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Determination of AM-binding proteins and the association of adrenomedullin (AM) therewith

a technology of adrenomedullin and am, which is applied in the field of detection of adrenomedullin (am)binding proteins, can solve the problems of reducing the levels of insulin in the bloodstream, and achieve the effects of inhibiting am/fh activity, affecting am/fh activity, and affecting am/fh activity

Inactive Publication Date: 2007-02-01
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0024] It is an additional object of the present invention to provide antagonist agents that inhibit AM, factor H, or AM / fH activity.
[0025] It is yet another object of the present invention to provide methods of treating cancer by administering one or more antagonist agents in amounts sufficient to inhibit AM, factor H, or AM / fH activity.
[0026] It is also an object of the present invention to provide methods of treating cancer by administering antibodies that specifically bind to the AM / factor H in amounts sufficient to bind to the AM / factor H complex and inhibit AM / fH activity.

Problems solved by technology

Additionally, intravenous injection of AM reduces the levels of insulin in the bloodstream with a concomitant increase in circulating glucose (Martinez, 1996, supra).

Method used

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  • Determination of AM-binding proteins and the association of adrenomedullin (AM) therewith
  • Determination of AM-binding proteins and the association of adrenomedullin (AM) therewith
  • Determination of AM-binding proteins and the association of adrenomedullin (AM) therewith

Examples

Experimental program
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example 1

[0123] Non-radioactive labeling of AM was accomplished by conjugating synthetic AM comprising amino acids 1-52 (Phoenix Pharmaceuticals, Inc., Mountainview, Calif.) with succinimidyl esters linked to biotin (Pierce Chemical Co., Rockford, Ill.), fluorescein (Molecular Probes, Inc., Eugene, Oreg.), or dinitrophenol (DNP; Molecular Probes, Inc.). Briefly, 100 μg of AM (16 μM) was dissolved in 1 ml of 50 mM sodium bicarbonate, pH 8.5, and the succinimidyl ester was added to yield a final molar concentration of 10:1 (linker:AM). The mixture was incubated with slow agitation for 1 hr at room temperature and the reaction was terminated by the addition of 0.1 M ethanolamine followed by another incubation for 1 hr. Unincorporated ligand was removed by extracting the AM using reverse phase Sep-Pak C-18 cartridges (Waters, Milford, Mass.) and eluting the sample with acidic-isopropanol. The extract was lyophilized and reconstituted in 1 ml of TBS (0.05 M Tris-HCl, 0.15 M NaCl), 0.1% alkali-tre...

example 2

[0124] AM-binding proteins were identified using an in vitro screening method. Total proteins derived from human plasma (2 μl) were electrophoretically fractionated on 3-8% Tris-acetate gel (Novex, San Diego, Calif.) under non-reducing conditions. For the ligand blotting experiment, the proteins were transferred to a 0.2 mm nitrocellulose membrane that was incubated for 15 min with 1% Nonidet P-40 (NP-40). Non-specific binding was further blocked with a 2 hr incubation in TBS containing 0.1% ATC. Incubations with 70 nM AM labeled with biotin, fluorescein, or DNP were carried out overnight at 4° C. in blocking buffer containing 0.1% Tween 20. For biotin detection, the ligand blot was incubated with an avidin-biotin-peroxidase complex and the AEC reagent (Vector Laboratories, Inc., Burlingame, Calif.). For fluorescein and DNP, the ligand blots were incubated 1 hr at room temperature with rabbit anti-fluorescein or anti-DNP IgG, respectively, (1:1000 in incubation buffer; Molecular Pro...

example 3

[0125] Competition of full-length AM binding to AM-binding proteins was tested by pre-incubating a membrane having plasma derived proteins bound thereto with 7 μM of unlabeled peptides; e.g., CGRP, amylin, insulin, IGF-1, PAMP, or AM truncated polypeptides (AM1-12, AM16-21, AM22-52, AM13-52, AM34-52) for 6 hr at 4° C. Following this preincubation, labeled full-length AM was introduced and incubated overnight at 4° C. Competition studies indicated that only full-length AM was able to dissociate the AM / AMBP-1 (AM / fH) complex. FIGS. 2 and 3.

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Abstract

The present invention provides methods for the isolation, identification, and purification of adrenomedullin (AM)-binding proteins. Also, provided are methods for utilizing the purified AM-binding proteins, or functional portions thereof, to diagnose, treat, and monitor AM-related diseases, for example, diseases or disorders associated with abnormally elevated AM levels. In addition, the present invention provides a newly identified complex between AM and a specific AM-binding protein 1 (AMBP-1); which has been isolated and identified herein as factor H (fH). The invention also provides AM / AMBP complexes, particularly AM / FH complexes, and antibodies specifically reactive with this complexes. Further provided are methods for identifying and purifying complexes of AM and an AM binding protein using anti-AM / fH antibodies, and methods for treating conditions such as cancer or diabetes utilizing compositions comprising these antibodies. The present invention additionally provides methods for identifying antagonists agents that inhibit the function of AM, factor H, or the AM / factor H complex. The invention also provides methods for treating conditions such as cancer or diabetes using these antagonist agents.

Description

RELATED APPLICATION [0001] This application is related to U.S. Provisional Application Ser. No. 60 / 153,397 filed Sep. 10, 1999, the contents of which are herein incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to newly developed methods for the detection of adrenomedullin (AM)-binding proteins. The invention further relates to the isolation, identification, and use of such AM-binding proteins, and to compositions and methods including these proteins. The AM-binding proteins can be employed in the diagnosis, treatment and monitoring of AM-related diseases. The present invention also relates to the isolation of AM / AM-binding protein complexes, the generation of antibodies to these complexes, and the use of these antibodies to detect AM / AM-binding protein complexes and treat AM-related diseases. The invention further relates to a newly discovered complex comprising AM and a newly identified AM binding protein, human complement factor H (fH; factor H...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K51/00G01N33/574A61K39/395C07K16/30C07K16/46A61K38/17C07K16/18G01N33/74
CPCA61K38/1709A61K2039/505G01N2500/00G01N33/74C07K16/18A61P43/00
Inventor CUTTITTA, FRANKELSASSER, TED H.MARTINEZ, ALFREDOPIO, RUBEN
Owner UNITED STATES OF AMERICA
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