Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2001-08-10
2004-08-10
Barts, Samuel (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S317000, C514S277000, C514S281000, C546S219000, C435S208000
Reexamination Certificate
active
06774135
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method of enhancing the activity of lysosomal &agr;-Galactosidase A (&agr;-Gal A) in mammalian cells and for treatment of glycosphingolipid storage diseases, in particular Fabry disease, by administration of 1-deoxy-galactonojirimycin and related compounds.
2. Background Information
Fabry disease (1) is a glycosphingolipid lysosomal storage disease caused by an X-linked inherited deficiency of lysosomal &agr;-galactosidase A (&agr;-Gal A), an enzyme responsible for the hydrolysis of terminal &agr;-galactosyl residue from glycosphingolipids. A deficiency in the enzyme activity results in a progressive deposition of neutral glycosphingolipids, predominantly globotriaosylceramide (ceramide trihexoside, CTH), in vascular endothelial cells causing renal failure along with premature myocardial infarction and strokes in patients with this condition (2). This disorder is classified by clinical manifestations into two groups: a classic form with generalized vasculopathy and an atypical variant form, with clinical manifestations limited to heart. Recently, the atypical variant of the disease was found in 10% of adult male patients with unexplained left ventricular hypertrophy, increasing the estimation of frequency for the disorder (3). Like other glycosphingolipid lysosomal storage diseases, enzyme replacement therapy, gene therapy, bone marrow transplantation, and substrate deprivation are suggested as potential strategies for the treatment of this disease (4). However, at the moment the only treatment for this disorder is symptomatic management. Therefore, development of a new therapeutic strategy for this disease is urgently needed.
Studies (5) on residual &agr;-Gal A activity of mutant enzymes revealed that some of mutant enzymes have similar kinetic properties to normal &agr;-Gal A but with significant instability. This is considered as the case for most of atypical variant patients who generally showed higher residual &agr;-Gal A activity than classical Fabry patients. For example (6), a purified mutant &agr;-Gal A with a genotype of Q279E, found in a patient with atypical variant of Fabry disease, had the same Km and Vmax as the normal enzyme, but lost most of the enzyme activity by incubating the enzyme at pH 7.0 at 37° C. for 30 min while the normal enzyme was stable under the same condition. Both mutant and normal enzymes were stable at pH 5.0 at 37° C. Furthermore, the majority of the mutant enzyme protein in cells formed aggregate in endoplasmic reticulum (ER) and was quickly degraded (7), suggesting that the deficiency of the enzyme activity in this mutant maybe primarily caused by the unsuccessful exit of ER leading to excessive degradation of the enzyme protein. The present invention focuses on the aid of smooth escape of the enzyme from ER to prevent the degradation of the mutant enzyme.
SUMMARY OF THE INVENTION
The strategy of the invention is based on the following model. The mutant enzyme protein tends to fold in an incorrect conformation in ER where the pH is around 7. As a result, the enzyme is retarded from the normal transport pathway from ER through the Golgi apparatus and endosome to the lysosome, but instead is subjected to degradation. On the other hand, the enzyme protein with a proper conformation is transported to the lysosome smoothly and remains in an active form because the enzyme is more stable at a pH of less than 5. Therefore, a compound which is able to induce a proper conformation in mutant enzyme may serve as an enhancer for the enzyme. The present inventors have unexpectedly found that strong competitive inhibitors for &agr;-Gal A at low concentrations enhance the mutant enzyme activity in cells, including mutant &agr;-Gal A gene transfected COS-1 cells, fibroblasts from a transgenic mouse overexpressing mutant &agr;-Gal A, and lymphoblasts from Fabry patients.
It is noted that while the above is believed to be the mechanism of operation of the present invention, the success of the invention is not dependent upon this being the correct mechanism.
Accordingly, it is one object of the present invention to provide a method of preventing degradation of mutant &agr;-Gal A in mammalian cells, particularly in human cells.
It is a further object of the invention to provide a method of enhancing &agr;-Gal A activity in mammalian cells, particularly in human cells. The methods of the present invention enhance the activity of both normal and mutant &agr;-Gal A, particularly of mutant &agr;-Gal A which is present in certain forms of Fabry disease.
In addition, the methods of the invention are also expected to be useful in nonmammalian cells, such as, for example, cultured insect cells and CHO cells which are used for production of &agr;-Gal A for enzyme replacement therapy.
Compounds expected to be effective in the methods of the invention are galactose and glucose derivatives having a nitrogen replacing the oxygen in the ring, preferably galactose derivatives such as 1-deoxygalactonojirimycin and 3,4-diepi-&agr;-homonojirimycin. By galactose derivative is intended to mean that the hydroxyl group at the C-3 position is equatorial and the hydroxyl group at the C-4 position is axial, as represented, for example, by the following structures:
wherein R
1
represents H, methyl or ethyl; R
2
and R
3
independently represent H, OH, a simple sugar (e.g. —O—galactose), a 1-3 carbon alkyl, alkoxy or hydroxyalkyl group (e.g. CH
2
OH).
Other specific competitive inhibitors for (x-galactosidase, such as for example, calystegine A
3
, B
2
and B
3
, and N-methyl derivatives of these compounds should also be useful in the methods of the invention. The calystegine compounds can be represented by the formula
wherein for calystegine A
3
: R
1
=H, R
2
=OH, R
3
=H, R
4
=H; for calystegine B
2
: R
1
=H, R
2
=OH, R
3
=H, R
4
=OH; for calystegine B
3
: R
1
=H, R
2
=H, R
3
=OH, R
4
=OH; for N-methyl-calystegine A
3
: R
1
=CH
3
, R
2
=OH, R
3
=H, R
4
=H; for N-methyl-calystegine B
2
: R
1
=CH
3
, R
2
=OH, R
3
=H, R
4
=OH; and for N-methyl-calystegine B
3
: R
1
=CH
3
, R
2
=H, R
3
=OH, R
4
=OH.
It is yet a further object of the invention to provide a method of treatment for patients with Fabry disease. Administration of a pharmaceutically effective amount of a compound of formula
wherein
R
1
represents H, CH
3
or CH
3
CH
2
;
R
2
and R
3
independently represent H, OH, a 1-6 carbon alkyl, hydroxyalkyl or alkoxy group (preferably 1-3), or a simple sugar;
R
4
and R
5
independently represent H or OH; or a compound selected from the group consisting of 2,5-dideoxy-2,5-imino-D-mannitol, &agr;-homonojirimycin, 3,4-diepi-&agr;-homonojirimycin, 5—O—&agr;-D-galactopyranosyl-&agr;-homonojirimycin, 1-deoxygalactonojirimycin, 4-epi-fagomine, and 1-Deoxy-nojirimycin and their N-alkyl derivatives, will alleviate the symptoms of Fabry disease by increasing the activity of mutant &agr;-Gal A in patients suffering from Fabry disease. Other competitive inhibitors of &agr;-Gal A, such as calystegine compounds and derivatives thereof should also be useful for treating Fabry disease.
Persons of skill in the art will understand that an effective amount of the compounds used in the methods of the invention can be determined by routine experimentation, but is expected to be an amount resulting in serum levels between 0.01 and 100 &mgr;M, preferably between 0.01 and 10 &mgr;M, most preferably between 0.05 and 1 &mgr;M. The effective dose of the compounds is expected to be between 0.5 and 1000 mg/kg body weight per day, preferably between 0.5 and 100, most preferably between 1 and 50 mg/kg body weight per day. The compounds can be administered alone or optionally along with pharmaceutically acceptable carriers and excipients, in preformulated dosages. The administration of an effective amount of the compound will result in an increase in &agr;-Gal A activ
Fan Jian-Qiang
Ishii Satoshi
Barts Samuel
Darby & Darby
Henry Michael C.
Mount Sinai School of Medicine of New York University
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