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Targeted Coagulation Factors and Method of Using the Same

a technology of coagulation factors and target coagulation, which is applied in the direction of fusions for specific cell targeting, peptide/protein ingredients, extracellular fluid disorder, etc. it can solve the problems of limited effect of biological drugs, and limited treatment options for hemophilia patients with fviii concentrates or recombinant fviii, etc., to achieve enhanced protein therapeutic effect and high local concentration

Inactive Publication Date: 2011-03-31
BAYER HEALTHCARE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014]The targeted coagulation factors according to the present invention comprise a coagulation factor linked with at least one domain that specifically binds to a membrane protein on a blood cell. A pharmaceutical composition comprising the newly disclosed targeted coagulation factors and a method for treating hematological diseases using the targeted coagulation factors is also provided. The present invention further provides a method for targeting a coagulation factor to the surface of a blood cell by using the newly disclosed targeted coagulation factors to increase the efficiency of treating hematological disease with coagulation factors.

Problems solved by technology

The effectiveness of biological drugs is often limited by their duration of action in patients, particularly when the disease requires constant modulation by the drug.
For example, current treatment of hemophilia A patients with FVIII concentrates or recombinant FVIII is limited by the high cost of these factors and their relatively short duration of action.
Unfortunately, this frequency is cost prohibitive for many patients.
For those who can afford the frequent dosaging recommended, it is nevertheless very inconvenient to frequently intravenously inject the protein.
An additional disadvantage to the current therapy is that about 25-30% of patients develop antibodies that inhibit FVIII activity (Saenko, et al., Haemophilia 8:1-11, 2002).
Antibody development prevents the use of FVIII as a replacement therapy, forcing this group of patients to seek an even more expensive treatment with high-dose recombinant Factor VIIa (FVIIa) and immune tolerance therapy.

Method used

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  • Targeted Coagulation Factors and Method of Using the Same
  • Targeted Coagulation Factors and Method of Using the Same
  • Targeted Coagulation Factors and Method of Using the Same

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0054]Modified RGD Peptides with High Affinity for GPIIb / IIIa Binding

[0055]Cyclic peptides have been described to bind potently and selectively to GPIIb / IIIa. One such peptide, integrilin, was used as a targeting domain to link with FVIII as it has been shown that integrilin can selectively bind to GPIIb / IIIa. Integrilin was modified by adding a short PEG linker ending in a maleimide moiety that can selectively couple to free cysteine residues in proteins. The modified integrilin is termed BHRF-1 with the linker only (FIG. 2A), and BHRF-3 with the linker and a fluorescein (FITC) (FIG. 2B). As shown in FIG. 3, the modified integrilins retain affinity for GPIIb / IIIa as they potently blocked fibrinogen (Fbn) binding to immobilized GPIIa / IIIb.

[0056]Peptide binding to GPIIb-IIIa was measured using a solid phase binding assay in which competition of fibrinogen binding by testing compounds is measured. The assay was performed as follows. Purified GPIIb-IIIa (Innovative Research, Novi, Mich...

example 2

[0058]Coupling GPIIb / IIIa Binding Peptides to FVIII

[0059]The polypeptide sequence of the full-length FVIII is known in the art (see, e.g., SEQ ID NO: 1, SEQ ID NO: 2, and as disclosed in WO 2006 / 053299.

Concentration of FVIII and uncapping of free sulfhydryl groups

[0060]The Cys residue located in the B-domain of recombinant FVIII can be capped by cysteine present in the media during protein expression, but it can be readily removed by treatment with reducing agents, such as TCEP, as follows. FVIII (20 mL) was thawed and concentrated in two Amicon®-15 cartridges (Millipore, Billerica, Mass.), spun at 2000×g (about 3153 rpm) for 25 minutes in the cold. The concentration of the 2.8 mL retentate is about 0.8-0.9 mg / mL by A280 using a NanoDrop® spectrophotometer (ThermoFisher Scientific, Waltham, Mass.). The buffer was then exchanged using a 10 mL Zeba desalting cartridge, pre-equilibrated with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl2 and 100 ppm Tween®-80 (polyoxyethylenesorbitan monooleate...

example 3

[0064]BHRF-1-FVIII Binds to Immobilized GPIIb / IIIa

[0065]To test the binding activity of BHRF-1-FVIII to GPIIb / IIIa, biotinylated GPIIb / IIIa was immobilized on streptavidin plates and treated with either BHRF-1-FVIII or unmodified FVIII, both in binding buffer (50 mM Tris, pH 7.5, 100 mM NaCl2, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2 and 1 mg / mL BSA). The unbound protein was removed by washing three times with binding buffer. Assay buffer (25 μL) was added to the plate, and FVIII activity was determined using a chromogenic assay kit (Coatest® SP4, Chromogenix, Lexington, Mass.). As shown in FIG. 4, there was binding of BHRF-1-FVIII, while only little binding of unmodified FVIII was detected. The increased binding of BHRF-1-FVIII was completely eliminated by addition of a cyclic RGD peptide (GpenGRGDSPCA; SEQ ID NO: 5) that competes for BHRF-1 binding to GPIIb / IIIa. Furthermore, only low background levels of either protein bound when no GPIIb / IIIa was immobilized on the plate. These data s...

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Abstract

Targeted coagulation factors comprising a coagulation factor linked with at least one domain that specifically binds to a membrane protein on a blood cell is provided. The disclosed targeted coagulation factors increase the efficiency of coagulation factors and prolong their duration of action and thus, are an improvement for the treatment of hematological diseases such as hemophilia A.

Description

[0001]This application claims benefit of U.S. Provisional application Ser. No. 61 / 053,932; filed on May 16, 2008, the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The invention relates to targeted coagulation factors having increased efficacy. The invention further provides methods of treating patients suffering from a coagulation factor deficiency disorder by selectively targeting coagulation factors to their biological sites of action, such as by targeting Factor VIII (FVIII) to red blood cells and platelets. Pharmaceutical compositions comprising the targeted coagulation factors according to the invention are also provided.BACKGROUND OF THE INVENTION[0003]The effectiveness of biological drugs is often limited by their duration of action in patients, particularly when the disease requires constant modulation by the drug. Consequently, enhancement of pharmacokinetic properties is often more critical to the success of a therape...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/36C07K14/745C07K14/755A61P7/00
CPCA61K38/37C07K14/755A61K38/4846A61K47/64A61K47/6811A61K47/6849C07K16/2848A61K38/00C07K2319/33A61P7/00A61P7/04C07K16/2839C07K2317/622C07K2319/30
Inventor FELDMAN, RICHARDKIM, JI-YUNJIANG, HAIYANMCLEAN, KIRKPAN, JUNLIANGPIERCE, GLENNWU, JAMESZHAO, XIAO-YAN
Owner BAYER HEALTHCARE LLC
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