presented as follows: TABLE 5 Treatment with solvent Iron in the total organ (mg) Hb = 15 g / dl 0.7 Concentration of iron in the 1.2 liver = 1.7 µmoles / g Iron concentration in the 0.002 spleen = 0.5 µmole / g Total 1.9 PR65: 50 nmoles Iron in the total organ (mg) Hemoglobin = 11.5 g / dl 0.5
    [142] [142] The resulting decrease in iron in plasma can also reduce the levels of toxic iron-bound non-transferrin substances (NTBI) and promote the mobilization of iron from the heart and endocrine organs where excess iron is not tolerated. Thus, in some embodiments, one or more mini-hepcidins may be administered to a subject, to reduce NTBI levels and / or promote the mobilization of iron from the heart and endocrine organs to other organs and tissues.
    [143] [143] Unlike phlebotomy or chelation, mini-hepcidins would not be expected to appreciably increase the body's iron losses. In a relatively mild model of iron overload in null HFE mice (Viatte L, et al. (2006) Blood 107 (7): 2952-2958), expression of transgenic hepcidin has been reported to cause significant redistribution of iron in liver macrophages, a place where the accumulation of iron is relatively non-toxic. In more overloaded Hamp1 - / - mice, red-fleshed macrophages in mice treated with mini-hepcidin retained iron,
    [144] [144] Thus, in established iron overload in human subjects, effective treatment with one or more mini-hepcidins may include more than one dose per day, a prolonged treatment period before a beneficial effect of iron on the liver can be detected, or can be combined with removal of iron by phlebotomy or chelation.
    [145] [145] The exclusive use of L-amino acids in PR65 has been shown to significantly reduce peptide production costs. In addition, the unnatural and highly aromatic residues in PR65 unexpectedly have been shown to substantially reduce the minimum effective dose in mice to 20 nmoles / d or 1.3 mg / kg / d.
    [146] [146] According to the US Food and Drug Administration's dosage adjustment guidelines, the difference in metabolic rates between mice and humans requires conversion based on the Km factor that normalizes doses to the body surface area (Reagan-Shaw S, et al. (2008) FASEB J 22 (3): 659-661). An equivalent human dose (HED) can be estimated by HED = animal dose (mg / kg) x (animal km / human km), where the km for the mouse and an adult human being is 3 and 37, respectively. Thus, according to the present invention, a subcutaneous dose of mini-hepcidin in a human can be up to about 50-100 µg / kg / d, about 75-125 µg / kg / d, or about 90 -110 µg / kg / d, preferably about 100 µg / kg / d (this dose is an easily administered amount of the peptide about three times the average basal dose of the most widely used peptide drug, subcutaneous insulin, commonly used in 0 , 75 U / kg / d or 33 µg / kg / d in type 2 diabetics (Rosenstock J, et al. (2001) Diabetes Care 24 (4): 631-636)). Of course, the lower doses, as well as the higher doses, depending on the particular mini-hepcidin, form of administration, formulation, subject and degree of iron overload, can be administered to the subject.
    [147] [147] Important differences between murine and human iron metabolism that can alter the effect of a minihepcidin, for example, PR65, in humans include a longer lifespan for human erythrocytes (120 days vs
    [148] [148] As provided herein, mini-hepcidins according to the present invention can be used to inhibit, reduce or treat iron overload in subjects at risk due to genetic defects or those who have already undergone iron depletion, but they no longer tolerate chelation therapy or venesection. The mini-hepcidins according to the present invention can be used to treat a subject having major β-thalassemia and / or a subject having levels of hepcidin that are higher than normal, but are lower than what is appropriate for the degree of iron overload, and the particular subject. For example, one or more mini-hepcidins according to the present invention can be used to treat a subject who suffers from dietary iron absorption but has normal levels of iron in order to reduce the amount of iron in the subject and compensate for hyperabsorption.
    [149] [149] Because of the relatively small size of the mini-hepcidins of the present invention, mini-hepcidins can be adequately formulated and optimized for oral administration or administration by other non-invasive means, such as those used for insulin administration (Roach P. (2008) Clinical Farmacokinetics 47 (9): 595-610), such as inhalation, or transcutaneous release, or release by nasal or oral mucosa.
    [150] [150] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as if each were individually so incorporated.
    [151] [151] Having thus described the exemplary embodiments of the present invention, it should be noted by those skilled in the art that the disclosures here are exemplary only and that various other alternatives, adaptations and modifications can be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments, as illustrated herein, but is only limited by the following claims.
    权利要求:
    Claims (18)
    [1]
    1. Isolated peptide characterized by the fact that it has the following structural formula IA or IB: A1-A2-A3-A4-A5-A6-A7-A8-A9-A10 IA A10-A9-A8-A7-A6-A5- A4-A3-A2-A1 IB where A1 is Asp, D-Asp, Glu, D-Glu, pyroglutamate, D-pyroglutamate, Gln, D-Gln, Asn, D-Asn, or an unnatural amino acid commonly used as a substitute for it, such as bhAsp, Ida, Ida (NHPal), and N-MeAsp, preferably Ida and N-MeAsp; A2 is Thr, D-Thr, Ser, D-Ser, Val, D-Val, Ile, D-Ile, Ala, D-Ala or an unnatural amino acid commonly used as a substitute for it, such as Tle, Inp, Chg , bhThr and N-MeThr; A3 is His, D-His, Asn, D-Asn, Arg, D-Arg, or an unnatural amino acid commonly used as a substitute for it, such as L-His (π-Me), D-His (π-Me ), L-His (τ-Me), or D-His (τ-Me); A4 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Trp, D-Trp, Tyr, D-Tyr, or an unnatural amino acid commonly used as a substitute for it, such as Phg, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, Amc, PheF5, hPhe, Igl or cyclohexylalanine, preferably Dpa; A5 is Pro, D-Pro, Ser, D-Ser, or an unnatural amino acid commonly used as a substitute for them,
    like Oic, bhPro, trans-4-PhPro, cis-4-PhPro, cis-5-PhPro,
    Idc and, preferably, bhPro;
    A6 is Arg, D-Arg, Ile, D-Ile, Leu, D-Leu, Thr, D-Thr,
    Lys, D-Lys, Val, D-Val, or an unnatural amino acid commonly used as a substitute for it, such as D-
    Νω, ω-dimethyl-arginine, L-Nω, ω-dimethyl-arginine, D-
    homoarginine, L-homoarginine, D-norarginine, L-norarginine,
    citrulline, a modified Arg, in which the guanidinium group is modified or substituted, Norleucine, norvaline, bhIle,
    Ach, N-MeArg, and N-MeIle, preferably Arg;
    A7 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, or an unnatural amino acid commonly used as a substitute for it, such as Cys (S-tBut), homoCys, Pen,
    (D) Pen, preferably S-tertiary butyl-cysteine,
    Cys (S-S-Pal), Cys (S-S-cysteamine-Pal), Cys (S-S-Cys-NHPal) and
    Cys (S-S-Cys);
    A8 is Arg, D-Arg, Ile, D-Ile, Leu, D-Leu, Thr, D-Thr,
    Lys, D-Lys, Val, D-Val, or an unnatural amino acid commonly used as a substitute for it, such as D-
    Νω, ω-dimethyl-arginine, L-Nω, ω-dimethyl-arginine, D-
    homoarginine, L-homoarginine, D-norarginine, L-norarginine,
    citrulline, a modified Arg, in which the guanidinium group is modified or substituted, Norleucine, norvaline, bheIle,
    Ach, N-MeArg, and N-MeIle, preferably Arg;
    A9 is Phe, D-Phe, Leu, D-Leu, Ile, D-Ile, Tyr, D-Tyr, Trp, D-Trp, Phe-Ra, D-Phe-Ra, Dpa-Da, D-Dpa- Ra, Trp-Ra, bhPhe-Ra, or a non-natural amino acid commonly used as a substitute for it, such as PheF5, N-MePhe, benzylamide, 2-aminoindane, bhPhe, Dpa, Bip, 1Nal, 2Nal, bhDpa, and cyclohexylalanine which may or may not have Ra attached to it, preferably bhPhe and bhPhe-Ra, where Ra one is palmitoyl-PEG, where PEG is PEG11 or miniPEG3, palmitoyl-PEG-PEG, where PEG is PEG11 or miniPEG3, butanoyl (C4) - PEG11-, octanoyl (C8, Caprylic) -PEG11-, palmitoyl (C16) - PEG11-, or tetracosanoyl (C24, Lignoceric) -PEG11-; and A10 is Cys, D-Cys, Ser, D-Ser, Ala, D-Ala, or an unnatural amino acid such as Ida, Ida (NHPal) Ahx, and Ida (NBzl2) Ahx; wherein the carboxy-terminal amino acid is in an amide or carboxy form; wherein at least one sulfhydryl amino acid is present as one of the amino acids in the sequence; and where A1, A1 to A2, A10, or a combination thereof are optionally absent, with the proviso that the peptide is not one of the peptides as defined in Table 1.
    [2]
    2. Peptide according to claim 1, characterized by the fact that the peptide contains at least one of the following:
    a) A1 = N-MeAsp, Ida, or Ida (NHPal); b) A5 = bhPro; c) A6 = D-Val, D-Leu, Lys, D-Lys, Arg, D-Arg, Ach, bhArg, from N-MeArg; d) A7 = Cys (S-S-Pal), Cys (S-S-cysteamine-Pal), Cys (S-S-Cys-NHPal), or Cys (S-S-Cys); and / or e) A8 = D-Val, D-Leu, Lys, D-Lys, Arg, D-Arg, Ach, bhArg, or N-MeArg.
    [3]
    3. Peptide according to claim 1 or 2, characterized by the fact that: i) when A1 is Ida and A9 is Phe, then A10 is not Ahx-Ida (NHPal); ii) when A1 is Ida, A9 is not bhPhe-Rb, where Rb is S- (palmitil) thioglycolic-PEG-; iii) when A4 is D-Phe, A7 is not D-Cys (SS-tBut) and A9 is not D-Trp-Rc, where Rc is butanoyl-PEG11-, Octanoyl-PEG11-, Palmitoil-PEG11-, or Tetracosanoyl-PEG11-; and iv) when A1 is Ida and A9 is bhPhe-Rd, where Rd is palmitoyl-PEG-miniPEG3-, A6 and A8 are not both D-Arg or both bhArg.
    [4]
    4. Peptide according to any one of claims 1 to 3, characterized in that A1 is D-Asp, N-MeAsp, Ida, or Ida (NHPal); A2 is Thr or D-Thr; A3 is His or D-His;
    A4 and Dpa or D-Dpa; A5 is Pro, D-Pro, bhPro, or Oic; A6 is Ile, D-Ile, Arg, D-Val, D-Leu, Ach, or N-MeArg; A7 is Cys, D-Cys, Cys (S-S-Pal), Cys (S-S-cysteamine-Pal), Cys (S-S-Cys-NHPal), or Cys (S-S-Cys); A8 is Ile, D-Ile, Arg, D-Val, D-Leu, Ach, or N-MeArg; A9 is Phe, D-Phe, Dpa, D-Dpa, Trp, D-Trp, bhPhe, Phe-Ra, D-Phe-Ra, Dpa-Ra, D-Dpa-Ra, Trp-Ra, bhPhe-Ra, where Ra is palmitoyl-PEG-, where PEG is PEG11 or miniPEG3, palmitoyl-PEG-PEG, where PEG is PEG11 or miniPEG3, butanoyl (C4) - PEG11-, octanoyl (C8, caprylic) -PEG11-, palmitoil (C16) - PEG11-, or tetracosanoil (C24, Lignoceric) -PEG11-; and A10, if present, is Ida (NHPal) Ahx or Ida (NBzl2) Ahx.
    [5]
    5. Peptide according to any one of the preceding claims, characterized by the fact that the peptide is selected from the group consisting of: PR42 ', PR47, PR48, PR49, PR50, PR51, PR52, PR53, PR56, PR57 , PR58, PR59, PR60, PR61, PR63, PR65, PR66, PR67, PR68, PR69, PR70, PR71, PR72, PR73, PR74, and PR82, preferably, PR47, PR48, PR49, PR50, PR51, PR52, PR53, PR56, PR57, PR58, PR59, PR60, PR61, PR63, PR65, PR66, PR67, PR68, PR69, PR70, PR71, PR72, PR73, PR74, and PR82.
    [6]
    6. Peptide according to any one of claims 1 to 5, characterized by the fact that the peptide has hepcidin activity.
    [7]
    7. Peptide according to any one of claims 1 to 5, characterized in that the peptide binds ferroportin.
    [8]
    8. Composition characterized by the fact that it comprises at least one peptide as defined in any one of claims 1 to 7.
    [9]
    9. Method for binding a ferroportin or inducing internalization and degradation of ferroportin characterized by the fact that it comprises contacting ferroportin with at least one peptide as defined in any one of claims 1 to 7 or the composition as defined in claim 8.
    [10]
    10. Method for treating an iron metabolism disorder in a subject characterized by the fact that it comprises administering at least one peptide as defined in any one of claims 1 to 7 or the composition as defined in claim 8 to the subject.
    [11]
    11. Method according to claim 10, characterized by the fact that the disease of iron metabolism is a disease of iron overload.
    [12]
    12. Kit characterized by the fact that it comprises at least one peptide as defined in any one of claims 1 to 7 or the composition as defined in claim 8, packaged together with a reagent, device, instructional material or a combination thereof .
    [13]
    13. Complex characterized by the fact that it comprises at least one peptide as defined in any one of claims 1 to 7 linked to a ferroportin or an antibody.
    [14]
    14. Use of one or more peptides as defined in any of claims 1 to 7 or the composition as defined in claim 8 characterized by the fact that it is for the manufacture of a medicament to treat an iron metabolism disease and / or lower the amount of iron in a subject in need of it.
    [15]
    15. Peptide according to any one of claims 1 to 7, or composition according to claim 8, characterized by the fact that it is for use in the treatment of an iron metabolism disease and / or to lower the amount of iron in a subject in need of it.
    [16]
    16. Use of one or more peptides according to any one of claims 1 to 7, or the composition according to claim 8, characterized by the fact that it is for the manufacture of a medicament to treat a metabolic disease iron and / or lower the amount of iron in a subject in need of it, in which the drug is prepared to be administered in an effective daily dose as a single daily dose or in divided daily doses.
    [17]
    17. Use, according to claim 16, characterized by the fact that the effective daily dose is about 10 to 500 µg / kg / day and the drug is formulated for subcutaneous injection.
    [18]
    18. Use according to claim 16, characterized by the fact that the effective daily dose is about 10 to 1000 µg / kg / day and the drug is formulated for oral, pulmonary or mucosal administration.
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    法律状态:
    2020-11-17| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2021-04-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
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    2021-07-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-09-28| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 9A ANUIDADE. |
    2021-10-19| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|
    2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|
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