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Combination therapy for treating protein deficiency disorders

a protein deficiency and therapy technology, applied in the field of protein deficiency disorders, can solve the problems of inability of proteins vectors infecting or transducing dividing cells, and inability to cross the blood-brain barrier, etc., to achieve the effect of enhancing the stability of purified proteins

Inactive Publication Date: 2007-08-02
FAN JIAN QIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides a method for improving the stability of purified proteins, which involves adding an active site-specific chaperone to a pharmaceutically acceptable carrier. This can be done with full-length or truncated proteins that retain activity. The invention also provides a method for increasing the shelf-life and activity in vivo of a protein replacement therapy. Additionally, the invention can be used to treat individuals with disorders that require protein replacement by adding the active site-specific chaperone to the replacement protein. The invention also provides a method for enhancing the stability of mutant proteins that are deficient due to defective folding or processing. The invention also includes a composition comprising a purified protein and an active site-specific chaperone in a pharmaceutically acceptable carrier."

Problems solved by technology

One of the main complications with protein replacement therapy is attainment and maintenance of therapeutically effective amounts of protein due to rapid degradation of the infused protein.
Protein replacement therapy has several additional caveats, such as difficulties with large-scale generation, purification and storage of properly folded protein, obtaining glycosylated native protein, generation of an anti-protein immune response, and inability of protein to cross the blood-brain barrier in diseases having significant central nervous system involvement.
Although promising, this approach is also limited by technical difficulties such as the inability of vectors to infect or transduce dividing cells, low expression of the target gene, and regulation of expression once the gene is delivered.
These heritable disorders are characterized by deficiencies in lysosomal enzymes that catalyze the breakdown of glycolipids in cells, resulting in an abnormal accumulation of lipids which disrupts cellular function.
This approach is also limited in that glycolipids are necessary for biological function, and excess deprivation may cause adverse effects.
If there are too few or too many glycolipids, the ability of the neuron to send signals is impeded.
In vivo, protein folding is complicated, because the combination of ambient temperature and high protein concentration stimulates the process of aggregation, in which amino acids normally buried in the hydrophobic core interact with their neighbors non-specifically.
Certain DNA mutations result in amino acid substitutions that further impede, and in many cases preclude, proper folding of the mutant proteins.
The high doses of this class of compounds required for efficacy makes them difficult or inappropriate to use clinically, although they are useful for the biochemical examination of folding defect of a protein intracellularly.
These compounds also lack specificity.

Method used

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  • Combination therapy for treating protein deficiency disorders
  • Combination therapy for treating protein deficiency disorders
  • Combination therapy for treating protein deficiency disorders

Examples

Experimental program
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Effect test

example 1

In Vitro Stabilization of (α-Gal A With ASSCs

[0120] Methods. The wild type α-Gal A was purified from culture medium of Sf-9 cells infected with recombinant baculovirus carrying human wild type α-Gal A cDNA and the mutant α-Gal A was collected as homogenates of hearts of transgenic mice overexpressing human mutant (R301Q) α-Gal A. The mice were treated with 0.5 mM DGJ as drinking water for one week prior to the experiment. The mutant and wild type enzymes were pre-incubated with 0.1 M citrate-phosphate buffer (pH 7.0) at 37° C. for the mutant enzyme and 42° C. for the wild type enzyme, respectively, in the presence of DGJ at a concentration of 1 μM, 0.1 μM, 0.03 μM or no DGJ. The wild type and mutant (R301Q) α-Gal A were incubated for a period of time in the absence or presence of DGJ (various concentrations), and the remaining enzyme activity was determined with 4-MU-α-Gal A as a substrate, after diluting the mixture with 5-volume of 0.1 M citrate buffer (pH 4.5). Enzyme activity i...

example 2

Intracellular Enhancement of Wild-Type α-Gal A With ASSCs

[0122] Methods. Human wild type α-Gal A purified from insect cells transfected with recombinant baculovirus or from recombinant CHO cells can be conjugated to α-2-macroglobulin (α-2-M), according to the previous reference (Osada et al., Biochem Biophys Res Commun. 1993; 142: 100-6). Since the conjugate of α-Gal A from coffee beans and α-2-M can be internalized by cultured fibroblasts derived from Fabry hemizygotes, the conjugate of α-Gal A and α-2-M is expected to be internalized by the cells as well. Alternatively, the wild type α-Gal A can be added into the culture medium of skin fibroblasts derived from Fabry patient with no residual enzyme activity as described in Blom et al., Am J Hum Gen. 2003; 72: 23-31.

[0123] Results. The half-life of the coffee bean α-Gal A is about 2 hr as described previously (Osada et al., Biochem Biophys Res Commun. 1987;143: 954-8). It is expected that the half-life of the α-Gal A / α-2-M conjuga...

example 3

Co-Administration of DGJ to Fabry Mice Treated by Infusion of Replacement Enzyme

[0124] Enzyme replacement therapy for Fabry disease has been developed by Genzyme Corporation as described above. It is expected that co-administration of DGJ to Fabry knock-out (KO) mice treated by infusion of the replacement enzyme increases the stability, e.g., half-life of the replacement enzyme in vivo, because the ASSC DGJ stabilizes the enzyme and prevents degradation. DGJ is orally administered to the KO mice after infusion of the wild type α-Gal A according to the protocol described previously (Ioannu et al., Am J Hum Genet. 2001; 68:14-25). The α-Gal A activity in various tissues including heart, kidney, spleen, liver, and lung as well as serum is determined over a period of time, and compared with those from the control mice that do not receive DGJ, and mice that receive only DGJ but no enzyme. The extended time will indicate that co-administration of ASSC can improve the efficiency of enzyme...

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Abstract

This application provides methods of improving protein replacement therapy by combining protein replacement therapy with active site-specific chaperones (ASSC) to increase the stability and efficiency of the protein being administered. The application further provides stable compositions comprising the purified protein and an ASSC, and methods of treatment by administering the compositions.

Description

[0001] This application is a divisional of U.S. application Ser. No. 11 / 317,404, filed on Dec. 23, 2005 (pending), which is a continuation of U.S. application Ser. No. 10 / 771,236, filed on Feb. 2, 2004 (abandoned), which claims priority benefit from U.S. Provisional Application Ser. No. 60 / 444,136, filed Jan. 31, 2003 (abandoned), each of which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION [0002] This application provides methods of improving protein replacement therapy by combining protein replacement therapy with active site-specific chaperones (ASSC) to increase the stability and efficiency of the protein being administered. The application further provides compositions comprising the purified protein and an ASSC. BACKGROUND Protein Deficiency [0003] Proteins are synthesized intracellularly according to the genomic nucleotide sequence of a particular gene through transcription, translation, and other processes. Protein deficiency can be caused by a m...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/47A61K31/445A61K31/46A61KC12N9/24C12N9/40C12N9/99
CPCA61K9/0019A61K38/47A61K31/46A61K31/445A61P3/00A61P3/02A61P3/08A61P43/00A61P9/00A61P9/14
Inventor FAN, JIAN-QIANG
Owner FAN JIAN QIANG
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