Treatment of alpha-galactosidase a deficiency

a technology of alpha-galactosidase and deficiency, which is applied in the field of treatment of alpha-galactosidase deficiency, can solve the problems of limited efficacy of current preparations of -gal a, extreme pain in the extremities, and pain in the extremities, and achieves the effects of increasing the sialic acid content of -gal, prolonging the circulating half-life, and increasing the phosphoryl

Inactive Publication Date: 2011-11-17
SELDEN RICHARD F +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The invention provides highly purified α-Gal A preparations, and various methods for purifying the α-Gal A glycoforms. The invention also provides α-Gal A preparations with altered charge and methods for making those preparations. Charge alterations are achieved by increasing the sialic acid content of α-Gal A and/or by increasing the phosphorylation of α-Gal A. The invention further provides α-Gal A preparations that have an extended circulating half-life in a mammalian host, and methods for making same. Finally,

Problems solved by technology

Fabry disease also affects the peripheral nervous system, causing episodes of agonizing, burning pain in the extremities.
A reduction in α-Gal A may be the cause of such cardiac abnormalities.
However, current preparations of α-Gal A have limited efficacy.
The use of proteinaceous lectin affinity resins and substrate analog resins is typically associated with the continuous leaching of the affinity agent from the solid support (Marikar et al., Anal. Biochem. 201: 306-310 (1992), resulting in contamination of the purified product with the affinity agent either free in solution or bound to eluted protein.
Such contaminants make the product unsuitable for use in pharmaceutical preparations.

Method used

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  • Treatment of alpha-galactosidase a deficiency
  • Treatment of alpha-galactosidase a deficiency
  • Treatment of alpha-galactosidase a deficiency

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation and Use of Constructs Designed to Deliver and Express α-Gal A

[0150]Two expression plasmids, pXAG-16 and pXAG-28, were constructed. These plasmids contain human α-Gal A cDNA encoding the 398 amino acids of the α-Gal A enzyme (without the α-Gal A signal peptide); the human growth hormone (hGH) signal peptide genomic DNA sequence, which is interrupted by the first intron of the hGH gene; and the 3′ untranslated sequence (UTS) of the hGH gene, which contains a signal for polyadenylation. Plasmid pXAG-16 has the human cytomegalovirus immediate-early (CMV IE) promoter and first intron (flanked by non-coding exon sequences), while pXAG-28 is driven by the collagen Iα2 promoter and exon 1, and also contains the β-actin gene's 5′ UTS, which contains the first intron of the β-actin gene.

[0151]1.1 Cloning of the Complete α-Gal A cDNA, and Construction of the α-Gal A Expression Plasmid pXAG-16

[0152]The human α-Gal cDNA was cloned from a human fibroblast cDNA library that was constru...

example 2

α-Gal A Purification

[0180]The following is a preferred method for producing, purifying, and testing α-Gal A. The purification process maintains α-Gal A in a soluble, active, native form throughout the purification process. The protein is not exposed to extremes of pH, organic solvents or detergents, is not proteolytically cleaved during the purification process, and does not form aggregates. The purification process is designed not to alter the distribution of α-Gal A glycoforms.

[0181]2.1 Purification of α-Gal A

[0182]Example 2.1 illustrates that α-Gal A may be purified to near-homogeneity from the conditioned medium of cultured human cell strains that have been stably transfected to produce the enzyme. α-Gal A is isolated from α-Gal A containing media using a series of five chromatographic steps. The five steps utilize various separation principles which take advantage of different physical properties of the enzyme to separate α-Gal A from contaminating material. Included are hydrop...

example 3

Pharmaceutical Formulation

[0219]Preparation of Buffer Solutions and Formulations

[0220]α-Gal A Purified Bulk is diluted to final concentration with α-Gal A Diluent. Based on the volume of purified bulk to be formulated, the concentration of α-Gal A (mg / mL), and the desired concentration of α-Gal A in the final formulation, the volume of α-Gal A diluent required is determined. α-Gal A diluent is prepared within 24 hours of use by mixing appropriate quantities of WFI, sodium chloride, and sodium phosphate monobasic, and adjusting the pH to 6.0 with sodium hydroxide solution. The composition of α-Gal A Diluent is listed in Table 8.

TABLE 8COMPOSITION OF α-GAL A DILUENT (per Liter)ComponentPart NumberQuantitySodiumchloride(USP)100-19168.8 gSodium hydroxide, 5N200-1903qs to adjust pH to 6.0Sodium phosphate,100-19133.5 gmonobasic (USP)Water for Injection (USP)100-2301qs ad 1.0 L

[0221]One liter or smaller volumes of α-Gal A Diluent are filtered by vacuum filtration using sterile 0.2 mm nylon...

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Abstract

The invention provides highly purified α-Gal A, and various methods for purifying it; α-Gal A preparations with altered charge and methods for making those preparations; α-Gal A preparations that have an extended circulating half-life in a mammalian host, and methods for making same; and methods and dosages for administering an α-Gal A preparation to a subject.

Description

RELATED APPLICATIONS[0001]The present application is a continuation-in-part of U.S. Ser. No. 08 / 928,881, filed on Sep. 13, 1996, and PCT / US97 / 16603, filed on Sep. 12, 1997, which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to methods and compositions for the treatment of α-galactosidase A deficiency.BACKGROUND OF THE INVENTION[0003]Fabry disease is an X-linked inherited lysosomal storage disease characterized by severe renal impairment, angiokeratomas, and cardiovascular abnormalities, including ventricular enlargement and mitral valve insufficiency. Fabry disease also affects the peripheral nervous system, causing episodes of agonizing, burning pain in the extremities. Fabry disease is caused by a deficiency in the enzyme α-galactosidase A (α-Gal A). α-Gal A is the lysosomal glycohydrolase that cleaves the terminal α-galactosyl moieties of various glycoconjugates. Fabry disease results in a blockage of the catabolism of the neutral...

Claims

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Application Information

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IPC IPC(8): A61K38/47A61P3/00C12N9/40A61K38/00A61K38/12A61K48/00C07K14/61C12N9/24C12N15/54C12N15/56
CPCA61K38/00C07K14/61C12Y302/01022C07K2319/02C12N9/2465C07K2319/00A61K38/47C12N15/85C12N2800/107A61P13/12A61P25/02A61P3/00A61P3/08A61P9/00
InventorSELDEN, RICHARD FBOROWSKI, MARIANNEKINOSHITA, CAROL M.TRECO, DOUGLAS A.
OwnerSELDEN RICHARD F