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Analogues of hepcidin mimetics with improved in vivo half lives

a technology of mimetics and hepcidin, which is applied in the field of analogues of hepcidin, can solve the problems of excessive absorption of iron from the diet, loss of iron-regulatory function, and development of iron overload, and achieves the effects of increasing serum half-life, enhancing solubility, and increasing molecular weigh

Inactive Publication Date: 2019-01-03
PROTAGONIST THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides new hepcidin analogues and dimers that have a half-life extension moiety. The half-life extension moiety can be selected from a variety of chemicals such as lauric acid, myristaric acid, palmitic acid, stearic acid, and more. Attaching the half-life extension moiety to the peptide increases its molecular weight by about 50 D to about 2 KD. The half-life extension moiety can also enhance the solubility and bioavailability of the hepcidin analogue.

Problems solved by technology

The N terminal region is required for iron-regulatory function, and deletion of 5 N-terminal amino acid residues results in a loss of iron-regulatory function.
This allows excessive absorption of iron from the diet and development of iron overload.
Currently, the only treatment for HH is regular phlebotomy, which is very burdensome for the patients.
Complications from iron overload are the main cause of morbidity and mortality for 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 chelation which is very burdensome, sometimes ineffective, and accompanied by frequent side effects.
Hepcidin has a number of limitations which restrict its use as a drug, including a difficult synthesis process due in part to aggregation and precipitation of the protein during folding, which in turn leads to high cost of goods.

Method used

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  • Analogues of hepcidin mimetics with improved in vivo half lives
  • Analogues of hepcidin mimetics with improved in vivo half lives
  • Analogues of hepcidin mimetics with improved in vivo half lives

Examples

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example 1

Synthesis of Peptide Analogues

[0301]Unless otherwise specified, reagents and solvents employed in the following were available commercially in standard laboratory reagent or analytical grade, and were used without further purification.

[0302]Procedure for Solid-Phase Synthesis of Peptides

[0303]Peptide analogues of the invention were chemically synthesized using optimized 9-fluorenylmethoxy carbonyl (Fmoc) solid phase peptide synthesis protocols. For C-terminal amides, rink-amide resin was used, although wang and trityl resins were also used to produce C-terminal acids. The side chain protecting groups were as follows: Glu, Thr and Tyr: 0-tButyl; Trp and Lys: t-Boc (t-butyloxycarbonyl); Arg: N-gamma-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; His, Gln, Asn, Cys: Trityl. For selective disulfide bridge formation, Acm (acetamidomethyl) was also used as a Cys protecting group. For coupling, a four to ten-fold excess of a solution containing Fmoc amino acid, HBTU and DIPEA (1:1:1.1)...

example 2

Activity of Peptide Analogues

[0329]Peptide analogues were tested in vitro for induction of internalization of the human ferroportin protein. Following internalization, the peptides are degraded. The assay measures a decrease in fluorescence of the receptor.

[0330]The cDNA encoding the human ferroportin (SLC40 A1) was cloned from a cDNA clone from Origene (NM_014585). The DNA encoding the ferroportin was amplified by PCR using primers also encoding terminal restriction sites for subcloning, but without the termination codon. The ferroportin receptor was subcloned into a mammalian GFP expression vector containing a neomycin (G418) resistance marker in such that the reading frame of the ferroportin was fused in frame with the GFP protein. The fidelity of the DNA encoding the protein was confirmed by DNA sequencing. HEK293 cells were used for transfection of the ferroportin-GFP receptor expression plasmid. The cells were grown according to standard protocol in growth medium and transfect...

example 3

In Vivo Validation of Peptide Analogues

[0334]Hepcidin analogues of the present invention were tested for in vivo activity, to determine their ability to decrease free Fe2+ in serum.

[0335]A hepcidin analogue (Compound 1) or vehicle control were administered to mice (n=3 / group) at 1000 nmol / kg either intravenously or subcutaneously. Serum samples were taken from groups of mice administered with the hepcidin analog at 30 min, 1 h, 2 h, 4 h, 10 h, 24 h, 30 h, 36 h, and 48 h post-administration. Iron content in plasma / serum was measured using a colorimetric assay on the Cobas c 111 according to instructions from the manufacturer of the assay (assay: IRON2: ACN 661). The data obtained from the cobas Iron2 analysis is presented in FIG. 1A (intravenous administration) and FIG. 1B (subcutaneous administration) as mean values+ / −SEM.

[0336]In another experiment, various hepcidin analogues or vehicle control were administered to mice (n=3 / group) at 1000 nmol / kg subcutaneously. Serum samples were...

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Abstract

The present invention provides hepcidin analogues with improved in vivo half-lives, and related pharmaceutical compositions and methods of use thereof.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Application No. 62 / 273,265, filed on Dec. 30, 2015, which is incorporated by reference herein in its entirety.STATEMENT REGARDING SEQUENCE LISTING[0002]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is PRTH_022_01WO_ST25.txt. The text file is 17 KB, was created on Dec. 29, 2016, and is being submitted electronically via EFS-Web.FIELD OF THE INVENTION[0003]The present invention relates, inter alia, to certain hepcidin peptide analogues, including both peptide monomers and peptide dimers, and conjugates and derivatives thereof, as well as compositions comprising the peptide analogues, and to the use of the peptide analogues in the treatment and / or prevention of a variety of diseases, conditions or disorders, in...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K14/575A61P3/02
CPCC07K7/08C07K14/575A61P3/02A61K38/00C07K14/00
Inventor BOURNE, GREGORY THOMASSMYTHE, MARK LESLIEFREDERICK, BRIAN TROYBHANDARI, ASHOK
Owner PROTAGONIST THERAPEUTICS INC
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