Abstract
The present invention relates to the use of galactose analogues of formula (I) with the capacity to stabilize the structure of the #-GalA enzyme, for the treatment of lysosomal storage diseases and, in a preferred embodiment, for the treatment of Fabry disease. In addition, the present invention relates to pharmacological compositions having an effective amount of at least one of the galactose analogues described in the present document for the treatment of lysosomal storage diseases and, in a preferred embodiment, for the treatment of Fabry disease.
Claims
1. A method of treating Fabry disease comprising administering a pharmaceutical composition comprising a galactose analogue represented by the following formula: ##STR00004## wherein R1 is selected from the group consisting of N.sub.3 and CN, NH.sub.2, NHCONH.sub.2, NHCH.sub.3, CH.sub.2NH.sub.2 and CH.sub.2NHCONH.sub.2, and wherein Fabry disease is caused by a mutation affecting the folding of the enzyme α-galactosidase A, wherein the mutation is selected from the group consisting of p.R301Q, p.Q279R, p.P205S and p.L131Q.
2. The method according to claim 1, wherein R1 is selected from the group consisting of N.sub.3 and CN.
3. The method according to claim 1, wherein R1 is selected from the group consisting of N.sub.3 and CN.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) FIGS. 1.A and 1.B. Assessment of the increase in the enzymatic activity of α-GalA in a human cell line (293T) transfected with plasmids expressing different mutants of the α-GalA: p.R301Q, p.Q279R, p.P205S, treated with galactose, PB48 and PB51. In the FIG. 1.A, the increase in the enzymatic activity of α-GalA is shown for the three indicated treatments at a concentration of 5 μM. In the case of the FIG. 1.B, the three treatments used are at a concentration of 10 μM. In both figures, 3 columns for each of the mutations are observed. Each one of them represent a treatment to which the human cell line (293T), which was transfected with the plasmids expressing different mutants of the α-GalA, has been subjected. The column on the left (the closest to the vertical coordinate axis), shows cells treated with galactose. The central column represents the cell line treated with PB48. The column on the right (the furthest to the vertical coordinate axis) represents the cell line treated with PB51. The increases in the activity have been calculated by subtracting from each obtained value the value of the activity of α-GalA obtained for the cells, transfected with the corresponding plasmid, which were not treated with any of the tested compounds (untreated cells).
(2) FIG. 2. Assessment of the enzymatic activity of α-GalA in cells (leukocytes) extracted from three hemizygous patients with Fabry disease carrying the mutation p.Q279R on the α-GalA. The mentioned cells were treated with PB48 at concentrations of 2.5 μM, 5 μM, 7.5 μM and 10 μM and with DGJ at concentrations of 2.5 μM, 5 μM and 10 μM.
(3) FIG. 3. Assessment of the enzymatic activity of α-GalA in cells (leukocytes) extracted from one hemizygous patient carrying the mutation p.Q279R on the α-GalA. The mentioned cells were treated with PB51 at concentrations of 2.5 μM, 5 μM, 7.5 μM and 10 μM and with DGJ at concentrations of 2.5 μM, 5 μM and 10 μM.
(4) FIG. 4. Assessment of the enzymatic activity of α-GalA in cells extracted from a hemizygous patient expressing the mutation p.Q279R, a heterozygous patient with the mutation p.Q279R and healthy volunteers, treated with PB48 at concentrations of 5 μM and 10 μM, PB51 at concentrations of 5 μM and 10 μM, DGJ at concentrations of 5 μM and 10 μM, and galactose at a concentration of 5 μM. For each one of the treatments 3 columns are shown, representing the cell groups that have been subjected to each one of the treatments. The column on the left (the closest to the vertical coordinate axis), represents leukocytes extracted from peripheral blood of one hemizygous patient for p.Q279R (group of hemizygous cell). The central column represents leukocytes extracted from peripheral blood of one heterozygous patient. The column on the right (the furthest of the vertical coordinate axis) shows leukocytes extracted from peripheral blood extracted of two healthy volunteers, one male and one female (controls). In the vertical axis, the activity of α-GalA, normalized to the untreated control, is represented.
(5) FIG. 5. Assessment of the enzymatic activity of the α-GalA with the mutatation p. L131Q in cells extracted (from a hemizygous patient and a heterozygous patient) treated with PB48 at concentrations of 2.5 μM, 5 μM, 7.5 μM and 10 μM or with DGJ at concentrations of 2.5 μM, 5 μM and 10 μM. For each one of the treatments at the specified concentration, 2 columns are observed, which represent the cell groups that were subjected to those treatments. The column on the left (the closest to the vertical coordinate axis), represents leukocytes extracted from peripheral blood of one hemizygous patient with the p.L131Q mutation. The column on the right (the furthest of the vertical coordinate axis) shows leukocytes extracted from peripheral blood of a heterozygous patient for with the p.L131Q mutation.
EXAMPLES
(6) Next, we proceed to present, as an explicative example and with no mean to limit the filed of the invention, some results of the efficacy assays, which support the invention, where the enzymatic activity of α-GalA was evaluated, following the treatment with the pharmacological chaperones of the formula (I) described herein.
Experiment 1
(7) PB48 and PB51 analogues were tested at the concentrations of 5 and 10 μM, in a human cell line (293T), transfected with different plasmids expressing different mutants of the α-GalA (p.R301Q, p.Q279R, p.P205S) and their effect were compared with the obtained by treating the same cells with galactose at the same concentration.
(8) As shown in the FIGS. 1.A and 1.B, PB48 and PB51 cause a positive increase in the enzymatic activity of the different α-GalA mutants. The increase of the enzymatic activity of α-GalA is particularly evident in the cells treated with PB48 in most of the cases.
Experiment 2
(9) Similar studies were carried out in leukocytes extracted from peripheral blood of 3 hemizygous patients for the mutation p.Q279R. In these studies, the activity of α-GalA was evaluated in cells treated with PB48 at the concentrations of 2.5 μM, 5 μM, 7.5 μM and 10 μM and with DGJ at the concentrations of 2.5 μM, 5 μM and 10 μM. As shown in FIG. 2, PB48 significantly increases the activity of α-GalA at the concentrations of 2.5 μM, 5 μM and 7.5 μM and is more effective than DGJ in cells of hemizygous patients with the mutation p.Q279R.
(10) On the other hand, the activity of α-GalA was also tested in cells of a hemyzigous patient with the mutation p.Q279R treated with PB51 at the concentrations of 2.5 μM, 5 μM, 7.5 μM and 10 μM and with DGJ at concentrations of 2.5 μM, 5 μM and 10 μM, see FIG. 3.
(11) As shown in FIG. 3, PB51 determines an increase of the enzymatic activity, which is higher than the one obtained for the treatment with DGJ. The treatment with PB51 at the concentration of 2.5 μM also determines an increase in α-GalA activity in comparison with untreated cells.
(12) Therefore, these results demonstrate that the analogues described in the present document, and in particular PB48, are a very suitable alternative for the patients suffering Fabry disease, and who cannot be treated with DJG, since a treatment based on DGJ do not determines the necessary increase of activity for the treatment of the disease on the mutated α-GalA, when the mutation of the GLA gene is the p.Q279R.
Experiment 3
(13) Studies were carried out on the activity of α-GalA in leukocytes extracted from peripheral blood of a hemizygous patient with the mutation p.Q279R (group of hemizygous cells), a heterozygous patient, and two healthy volunteers (a male and a female, indicated as control). In the presented studies the α-GalA activity was evaluated in cells treated with PB48 at the concentrations of 5 μM, and 10 μM. In a second group cells were treated with PB51 at concentrations of 5 μM, and 10 μM and with DGJ at concentrations of 5 μM, and 10 μM. In order to compare the results obtained for the treatments with the galactose analogues that are described herein, the assessment of α-GalA activity was performed for the same groups of patients' cells, following treatment with galactose at the concentration of 5 μM, and in patients' cells groups, which were not subjected to any treatment.
(14) As shown in the FIG. 4, PB48, at the concentration of 5 μM, causes a significant increase of the enzymatic activity in comparison with DGJ, at the same concentration, in the cells of the hemizygous patient and a significant increase of the activity with respect to the untreated cells. The values of the α-GalA activity were normalized to the untreated control, that is, the value of α-GalA enzymatic activity in the samples that have been subjected to each one of the pharmacological treatment (PB48, PB51 or DGJ) was divided by the α-GalA activity value obtained in the corresponding untreated control sample (value of α-GalA activity obtained in the cells of each patients who has not received the treatment).
(15) On the other hand, it has been observed that DGJ (5 μM) is more effective than PB48 (504) as stabilizer of the protein when tested in the leukocytes of the heterozygous patient. This leads to the conclusion that DGJ (504) can be a more efficient chaperone for the α-Gal A native form (wild type), while PB48 at the same concentration is more efficient than DGJ when the mutated form of the enzyme (p.Q279R) is exclusively present.
(16) The data obtained in healthy controls at the same concentration (5 μM) confirm this conclusion.
Experiment 4
(17) PB48 and DGJ were tested in cells (leukocytes) from a hemizygous patient with the p.L131Q mutation of the α-GalA, which produces the classic phenotype of the Fabry disease and a heterozygous patient with the same mutation (p.L131Q), who also expresses the wild type allele of the enzyme.
(18) As observed in FIG. 5, the enzymatic activity of the α-GalA is significantly higher in cells of the hemizygous patient treated with PB48 at the concentration of 5 μM when compared with untreated cells and with cells treated with DGJ at the same concentration. On the other hand, it can be verified that the enzymatic activity of the α-GalA is higher in the cells of the heterozygous patient treated with DGJ.
