Assays for diagnosing and evaluating treatment options for fabry disease

Abstract

To provide means to treat a patient with Fabry disease.SOLUTION: The present invention provides a pharmaceutical composition for treating a patient with Fabry disease, containing 1-deoxygalactonojirimycin or a salt thereof in a therapeutically effective dose of 150 mg, where the pharmaceutical composition is administered to the patient every other day.SELECTED DRAWING: None

Description

[Technical field]

[0001] Cross-reference to related applications This patent application claims priority to U.S. Provisional Patent Application No. 60 / 801,089, filed May 16, 2006, and U.S. Provisional Patent Application No. 60 / 853,631, filed October 23, 2006.

[0002] Technical Field The present invention provides methods for determining whether a patient suffering from Fabry disease would benefit from treatment with a specific pharmacological chaperone. The present invention illustrates two methods, an in vitro method and an in vivo method, for determining α-galactosidase A responsiveness to a pharmacological chaperone, such as 1-deoxygalactonojirimycin, in a patient's cells. The present invention also provides methods for diagnosing Fabry disease in a patient suspected of having Fabry disease.

[0003] Background technology Fabry disease is a glycosphingolipid (GSL) lysosomal storage disease caused by an X-linked inherited deficiency of lysosomal α-galactosidase A (α-GAL), the enzyme responsible for the hydrolysis of terminal α-galactosyl residues from the glycosphingolipid (Brady et al., N Engl J Med. 1967; 276: 1163-7). The deficiency in the enzyme activity leads to the gradual accumulation of neutral glycosphingolipids, predominantly composed of globotriaosylceramide (ceramide trihexoside; CTH, GL-3), in the cells of Fabry patients. Symptoms can be severe and debilitating, including kidney failure and high risk of heart attacks and strokes. Some of the mutations cause changes in the amino acid sequence of α-GAL, which can result in the production of less stable α-GAL that does not fold into the correct conformational shape. Although the α-GAL produced in the patient's cells often maintains some level of biological activity potential, the cell's performance regulation mechanisms recognize and maintain the misfolded α-GAL in the endoplasmic reticulum, or ER, until it is eventually transported to another part of the cell for degradation and elimination. As a result, little or no α-GAL is transported to the lysosomes, where it normally hydrolyzes GL-3. This leads to the accumulation of GL-3 within the cell, which is believed to be responsible for the symptoms of Fabry disease. Furthermore, accumulation of the misfolded α-GAL enzyme in the ER leads to cellular stress and an inflammatory-like response that may contribute to cellular dysfunction and disease.

[0004] Fabry disease is classified into three groups based on clinical manifestations: the classical form with systemic vasculopathy; an atypical variant with clinical manifestations restricted to cardiac tissue; and late-onset disease (with a moderate to severe form of the disease, including female carriers). The incidence of the classic disease is estimated to be about 1:40,000 to 1:60,000 in men, and has been reported worldwide in various ethnic groups. Typically, affected men have little or no detectable α-GAL concentration and are the most severely affected. The clinical manifestations include angiokeratomas (small, raised reddish purple spots on the skin), acroparesis (burning sensation in the hands and feet), hypohidrosis (reduced ability to sweat), and characteristic corneal and lenticular opacities (The Metabolic and Molecular Bases of Inherited Disease, 8th ed., 2001, Scriver et al. eds., pp. 3733-3774, McGraw-Hill, NY). Lipid storage can lead to impaired arterial circulation and increased risk of heart attack and stroke. The heart also becomes enlarged and the kidneys may become gradually affected. Other symptoms include fever and gastrointestinal distress, especially after meals. The life expectancy of the affected male is reduced and death occurs in the fourth or fifth decade as a result of vascular disease of the heart, brain and / or kidneys.

[0005] Individuals affected by late-onset Fabry disease can be male or female. Late-onset Fabry disease manifests as atypical variants, and gradually emerging evidence indicates that there may be a significant number of "atypical variants" that are unexplained in the world. Females who inherit an X chromosome containing an α-GAL mutation may show symptoms later in life, which significantly increases the prevalence of the disease. These patients typically experience disease symptoms for the first time in their adulthood, and often have disease symptoms localized to a single organ. For example, many men and women affected by late-onset Fabry disease suffer from left ventricular hypertrophy of the heart. Late-onset Fabry disease may also manifest as strokes of unknown etiology. As the patient ages, cardiac complications of the disease may progress and lead to death. In contrast, patients with milder "cardiac variants" of Fabry disease usually have 5-15% of normal α-GAL activity and exhibit left ventricular hypertrophy or cardiomyopathy. These patients remain essentially asymptomatic, even though their classically affected counterparts are at significant risk. Cardiac variants are seen in 11% of adult male patients with unexplained left ventricular hypertrophic cardiomyopathy, suggesting that Fabry disease may be more frequent than previously expected (Nakao et al., N. Engl. J. Med., 1995; 333: 288-293).

[0006] The α-GAL gene has been mapped to Xq22 (Bishop et al., Am. J. Hum. Genet., 1985; 37: A144), and the full-length cDNA and the complete 12-kb genomic sequence encoding α-GAL have been reported (Calhoun et al., Proc. Natl. Acad. Sci. USA. 1985; 82: 7364-7368; Bishop et al., Proc. Natl. Acad. Sci. USA. 1986; 83: 4859-4863; Tsuji et al., Eur. J. Biochem. 1987; 165: 275-280; and Kornreich et al., Nucleic Acids Res. 1989; 17: 3301-3302). There is a remarkable genetic heterogeneity of the mutations causing Fabry disease (The Metabolic and Molecular Bases of Inherited Disease, 8th ed., 2001, Scriver et al., eds., pp. 3733-3774, McGraw-Hill, NY; Eng et al., Am. J. Hum. Genet. 1993; 53: 1186-1197; Eng et al., Mol. Med. 1997; 3: 174-182; and Davies et al., Eur. J. Hum. Genet. 1996; 4: 219-224). To date, a variety of missense, nonsense, and splicing mutations, as well as small deletions and insertions, and large gene rearrangements have been reported, although the majority of mutations are missense mutations. Fabry disease is heterogeneous and it is often difficult to correlate genotype with phenotype. People with the same genotype often show different clinical symptoms and disease pathology. However, a correlation between residual enzyme activity and disease severity has been found, with lower α-GAL activity resulting in the most severe disease. The majority of α-GAL mutations are missense mutations, most of which are outside the catalytic site, but it is difficult to predict which mutations will result in an unstable enzyme that can be rescued by specific pharmacological chaperones (SPCs) that stabilize the unstable enzyme, and which mutations will not be stabilized by the SPCs.

[0007] Diagnosis of Fabry Disease Fabry disease is rare, involves multiple organs, has a wide age range of onset, and is heterogeneous, so obtaining a proper diagnosis is one of the challenges. It is rarely noticed by health professionals and is frequently misdiagnosed. Some examples of diagnoses that may be important in patients who are eventually diagnosed as having Fabry disease include mitral valve prolapse, glomerulonephritis, idiopathic proteinuria, systemic lupus erythematosus, Whipple's disease, acute abdominal disease, ulcerative colitis, acute intermittent porphyria, and occult malignancies. Thus, even for typical affected men, diagnosis typically takes about 5-7 years or even longer. This is a major problem because the longer a person is affected by Fabry disease, the more damage the affected organs are likely to cause and the more severe the person's condition may become. The diagnosis of Fabry disease is most frequently confirmed on the basis of low α-GAL activity in plasma or peripheral white blood cells (WBCs), combined with mutation analysis once the patient is symptomatic. Diagnosis in females is much more challenging, since enzymological identification of carrier females is unreliable due to random X-chromosome inactivation in some cells of carriers. For example, some obligate carriers (typically daughters of affected men) have α-GAL enzyme activity ranging from normal to very low activity. Since carriers can have normal α-GAL enzyme activity in white blood cells, only identification of the α-GAL mutation by genetic testing provides accurate carrier identification and / or diagnosis.

[0008] Treatment of Fabry disease The only therapy approved for treating Fabry disease is enzyme replacement therapy, which typically involves intravenous infusion of the corresponding wild-type protein (Fabrazyme™, Genzyme Corp.). One of the main problems with protein replacement therapy is achieving and maintaining a therapeutically effective amount of protein in vivo, as the injected protein is rapidly degraded. A common method to overcome this problem is to perform multiple injections of high doses, which is costly. Protein replacement therapy has several associated problems, such as difficulties with large-scale production, purification, and storage of properly folded protein; problems with the availability of glycosylated native protein; development of anti-protein immune responses; and inability of the protein to cross the blood-brain barrier and ameliorate central nervous system pathology (i.e., low bioavailability). Furthermore, the replacement enzyme cannot enter the heart or kidney in sufficient quantities to reduce substrate accumulation in renal podocytes and cardiac myocytes, which is prominent in the pathology of Fabry disease.

[0009] Gene therapy using recombinant vectors containing nucleic acid sequences encoding functional proteins or genetically modified human cells expressing the functional proteins has also been evaluated to treat protein deficiencies and other diseases that would benefit from protein replacement. Although promising, this approach is limited by technical difficulties, such as the inability of vectors to infect or transduce dividing cells, low expressibility of the target gene, and regulation of its expression once the gene is delivered. A third, more recent approach to treating some enzyme deficiencies involves the use of small molecule inhibitors to reduce the production of the natural substrate of the defective enzyme protein, thereby ameliorating the pathology. This "substrate reduction" approach has been specifically described for a group of about 40 related enzyme disorders called lysosomal storage disorders, including glycosphingolipid storage disorders. The small molecule inhibitors proposed for use as therapeutic agents reduce the amount of cellular glycolipids that are involved in the synthesis of cellular glycolipids and need to be destroyed by the defective enzyme. This approach is also limited in that glycolipids are necessary for biological functions, especially neurological functions, and excessive deficiency can cause deleterious effects.

[0010] It has been previously shown that the binding of small molecule inhibitors of enzymes related to LSDs can increase the stability of both mutant and corresponding wild-type enzymes (U.S. Patent Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; 6,916,829; and 7,141,582; all of which are incorporated herein by reference). In particular, it has been found that administration of small molecule derivatives of glucose and galactose, which are specific, selective, and competitive inhibitors of some target lysosomal enzymes, effectively increases the stability of the enzymes in cells in vitro and, as a result, increases the transportability of the enzymes to the lysosomes. Thus, by increasing the amount of enzymes in the lysosomes, it is expected that the hydrolysis of the enzyme substrates will be increased. The original theory behind this method is as follows: since the mutant enzyme protein is unstable in the ER (Ishii et al., Biochem. Biophys. Res. Comm. 1996; 220: 812-815), the enzyme protein is delayed in the normal transport pathway (ER → Golgi apparatus → endosomes → lysosomes) and is degraded early. Therefore, compounds that bind to and increase the stability of the mutant enzyme can function as "chaperones" for the enzyme and increase the amount of it that can exit the ER and travel to the lysosomes. Furthermore, since the folding and trafficking of some wild-type proteins is defective and up to 70% of some wild-type proteins are degraded, in some cases, before reaching their final cellular location, the chaperones can be used to stabilize the wild-type enzyme and increase the amount of it that can exit the ER and travel to the lysosomes. This method has been shown in vitro and in vivo to increase several lysosomal enzymes, including β-glucocerebrosidase and α-glucosidase, deficiencies of which are associated with Gaucher and Pompe diseases, respectively.

[0011] However, as shown above, successful candidates for SPC therapy must have certain mutations that result in the production of an enzyme that has the potential to stabilize and fold into a conformation that allows export from the ER. Mutations that cause significant truncation of the enzyme, such as nonsense mutations, or mutations in the catalytic domain that prevent binding of the chaperone, are not likely to be "rescuable" or "enhanced" using SPC therapy. Missense mutations outside the catalytic site are quite likely to be rescued using SPCs, but do not necessarily require screening for responsive mutations. This makes it extremely difficult, if not impossible, to predict whether a particular Fabry patient will respond to treatment with SPC, even when Fabry disease is diagnosed by detecting deficient α-GAL activity in WBCs. Moreover, WBCs only survive in culture (in vitro) for a short period of time, making it difficult to screen for SPC enhancement of α-GAL. In order to effectively apply SPC therapy, a widely applicable, rapid and efficient method for screening patients for responsiveness to SPC therapy must be employed prior to initiating treatment. Thus, there remains a need in the art for a relatively non-invasive method for rapidly assessing enzyme enhancement for potential therapeutic approaches before a treatment decision is made, taking into account both the cost and emotional benefits to the patient.

[0012] Means for solving the problem Summary of the Invention The present invention provides two methods for determining whether a patient is a candidate for SPC therapy. Specifically, the present invention provides in vivo and in vitro assays for evaluating α-GAL activity in blood cells from patients with Fabry disease in the presence or absence of SPC. The present invention also includes criteria for the evaluation of SPC as a treatment option for any number of other protein disorders and / or enzyme deficiencies. The present invention also provides diagnostic kits that include the components necessary to perform the assay. The present invention further provides an improved method for the diagnosis of Fabry disease by measuring α-GAL activity in T cells from patients who may be affected by Fabry disease. [Brief description of the drawings]

[0013] [Figure 1] Time course of enhancement for A97V. T-cells carrying the A97V mutation in α-GAL A were cultured in the absence (open circles) or presence (closed circles) of 20 μM DGJ for 1-4 days and then assayed for α-GAL activity. Change of medium after 2 days and replacement with fresh medium with (open triangles) or without (closed triangles) DGJ had no effect on the observed enzyme activity. [Figure 2A] Concentration dependence of DGJ in T-cells from normal controls and Fabry patients. T-cells from a normal individual (2A) were incubated with DGJ ranging from 2 nM to 200 μM for 3 days and then assayed for α-GAL activity. Results of these three experiments at different days are shown. T-cells from Fabry patients carrying the A97V (2B), R112H (2C), or R301Q (2C) mutations were incubated with DGJ ranging from 2 nM to 200 μM for 3 days and then assayed for α-GAL activity. Three independent DGJ treatment experimental groups, each performed in triplicate, are shown. [Figure 2B]Concentration dependence of DGJ in T-cells from normal controls and Fabry patients. T-cells from a normal individual (2A) were incubated with DGJ ranging from 2 nM to 200 μM for 3 days and then assayed for α-GAL activity. Results of these three experiments at different days are shown. T-cells from Fabry patients carrying the A97V (2B), R112H (2C), or R301Q (2C) mutations were incubated with DGJ ranging from 2 nM to 200 μM for 3 days and then assayed for α-GAL activity. Three independent DGJ treatment experimental groups, each performed in triplicate, are shown. [Figure 2C] Concentration dependence of DGJ in T-cells from normal controls and Fabry patients. T-cells from a normal individual (2A) were incubated with DGJ ranging from 2 nM to 200 μM for 3 days and then assayed for α-GAL activity. Results of these three experiments at different days are shown. T-cells from Fabry patients carrying the A97V (2B), R112H (2C), or R301Q (2C) mutations were incubated with DGJ ranging from 2 nM to 200 μM for 3 days and then assayed for α-GAL activity. Three independent DGJ treatment experimental groups, each performed in triplicate, are shown. [Diagram 3] T-cells from various Fabry patients were incubated for 3 days in the absence or presence of 20 μM DGJ and then assayed for α-GAL activity. The specific activity of α-GAL was plotted as a percentage of the mean normal value to show the rescue effect of DGJ on the various genotypes of Fabry patients. The lower panel shows the results of Western blots for each mutation probed with a polyclonal rabbit antibody specific for α-GAL. This demonstrates high protein stability for the enhanceable mutations A97V and R301Q, and no increase in protein amount for the mutations R356W, G132R, and A143P. [Figure 4]FIG. 1 is a graphical representation of in vivo enhancement of α-GAL activity in WBCs of Fabry patients following treatment with DGJ. [Diagram 5] FIG. 1 is a graphical representation of a comparison of in vitro and in vivo enhancement of α-GAL for ten genotypes.

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Detailed Description The present invention provides two methods that allow accurate determination of whether SPC enhances the enzyme activity of cells from patients with Fabry disease. These assays allow the determination of whether the patient is a candidate for SPC therapy. This novel in vitro assay is highly sensitive and can be performed on isolated T cells without the need for extensive in vivo culture and maintenance, which reduces the time required to perform the assay (compared to using fibroblasts). This assay can also be used as a diagnostic assay for patients suspected of having Fabry disease, especially female patients, because it is more sensitive than the WBC assay typically used to detect α-GAL activity. This novel in vivo assay is also non-invasive and provides a highly reliable method to determine whether SPC therapy will be effective in a particular patient. Furthermore, in combination with genotyping, both of these assays provide a method to determine whether a newly discovered α-GAL mutation (e.g., a spontaneous mutation) produces a misfolded α-GAL and, as a result, is potentially "rescueable" using SPC.

[0015] definition The terms used herein generally have their ordinary meaning in the art, within the context of this invention and the specific context in which each term is used. Some terms are described below or explained throughout the specification to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use the compositions. The term "Fabry disease" refers to an X-linked inborn error of glycosphingolipid catabolism due to a deficiency in lysosomal α-galactosidase A activity. This defect results in the accumulation of globotriaosylceramide (ceramide trihexoside) and related glycosphingolipids in vascular endothelial lysosomes of the heart, kidney, skin, and other tissues. The term "atypical Fabry disease (patient)" refers to a patient with the predominantly cardiac manifestation of the α-GAL deficiency, i.e., the gradual accumulation of globotriaosylceramide (GL-3) in cardiomyocytes leading to marked hypertrophy of the heart, particularly the left ventricle.

[0016] A "carrier" is a female who has one X chromosome with a deleted α-GAL gene and one X chromosome with a normal gene, and in which X chromosome inactivation of the normal allele is present in one or more cell types. Carriers are often afflicted with Fabry disease. "Patient" refers to a subject who has been diagnosed with a particular disease. The patient may be a human or an animal. "Fabry disease patient" refers to a subject (individual) who has been diagnosed with Fabry disease and has a mutated α-GAL as further defined below. Characteristic markers of Fabry disease typically affect women more mildly, but may occur in male hemizygous carriers and female carriers with the same incidence. Human α-galactosidase A (α-GAL) refers to the enzyme encoded by the human Gla gene, which consists of 429 amino acids and has the GenBank Accession No. U78027.

[0017] In one embodiment, the term "mutant α-GAL" as used herein includes α-GAL having a mutation in the gene encoding α-GAL that renders the enzyme unable to achieve a stable conformation under conditions normally present in the ER. Failure to achieve a stable conformation results in the enzyme not being delivered to the lysosome, but rather in substantial amounts of the enzyme being degraded. Such mutations are often referred to as "conformational mutations." Non-limiting examples of α-GAL mutations associated with Fabry disease that result in unstable α-GAL include L32P; N34S; T41I; M51K; E59K; E66Q; I91T; A97V; R100K; R112C; R112H; F113L; T141L; A143T; G144V; S148N; A156V; L166V; D170V; C172Y; G183D; P205T; Y207C; Y207S; N215S; A228P; S235C; D244N; P259R; N263S; N264A; G272S; S276G; Q279E; Q279K; Q279H; M284T; W287C; Includes I289F; M296I; M296V; L300P; R301Q; V316E; N320Y; G325D; G328A; R342Q; E358A; E358K; R363C; R363H; G370S; and P409A.

[0018] As used herein, the term "specific pharmacological chaperone" (SPC) or "pharmacological chaperone" refers to any molecule, including small molecules, proteins, peptides, nucleic acids, carbohydrates, etc., that specifically binds to a protein and has one or more of the following effects: (i) enhances the formation of a stable molecular conformation of the protein; (ii) induces transport of the protein from the ER to another cellular location, preferably the native cellular location, i.e., prevents ER-associated degradation of the protein; (iii) prevents aggregation of misfolded proteins; and / or (iv) at least partially restores or enhances wild-type function and / or activity of the protein. For example, a compound that specifically binds to α-GAL means that it binds to and chaperones α-GAL, but does not bind to and chaperone a general group of related or unrelated enzymes. More specifically, the term does not refer to endogenous chaperones, such as BiP, or non-specific agents, such as glycerol, DMSO, or deuterated water, i.e., chemical chaperones, which have been shown to be non-specific chaperones for a variety of proteins (Welch et al., Cell Stress and Chaperones, 1996; 1(2):109-115; Welch et al., Journal of Bioenergetics and Biomembranes, 1997; 29(5):491-502; U.S. Pat. Nos. 5,900,360, 6,270,954, and 6,541,195). In the present invention, the SPC is a reversible, competitive inhibitor.

[0019] A "competitive inhibitor" of an enzyme refers to a compound that is structurally similar to the chemical structure and molecular geometry of the enzyme substrate and binds at approximately the same position as the substrate. Thus, the inhibitor competes for the same active site as the substrate molecule, thereby increasing its Km. Competitive inhibitors are usually reversible, i.e., they can bind reversibly, if sufficient substrate molecules are available to replace the inhibitor. Thus, the amount of enzyme inhibition depends on the concentration of the inhibitor, the concentration of the substrate, and the relative affinities of the inhibitor and substrate for the active site. The following is a description of some of the specific pharmacological chaperones contemplated in the present invention. 1-deoxygalactonojirimycin refers to a compound having the structure:

[0020] [ka]

[0021] The term encompasses the free base and any salt forms. The hydrochloride salt of DGJ is known as migalastat hydrochloride (Migalastat). Additional SPCs against α-GAL are described by Fan et al. in U.S. Pat. Nos. 6,274,597, 6,774,135, and 6,599,919, and include α-3,4-di-epi-homonojirimycin, 4-epi-fagomine, and α-allo-homonojirimycin, N-methyl-deoxygalactonijirimycin, β-1-C-butyl-deoxygalactonijirimycin, and α-galacto-homonojirimycin, calystegine A3, calystegine B2, calystegine B3, N-methyl-calystegine A3, N-methyl-calystegine B2, and N-methyl-calystegine B3.

[0022] The term "specifically binds" as used herein refers to the interaction of a pharmacological chaperone with a protein, such as α-GAL, specifically with amino acid residues of the protein that are directly involved in contact with the pharmacological chaperone. A pharmacological chaperone specifically binds to a target protein, such as α-GAL, and exerts a chaperone effect on α-GAL, but not on a general group of related or unrelated proteins. The amino acid residues of a protein that interact with any given pharmacological chaperone may or may not be within the "active site" of the protein. Specific binding can be assessed through routine binding assays or through structural studies, such as co-crystallization, NMR, etc. The active site for α-GAL is the substrate binding site. "Deficient α-GAL activity" means α-GAL activity in cells from a patient that is below the normal range compared to α-GAL activity (using the same method) in normal individuals not suffering from or at risk of suffering from Fabry disease or any other disease (particularly a blood disorder).

[0023] The term "enhancing (enhancing) α-GAL activity" or "increasing α-GAL activity" as used herein refers to an increase in the amount of α-GAL in a stable conformation in a cell contacted with a pharmacological chaperone specific for α-GAL, relative to the amount in a cell (preferably the same cell type or the same cell, e.g., at an earlier time point) that has not been contacted with the pharmacological chaperone specific for α-GAL. The term also refers to increasing the amount of α-GAL delivered to the lysosome in a cell contacted with a pharmacological chaperone specific for α-GAL, relative to the delivery of α-GAL not contacted with a pharmacological chaperone specific for the protein. These terms relate to both wild-type and mutant α-GAL. In one embodiment, the increase in the amount of α-GAL in the cell is determined by measuring the amount of hydrolysis of an artificial substrate in a lysate from cells treated with the SPC. An increase in the amount of hydrolysis is indicative of high α-GAL activity. The term "α-GAL activity" refers to the normal physiological function of wild-type α-GAL in a cell. For example, α-GAL activity includes hydrolysis of GL-3.

[0024] A "responder" is a subject (diagnosed with or suspected of having Fabry disease) whose cells show sufficiently high α-GAL activity and / or symptomatic improvement or improvement in surrogate markers in response to contact with SPC. Non-limiting examples of improvement in surrogate markers for Fabry disease include increased α-GAL concentration or activity in cells (e.g., fibroblasts) and tissues; decreased accumulation of GL-3; decreased plasma concentrations of homocysteine ​​and vascular cell adhesion molecule-1 (VCAM-1); decreased accumulation of GL-3 in cardiomyocytes and valvular fibrocytes; decreased cardiac hypertrophy (especially the left ventricle), improved valvular insufficiency, and decreased arrhythmias; improved proteinuria; decreased urinary concentrations of lipids such as CTH, lactosylceramide, ceramide, and increased urinary concentrations of glucosylceramide and sphingomyelin (Fuller et al., Clinical Chemistry. 2005; 51: 688-694); absence of laminated inclusion bodies (Zebra bodies) in renal glomerular epithelial cells; improvement of renal function; subsidence of oligohidrosis; absence of angiokeratoma; and improvement of hearing abnormalities such as high frequency sensorineural hearing loss, gradual hearing loss, sudden hearing loss, or tinnitus. Improvement of neurological symptoms includes prevention of transient ischemic attacks (TIAs) or strokes; and improvement of neuropathic pain manifested as acroparesthesia (burning or tingling sensations in the extremities).

[0025] A dose that achieves one or more of the above responses is a "therapeutically effective dose." The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that are physiologically acceptable and typically do not cause untoward reactions when administered to humans. Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency listed in the Federal or State government or the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous solutions, saline solutions, and aqueous dextrose and glycerol solutions are preferred for use as carriers, particularly injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences", EW Martin, 18th Edition, or other editions.

[0026] The term "isolated" as used herein means that the specified material is removed from the environment in which it is normally found. Thus, an isolated biological material may be free of cellular components, i.e., components of the cell in which it is found or produced. In the case of a nucleic acid molecule, an isolated nucleic acid includes a PCR product, an mRNA band on a gel, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it can be found, and more preferably, the nucleic acid is no longer joined to non-regulatory, non-coding regions, or other genes located upstream or downstream of the gene contained in the isolated nucleic acid molecule, if found in the chromosome. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acids include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in one particular embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it is associated within the cell, or, if the protein is a membrane-associated protein, with the cell membrane. Isolated organelles, cells or tissues are removed from the anatomical site they are found in an organism. An isolated material can be, but need not be, purified.

[0027] The terms "about" and "approximately" generally refer to a tolerance of error in the measured quantity given the nature or precision of the measurement. Typically, an example of the tolerance of error is within 20 percent (%) of a given value or range, preferably within 10%, and more preferably within 5%. Alternatively, and particularly in biological systems, the terms "about" and "approximately" can refer to values ​​within a certain range of magnitude, preferably within 10-fold or 5-fold of a given value, and more preferably within 2-fold of it. Numerical quantities given herein are approximate values ​​unless otherwise stated, and the terms "about" and "approximately" can be presumed to be, unless expressly stated.

[0028] method To easily determine whether SPC therapy is an effective treatment for Fabry disease patients, including female carriers, we developed a simple, non-invasive DGJ rescue assay for α-GAL activity in WBCs, or a subpopulation of WBCs, from Fabry disease patients. I. In Vitro Assays In one embodiment, the diagnostic method of the present invention comprises purifying T cells and establishing a T cell culture from a blood sample from a Fabry patient (or a patient suspected of having Fabry disease). The T cell culture is then treated with an SPC, such as DGJ, for a sufficient period of time or not to reveal an enhanced (i.e., increased) α-GAL activity. The T cells are then lysed and the resulting lysate is used in an assay to measure enzyme activity. A sufficient increase in α-GAL activity in the lysate from cells treated with the SPC compared to α-GAL activity in the lysate from untreated cells indicates that the patient will have responded to SPC therapy (i.e., the patient is a "responder"). This aspect can be implemented as follows:

[0029] Leukocyte isolation WBCs (white blood cells) are prepared using standard techniques, such as collection, centrifugation, sorting, and washing. More specifically, they can be prepared according to the following steps: 1. A blood sample is taken from a Fabry patient. In a particular embodiment, a sample of about 8-10 mL is removed into a suitable container, such as a CPT tube from Becton-Dickenson (containing anti-coagulant and separation medium). 2. The blood sample is centrifuged to separate red blood cells from white blood cells and plasma. Typically, this step is performed in a tabletop centrifuge for about 20-30 minutes at about 1800xg and room temperature, or until the red blood cells are separated from the plasma and white blood cells (WBCs). However, other known white blood cell separation techniques can be used, such as Ficoll-Hypaque, Percoll, or other similar density gradient methods. In another embodiment, T cells are enriched from WBCs by magnetic separation using antibody-mediated or negative selection to remove other cell types to obtain unbound T cells. Any known method for enriching T cells can be used, but the more convenient and least expensive method is preferred.

[0030] 3. Discard half of the plasma layer (without disturbing the white blood cell layer) and transfer the remaining fluid containing the white blood cells to a centrifuge tube. 4. The WBCs are then pelleted and the pelleted cells are resuspended in an appropriate isotonic buffer, such as PBS, and centrifuged at about 320 x g for about 15-20 minutes, two or more times. 5. The pellet is then resuspended in a small volume of a suitable isotonic buffer, such as PBS. Half of the pellet is transferred to a labeled cryo-vial for freezing. The other half is used to establish T cell cultures, as described below. The samples to be frozen are centrifuged and then resuspended in a small volume of a suitable isotonic buffer, such as RPMI 1640+DMSO, before freezing.

[0031] T cell culture In one embodiment, T cell cultures are established by stimulating T cells present in the WBC preparation, for example according to a procedure such as: 1. The washed cells are resuspended in a suitable cell culture medium, for example, RPMI supplemented with T cell stimulating cytokines and / or mitogens. Suggested stimulating cytokines include IL-2, IL-12, IL-15, phytohemagglutinin (PHA), concanavalin A (con A), and pokeweed-derived mitogen. In one particular embodiment, the WBC are resuspended in a suitable volume of RPMI 1640 medium supplemented with FBS, IL-2, and a stimulating concentration of PHA. They are then transferred to a suitable culture vessel and incubated for a sufficient time to grow, for example, about 2-3 days. 2. The T cells can be expanded and then frozen at approximately 3×106 cells / vial using RPMI 1640 medium supplemented for cryopreservation, e.g., containing FCS and DMSO, sufficient to thaw and obtain a 5 mL culture of 5×105 viable cells / mL.

[0032] Although it is noted that one of skill in the art will be able to ascertain appropriate amounts of T cell stimulatory cytokines or mitogens, typically such agents are added in amounts of about 1 ng / mL to about 25 ng / mL (or about 100 U / mL) for cytokines, and concentrations of about 10 ng / mL to about 10 μg / mL for mitogens, with the low μg / mL range being most effective. Enzyme activity / enhancement assays Typically, the T cells isolated above (e.g., about 2.5×106) are grown in the absence or presence of the SPC, such as DGJ, in a suitable culture vessel in medium (previously thawed if frozen) for a time sufficient to estimate changes in α-GAL activity, e.g., 2 or 3 days. The dose of DGJ expected to enhance α-GAL is in the range of about 2 nM to about 150 nM, preferably about 1 nM to about 100 nM, and more preferably about 5 nM to about 50 nM. In one particular embodiment, DGJ is added at a dose of about 20 μM. Cells can be harvested by centrifugation and washed twice with PBS. The pellet can be stored frozen at −80° C. until assayed for enzyme activity. The cells are then lysed at room temperature or on ice by addition of lysis buffer (or deionized water) and physical disruption (pipetting, vortexing and / or stirring, and / or sonication), and the resulting lysates are then pooled on ice, and the pooled lysates are then divided into small aliquots and frozen.

[0033] The lysate can be thawed immediately prior to the assay and should be suspended and sonicated using a vortex mixer before being added to the appropriate wells of a microplate or the like. N-acetylgalactosamine (GalNAc) is then added to each well (to inhibit α-galactosidase B) followed by a brief incubation. 4-Methylumbelliferyl-α-D-galactopyranoside (4-MU Gal) or other suitable labeled DGJ substrate is then added, the plate is briefly mixed gently, covered and incubated at 37° C. for a period sufficient to hydrolyze the substrate, usually about 1 hour. To stop the reaction, NaOH-glycine buffer at a pH of 10.7 is added to each well and the plate is read in a fluorescent plate reader (e.g., a Wallac 1420 Victor3™ or similar instrument). Excitation and emission wavelengths are typically set at 355 nm and 460 nm, respectively. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 nM of 4-methylumbelliferone per hour. For each patient sample, at least three normal samples should also be tested simultaneously.

[0034] Those skilled in the art will be able to easily make various modifications to this assay. Examples of artificial substrates that can be used to detect α-GAL activity include, but are not limited to, p-nitrophenyl-α-D-galactopyranoside and 4-MU GAL. Obviously, only substrates that can be cleaved by human α-GAL are suitable for use. Although the use of fluorogenic substrates is preferred, other methods of measuring α-GAL activity are contemplated for use with this method, examples of which include the use of chromogenic substrates or immunoassay techniques.

[0035] Diagnosis and Prognosis The T cell assay can be easily improved to be used as a diagnostic assay for diagnosing Fabry disease by simply eliminating the step of culturing the T cells in the presence of DGJ before performing the enhancement assay. The activity of α-GAL in T cells established from an individual suspected of having Fabry disease can be quantified using T cells from a normal individual as a control instead. Furthermore, both α-GAL activity and SPC enhancement assays can be performed almost simultaneously using the same T cells from one patient sample. Since T cells can express more α-GAL (α-GAL activity in normal T cells is much higher compared to WBC) and there is less margin of error, it will be easier to confirm with higher certainty whether a patient has α-GAL activity below the normal range. Thus, the use of the T cell assay will likely allow misdiagnosis to be avoided. Furthermore, the improved assay also makes it possible to continuously monitor the progress of patients who have begun SPC therapy and to confirm that α-GAL activity is increased relative to that before the initiation of the therapy.

[0036] II. In vivo assays In a second embodiment, WBCs are assessed for enhancement of α-GAL by SPCs in vivo. In this embodiment, α-GAL activity is assessed in WBCs from a patient prior to administration of SPCs to obtain a baseline value. The patient is then administered DGJ daily (e.g., 150 mg / day) for a sufficient period of time, e.g., about 10 days to about 2 weeks, and then blood is drawn and changes in α-GAL activity from baseline values ​​are determined. No culturing of the cells is required prior to or after administration. During the in vivo evaluation period, the dose and mode of administration of DGJ can vary depending on the patient (since there is a high degree of heterogeneity in mutations) and on the patient's residual α-GAL activity. As non-limiting examples, the following doses and modes of administration are expected to be sufficient to increase α-GAL in many "salvageable" individuals: 25mg (twice a day (bid)); 50mg (once a day); 50mg (bid); 50mg (once every other day); 75mg (once a day); 75mg (bid); 100mg (once a day); 100mg (bid); 150mg (once a day); 150mg (bid); 150mg (once every other day); 250mg (once a day); 250mg (bid) and 250mg (once every other day). In particular embodiments, the dosage is 50 mg once per day; 50 mg once every other day; 150 mg once per day; 150 mg once every other day.

[0037] The dosage form of DGJ according to the present invention is in a formulation suitable for any administration route, but is preferably administered orally as an oral dosage form, such as tablet, capsule or solution.As an example, a patient is orally administered capsules containing 25mg, 50mg, 75mg or 100mg or combinations thereof.In the context of this assay, when administered orally, it is preferred that the patient is administered the DGJ without food (e.g., without food for 2 hours before and 2 hours after administration).Because when taken with food, bioavailability may be reduced, which may lead to inaccurate results. Patients receiving other treatments, such as ERT, should discontinue such treatment for at least about 28 days prior to the in vivo assay to ensure the most accurate results. Leukocyte isolation For the T cell in vivo assay, WSCs are isolated and sorted as described above, but no RPMI medium or DMSO should be added to the pellet prior to freezing (per step 5 above).

[0038] Enzyme activity / enhancement assays The pellet is thawed on ice, then the cells are lysed by addition of lysis buffer and physical disruption (e.g., vortex mixing and stirring, and / or sonication at room temperature) for a sufficient period of time, then the lysate is pooled in polypropylene tubes on ice, and the pooled lysate is further divided into aliquots for freezing. The WSC lysate is then thawed on ice and mixed (again by sonication and / or vortexing). Each lysate sample as well as standards and negative controls are then added to appropriate wells, for example in a 24- or 96-well microplate. For example, an equal amount of GalNAc in pH 4.6 citrate / phosphate buffer is added to each well, and then a labeled substrate, for example 4-MU Gal (also in pH 4.6 citrate / phosphate buffer), is added to all wells and incubated for a short period at ambient temperature. The plate is then mixed briefly and incubated at 37° C. for a sufficient time to hydrolyze the substrate, for example about 1 hour. After the sufficient time has elapsed, the reaction is stopped by adding stop buffer, and the plate is read in a fluorescent plate reader (for example, Wallac 1420 Victor3™) to determine the enzyme activity per well.

[0039] Those skilled in the art can easily identify various modifications of this assay. Examples of artificial substrates that can be used to detect α-GAL activity include, but are not limited to, p-nitrophenyl α-D-galactopyranoside and 4-MU Gal. Obviously, only substrates that can be cleaved by human α-GAL are suitable for use. Although the use of fluorogenic substrates is preferred, other methods of measuring α-GAL activity are contemplated for use with this method, examples of which include the use of chromogenic substrates or immunoassay techniques. Appropriateness criteria The criteria for determining suitability for SPC therapy depend on the patient's residual enzyme activity at baseline, i.e., activity determined in the naive T cells in the in vitro assay described above, or in WSCs before administration of SPC in the in vivo assay. The lower the residual activity, the higher the degree of enhancement required to predict that the patient will be a responder to treatment. In one embodiment, the criteria for determining suitability for the in vitro assay are as follows:

[0040] · If the residual α-GAL activity in lymphocytes is less than 1% of the normal value, the α-GAL activity after incubation with DGJ should be at least 2% of the normal value; If residual α-GAL activity in lymphocytes is between 1% and <3% of normal, α-GAL activity after incubation with DGJ should be at least 2x the baseline level; If the residual α-GAL activity in lymphocytes is between 3% and <10% of normal, the α-GAL activity after incubation with DGJ should be at least 3% higher than the baseline level in healthy individuals; If residual α-GAL activity in lymphocytes is 10% or greater than normal, α-GAL activity after incubation with DGJ should be at least 1.3x the baseline level.

[0041] In another embodiment, a patient with Fabry disease can be classified as suitable for SPC therapy if the α-GAL activity in T cells is at least about 35-40nM / hr / mg protein (which is about 58% of normal) in the presence of SPC such as DGJ. According to the present invention, the average profile is too variable to be reported as a comprehensive measure. Therefore, the activity of the patient's T cell sample was compared to at least three normal controls, which were collected within 48 hours from the date of collection of the patient specimen and grown under equivalent conditions (see Example 1). In comparison, the α-GAL activity in T cells from a Fabry patient containing A97V, R301Q and R111H at baseline was 8nM / hr / mg protein, 4nM / hr / mg and 1.8nM / hr / mg. T cells express α-GAL at high levels compared to other WBCs, and therefore α-GAL activity in T cell-enriched cultures is expected to be significantly higher than what is considered normal in total WBCs (21 nM / h / mg protein to approximately 50 nM / h / mg protein; Desnick et al., The Metabolic and Molecular Bases of Inherited Diseases, 8th ed., 2001, Scriver et al., eds., pp. 3733-3774, McGraw-Hill, NY). For comparison, three Fabry disease patients with the mutations R220X, R356W, and G132R had WBC α-GAL activities of 0.22, 0.18, and 0.26 nM / hr / mg protein, respectively.

[0042] In one embodiment, the following criteria are used to determine suitability in connection with in vivo assays: · If baseline α-GAL is less than 1% of normal, α-GAL activity on day 15 after treatment with DGJ should be at least 2% of normal; If baseline α-GAL is between 1% of normal and <5% of normal, α-GAL activity must be at least 2x the baseline level after the treatment period; If baseline α-GAL is between 5% and <10% of normal, the α-GAL activity must be at least 5% higher than the baseline level after the treatment period; If baseline α-GAL is 10% or greater of normal, the α-GAL activity must be at least 1.5x the baseline level after the treatment period. In another embodiment, an increase in activity of at least about 20% in cells cultured with SPCs compared to activity in cells not cultured with SPCs, either in in vitro or in vivo assays, may be an indication that the patient will have a clinically relevant (therapeutically effective) response to SPC therapy.

[0043] This discovery provides a method for improving diagnostics and facilitating clinical treatment decisions for Fabry disease in particular and lysosomal storage diseases in general. Moreover, this method can be extended to a wide range of genetically defined diseases in appropriate cell types. This group of diseases includes, among others, other lysosomal storage diseases, cystic fibrosis (CFTR) (respiratory or sweat gland epithelial cells), familial hypercholesterolemia (LDL receptor, LPL - adipocytes or vascular epithelial cells), cancer (p53; PTEN - tumor cells) and amyloidosis (transthyretin). kit The present invention also provides diagnostic test kits for sale for making therapeutic treatment decisions, which provide, in one convenient package, all of the materials for preparing and conducting each of the assays discussed above and more specifically in the Examples below, except, of course, for the patient's blood, optionally including instructions for use and analytical guidelines.

[0044] As a non-limiting example, a kit for assessing α-GAL activity would minimally include the following: a. at least one T cell stimulatory agent; b. specific pharmacological chaperones; c. a chromogenic or fluorogenic substrate for the enzyme assay (including appropriate standards); and d. GalNAc. The kit may also include instructions for optimally performing the protein enhancement assay. In another embodiment, the kit includes appropriate tubes, buffers (e.g., lysis buffer), and microplates. In one embodiment, the SPC is supplied in a dry state, which will be regenerated prior to addition. In another embodiment, the invention provides a kit for diagnosing Fabry disease. In this embodiment, the SPC is not included in the kit and the instructions are written specifically for diagnosis. Patients who test positive for enzyme augmentation with SPC are then treated with that agent, while patients who do not demonstrate enzyme augmentation with a particular agent can avoid treatment, saving money and avoiding the emotional cost of not responding to therapeutic modalities.

[0045] The present invention is further illustrated by the examples given below. Such examples are used for illustrative purposes only and are not intended to limit the scope and meaning of the present invention or any exemplified terms in any way. Likewise, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many improvements and modifications of the present invention will be apparent to those skilled in the art upon reading this specification. Therefore, the present invention is limited only by the appended claims and the full scope of equivalents to which such claims are entitled. Example 1: In vitro method to evaluate the effect of SPC on α-GAL activity This example provides an in vitro diagnostic assay to determine the responsiveness of Fabry disease patients to specific pharmacological chaperones. A. Preparation of human WBC pellets for growing T lymphocytes

[0046] 1. Material: · CPT tubes; Becton-Dickenson (containing sodium citrate, BD Vacutainer™ CPT™ cell preparation tubes, catalog # 362761); Human IL-2 (recombinant), PreProTECH, Cat# 200-02; Phytohemagglutinin (M type) (PHA), liquid, Invitrogen, catalog # 10576-015; RPMI-1640 medium, Mediatech Inc., catalog # 10-040-CV; · Fetal bovine serum, Mediatech, Cat# 35-010-CV; Citric acid monohydrate, ACS, Mallinckrodt, catalog cat # 0627; · Sodium phosphate dibasic (Na2HPO4), ACS, Mallinckrodt, Catalog # 7917; · Volumetric standard solution of sodium hydroxide 10N, Mallinckrodt, catalogue # H385; · Phosphoric acid, ACS, Mallinckrodt, catalog # PX0995-3; 4-MU α-D-galactopyranoside (4-MU-Gal), Sigma, catalog # M-7633; ·N-Acetyl-D-galactosamine (GalNAc), Sigma, Cat# A-2795; · 4-Methylumbelliferone (4-MU), Sigma, Cat# M-1381; Glycine, tissue culture grade, Fisher, catalog # BP381; ·Double deionized water; ·Durbeco Phosphate Buffered Saline, PBS (Ca & Mg free), Mediatech Inc., Cat# 21-031-CV; Micro BCA Protein Assay Kit, Pierce, Cat# 23235; · 96-well microtiter plate, round bottom Costar black polystyrene 96-well, catalog # 3792; · Costar 24-well tissue culture treated microplates, Corning Life Sciences, Catalog # 3526; · 15mL polypropylene Falcon tubes, Becton Dickinson, catalog # 352097; · Sterile cryovials; · Humidified 5% CO2, 37°C incubator; · 37°C water bath; Fluorescence plate reader

[0047] 2. WBC separation: · Patient blood was collected into 8mL CPT tubes and stored at 18-25°C; Immediately after blood collection, the tubes were mixed by inverting 8-10 times; The tubes were centrifuged at 1800 x g for 30 minutes at room temperature (18-25°C) in a swinging bucket tabletop centrifuge. Standard precautions for handling blood specimens were employed, including the use of closed canister-type buckets for centrifugation. Following the centrifugation, several layers of the blood composition should be separable, indicating that red blood cells have separated from plasma and white blood cells. If this does not occur, warm in your hands for 5 minutes and centrifuge again. 3. Washing of WBCs Half of the plasma layer was aspirated using vacuum and discarded without disturbing the white blood cells. All remaining fluid, including the cell layer, was transferred with a Pasteur pipette to a 15 mL conical screw-cap Falcon centrifuge tube. PBS was added to bring the volume to 14 mL and mixed by inverting the tube. The tubes were centrifuged at 1300 rpm (approximately 320 x g) for 20-30 minutes at room temperature. Immediately following the centrifugation, as much of the supernatant as possible was aspirated using vacuum and discarded without disturbing the resulting cell pellet.

[0048] 4. Optional Washing The cell pellet was resuspended in the remaining liquid by gently tapping the bottom of the tube. · 10mL of PBS was added to the resuspended cells and centrifuged at 1300 rpm for 20 minutes at room temperature. Immediately following the centrifugation, as much of the supernatant as possible was aspirated using vacuum and discarded without disturbing the resulting cell pellet. 5. Optional step: Freezing of WBC pellets The cell pellet was mixed in the remaining liquid by gently tapping the bottom of the tube with a finger. · 0.5-1 mL of PBS was added to the resuspended cells and half of the pellet was transferred (using a sterile tip of a micropipette) to a labeled 1.8 mL cryovial. The vials were centrifuged at 5000 rpm (approximately 2250 g) for 5 minutes at room temperature. All of the supernatant was discarded using a Pasteur pipette without disturbing the cell pellet. · 0.5-1mL of RPMI 1640 containing 10% FBS and 5% DMSO was then added to the tubes and frozen overnight at -80°C before transferring to a liquid nitrogen cell storage freezer.

[0049] B. Establishment of T-cell cultures from blood samples 1. The washed cells were resuspended in 3.0 mL of RPMI 1640 containing 10% Cosmic Calf Serum (CCS, Hyclone Laboratories, Logan, UT), approximately 25 ng / mL IL-2 (PreProTECH, Rocky Hill, NJ), and the manufacturer's recommended concentration of PHA (Life Technology, Gaithersburg, MD). The cells were then transferred to an upright 25 cm3 culture flask and incubated at 37°C, 5% CO2 for 3-4 days. 2. The cell culture was diluted with 5 mL of growth medium (RPMI 1640, 10% FBS, 25 ng / mL IL-2). The cell concentration was then adjusted to approximately 5×105 cells / mL in the flask. 3. The growth of the cells was monitored daily. Cells were maintained at 5×105 to 1.5×106 cells in upright flasks. The depth of the medium in the flasks was maintained at 1 cm (approximately 7 mL in T25 and 20 mL in T75). Cultures were maintained for approximately 21 days with a doubling time of approximately 24 hours. The senescence phase of the cultures was evident by a rapid decrease in growth rate. Culture times could be extended, possibly by re-stimulation with PHA. 4. Optional-freezing T-lymphocytes: T-lymphocytes can be frozen at 3x106 cells / vial using RPMI 1640 medium with 20% FCS and 7.5% DMSO. On days 5, 6 or 7, cryopreserve as many vials as possible at 3x106 cells / vial. This is enough to thaw 5mL of culture at 5x105 viable cells / mL.

[0050] When establishing T-cell cultures, the following points should be noted: Fresh blood specimens should be collected in heparinized tubes (or tubes containing an appropriate anticoagulant) and used the same day. If the specimen is not processed within 24 hours, ACD tubes should be used (Clin. Chem., 1988, Jan;34(1):110-3; Clin. Diagn. Lab. Immunol., 1998, Nov;5(6):804-7). · Typically, 8 blood samples of 10 mL each are sufficient to establish 2 x 107 cells by 5 days. T lymphocytes are a specific target of the HIV virus. Extreme caution should be exercised when the patient's HIV status is unknown. Each new lot of IL-2 should be tested to determine the optimal concentration. The lot from PreProtec used for these experiments was optimal at 25 ng / mL and showed a slight decrease in cell proliferation at concentrations up to 50 ng / mL. · Each lot of mitogen, e.g., phytohemagglutinin A (PHA), is assayed by the supplier (Invitrogen) and should be used at their recommended dilution. All cultures are maintained at 37°C, 5% CO2, in a water-saturated atmosphere. · Mononuclear cells and lymphocytes may also be harvested using lymphocyte separation medium (Ficoll-Hypaque) or Lymphoprep tubes according to the manufacturer's standard procedures.

[0051] When analyzed by fluorescence-activated cell sorting, the IL-2 and PHS stimulation regimen resulted in 99% CD3-positive cells (staining all T cell subsets) with equal numbers of CD4-positive and CD4-negative cells (data not shown). C. Chaperone Treatment The density of the T cells is adjusted to 1×106 cells per 3 mL of medium (RPMI 1640, 10% FBS, 25 ng / mL IL-2). 3 mL (~1×106 cells) is then pipetted into each of six wells of a labeled 6-well culture plate and incubated overnight at 37° C., 5% CO2. 3 mL of additional medium is then added to three wells for a final volume of 6 mL / well. The remaining three wells are incubated with 3 mL of medium containing DGJ (Cambridge Major Laboratories, Inc., Germantown, WI) at a concentration of approximately 40 μM (2x; final concentration is 20 μM) for 4-5 days. Cells are harvested by centrifugation (400×g for approximately 10 min) and washed once with 10 mL of PBS. The resulting pellet was resuspended in 1 mL of PBS, transferred to a 1.7 mL microfuge tube, and centrifuged at 3000 rpm for 5 minutes in a refrigerated microcentrifuge, the resulting supernatant was aspirated, and the resulting pellet was stored frozen at -80°C until assayed for enzyme activity. Prior to performing the enhancement assay, the optimal concentration of DGJ was determined using concentrations in the range of 2 nM-200 μM, with 20 μM found to be optimal.

[0052] D. Preparation of Fibroblast Cells For comparison, fibroblast cultures were prepared as previously described (e.g., U.S. Patent No. 6,274,597). Briefly, fibroblast cultures were derived from skin biopsies of patients and grown in DMEM containing 10% FBS until the cultures were established (3-4 weeks). E. Activity Assays Prior to the assay, the T cells were thawed on ice and sonicated for 2 minutes, and all other assay reagents were thawed at room temperature. Fluorometric assay of α-GAL activity was performed essentially as previously described (Kusiak et al., J. Biol. Chem., 1978; 253(1), 184-190). The cells were lysed in 0.2 mL of deionized water with vigorous pipetting and vortexing. The supernatant obtained after centrifugation at 13000 rpm for 2 minutes at 4°C was placed in a new tube and used as the source of α-GAL. α-GAL activity was determined by incubating 50 μL aliquots of the supernatants (containing protein comparable to that measured using 20 μL in a standard protein quantification assay) with 3.75 mM 4-methylumbelliferyl-α-D-galactopyranoside (4-MU Gal) (Research Products International, Mount Prospect, IL) in citrate / phosphate buffer (27 mM citrate / 46 mM phosphate buffer, pH 4.6) without taurocholate and containing BSA (3 mg / mL) at 37° C. in 24-well microplates. The percentage of α-GAL was determined by comparing the total activity to that observed in the presence of 117 mM N-acetylgalactosamine (GalNAc) (Sigma Chemical Co., St. Louis, MO), a specific inhibitor of N-acetylgalactosaminidase. Released 4-MU was measured at excitation and emission wavelengths of 355 nm and 460 nm, respectively, using a Wallac 1420 Victor3™ fluorescence detection reader (Perkin Elmer, CA). Appropriate wells for fluorescence standards and negative samples (no substrate or no lysate) were also included. At least three normal samples were tested simultaneously for each patient sample.

[0053] Incubations were typically carried out for 30 minutes, although longer or shorter times can be used with similar results. Enzyme activity (nM / hr / mg protein) was calculated according to the following formula: [(fluorescence of sample) / (fluorescence of standard)] x [(60 min) / (incubation time (min))] x [(1000 μL) / (assayed volume (μL)] x [1 / (protein value (mg / mL))] One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 nM of 4-methylumbelliferone per hour. The baseline "noise" in the fluorescence output was obtained by evaluating the average value of six blanks. The activity associated with SPC treatment was considered to be responsive and not noise if it exceeded the baseline by at least two standard deviations. In relation to the comparative fibroblast enhancement assay, fibroblasts (~1.5x106) were grown in 12mL of medium in T75 tissue culture flasks in the absence or presence of 20μM DGJ for 3 days. At the end of the incubation period, cells were treated with trypsin-EDTA solution, removed from the flask, collected by centrifugation, and washed three times with phosphate-buffered saline. Cell pellets were frozen at -80°C until assayed for α-GAL activity. All steps for processing the cell pellets, including the extraction buffer, sonication time, and volumes used were the same as those used for the T-cells assayed above.

[0054] F. Western Blot The levels of α-GAL protein were measured by Western blot. Protein was measured using the Micro BCA Protein Assay kit (Pierce, Rockford, IL) with bovine serum albumin (BSA) as a standard. Absorbance at 562 nm was measured using a Molecular Devices VersaMax absorbance reader in a 96-well format. For gel electrophoresis prior to Western blot, proteins were separated using Novex Triglycine neat or SDS-PAGE in 8-16% gradient gels (Invitrogen). Western blots were developed with a rabbit polyclonal antibody against α-GAL and were performed as previously described (Park et al., Proc. Natl. Acad. Sci. USA. 2003; 100: 3450-54). result: The above method using T cells is rapid and effective when compared to a fibroblast-based α-GAL assay performed in a substantially similar manner, except that approximately 1.5×10 fibroblasts were plated in each well instead of 2.5×10 T cells.

[0055] Using this method, T cells from Fabry patients were incubated for 1, 2, and 4 days in the presence or absence of 20 μM DGJ, respectively, and the α-GAL activity in cell homogenates was measured and compared to normal control values. When the medium was refreshed after 2 days and the cells were incubated for an additional 2 days, the α-GAL activity of A97V was 13% of the normal control (Figure 1, open circles). However, when 20 μM DGJ was added to the medium containing the T cells, the α-GAL activity increased to about 40% of normal values ​​after only 1 day of incubation, and continued to increase to about 80% of normal values ​​after 4 days of incubation (Figure 1, filled circles). Addition of fresh DGJ, and addition of medium after 2 days and incubation for an additional 2 days did not show any change in the profile relative to that observed for the addition of DGJ once. The increase in activity observed after 3 days to levels clearly distinguishable from α-GAL activity observed in the absence of added DGJ led us to adopt 3 days after incubation with 20 μM DGJ as the standard measurement time in subsequent experiments. The use of a 3-day time period eliminates the need to use fresh medium and / or separate cells in culture after 3 days.

[0056] To determine the dose effect of DGJ in T cells from normal controls, α-GAL activity was measured in patient cells using DGJ in the range of 2 nM to 200 μM (FIG. 2A) and compared to untreated normal control values ​​assayed on the same day. α-GAL activity was increased by DGJ in the range of 2 nM to 20 μM. At 200 μM, DGJ inhibited normal α-GAL activity to about 40% of the mean value of untreated normal controls. Within this same DGJ concentration range, the optimal enhancement of mutated α-GAL activity was determined for the A97V mutation and compared to normal controls (FIG. 2B). Three separate experiments were performed for the dose effect on A97V. DGJ in the range of 2 nM to 20 μM increased the α-GAL activity of A97V in a dose-dependent manner. However, at a DGJ concentration of 200 μM, a decrease in α-GAL activity was observed when compared to the highest level of activity when cells were grown in 20 μM DGJ. In all three experiments, the optimal enhancement of the activity of the A97V mutation was observed at 20 μM DGJ, with slightly lower activity at 2 and 200 μM. A similar pattern emerged when mutations R112H and R301Q were tested in the same concentration range, with the highest level of enhanced activity observed at 20 μM DGJ (FIG. 2C). These results indicated that the various mutations had similar dose-response profiles but differed in the enhancement levels. Among the three α-GAL mutant genotypes tested for the dose effect, the 20 μM DGJ resulted in an increase in α-GAL to at least 50% of the normal control.

[0057] The rescue effect of mutant α-GAL from at least 11 distinct genotypes of Fabry disease patients was observed using this T-cell-based α-GAL assay and using pharmacological chaperones. The results shown in Table 1 below showed that DGJ enhanced the activity of at least five different α-GAL mutant forms in T cells and fibroblasts (F). However, the pharmacological chaperone did not enhance the activity of four different α-GAL mutant forms. The α-GAL activity of one classical Fabry disease patient was enhanced by intermediate levels of DGJ. The importance of this assay lies in the fact that it can be used to screen for patients who may benefit from pharmacological chaperone administration, thus avoiding the expense and frustration of unnecessary treatments and tissue biopsies.

[0058] [Table 1] TIFF2025060691000003.tif200170TIFF2025060691000004.tif179170Abbreviations used: PT: Patient; NL: Normal individual; E: Enhanced; I: Enhanced (intermediate); NE: Non-enhanced

[0059] In the in vitro assay, it was determined that optimal enhancement of mutated α-GAL activity was achieved with approximately 20 μM DGJ, which resulted in an increase in α-GAL to at least 50% of the normal control for each of the three α-GAL mutant genotypes tested for dose effect (FIG. 3). In Table 1 above, the enhanceable group included patients whose α-GAL activity was at least 50% of normal control when cultured in the presence of DGJ (e.g., R112H was enhanced to 60% of normal control activity). In fact, in the enhanceable group, there are the following mutations: A97V, R301Q, R112H and L300P. For example, the activity of the A97V mutation increased from 14% to 75% in the presence of DGJ, which is a 6-fold increase. Similarly, the activity of R301Q increased 12-fold from 7% to 80% of normal, and the activity of R112H increased from 3% to 60%, which is an almost 20-fold change. Furthermore, the activity of the L300P mutation increased from 2% to 72% of normal, which is a 37-fold increase, which is the largest among the enhanceable mutations examined. L300P was exceptional in that some of the activity in the absence of DGJ was below the lowest threshold of detection. These results demonstrate mutation-dependent enhancement levels and ratios.

[0060] Western blots showed that treatment with DGJ significantly increased the band intensity in normal control cells and cells carrying the A97V and R301Q mutations, while R356W, G132R, and A143P did not show any increase (Figure 3). The protein appeared to shift to lower molecular weights, indicating maturation of the enzyme by translocation from the endoplasmic reticulum, through the Golgi apparatus, to lysosomes. The Western blots show that the enhancement of α-GAL activity by DGJ correlates with high concentrations of α-GAL protein. No cases have yet been observed in which high protein levels, as measured by Western blot, did not result in higher enzyme activity.

[0061] Essay The use of T cells in a test system for the enhancement of the enzyme by SPC offers significant advantages in the speed of the assay and convenience over other culture systems. A critical step in determining which patients could benefit from SPC therapy was to develop a rapid and reliable method to screen patient-derived cells for enhancement of α-GAL activity by DGJ. The above results demonstrate that a method for the rapid generation of short-lived cell cultures allows testing for such enhancement and provides a useful system for future studies of the mechanism of action and for screening additional chaperone molecules. Traditionally, white blood cells used to diagnose the status of affected individuals and carriers do not survive long enough to be assayed repeatedly, if necessary. Epstein-Barr virus-transformed B lymphoblasts (Fan et al., Nat Med., 1999; 5(1), 112-115) and primary fibroblast cultures (Fan, supra; Mayes et al., Clin. Chim. Acta., 1981; 112(2), 247-251) have been tested, but these are inconvenient to use on a large scale to screen patients for clinical studies. Primary fibroblast cultures require invasive skin punch biopsies and generally require at least 3-4 weeks to grow enough cells for the assay. B cell lymphoblasts require Epstein-Barr virus transfection and selection steps that, in addition to having unknown effects on enzyme activity, are time- and labor-intensive.

[0062] The present invention provides a method for establishing T cell cultures from fresh blood from normal control individuals and patients with Fabry disease. These cultures can be expanded for 7-10 days for use in enhancement assays for α-GAL. These data also show that the effectiveness of DGJ enhancement is evident after approximately 3 days in the T cell growth medium. The data obtained provide a reproducible measure of the degree of enhancement of enzyme activity by SPC for a particular genotype. This method can be used for other SPC-based enrichment assays of other genetic diseases, including glycosphingolipidosis, mucopolysaccharidoses, and glycogen storage diseases (Pompe disease), and can be expanded into research and clinical protocols in a wide range of genetically defined diseases, such as cystic fibrosis (CFTR) and cancer (p53, PTEN).

[0063] Example 2: In vivo method to assess the effect of SPC on α-GAL activity This example describes the results obtained in an open label Phase II study of DGJ in Fabry disease patients (n=11) bearing 10 different α-GAL mutations and confirms the effectiveness of the use of in vivo assays. The patients were selected for the Phase II study based on increased α-GAL activity in the T-cell assay described above. The genotypes were as follows: T41L (2 patients); A143T; A97V; M51K; S276G; L300P; G328A; P205T; N215S; and L415P. Some patients (8) received DGJ according to the following dosing schedules: 25 mg bid (twice daily) for 2 weeks; 100 mg bid for 2-4 weeks; 250 mg bid for 4-6 weeks; and 25 mg bid for 6-12 weeks. Three patients received 150 mg of DGJ every other day throughout the study. Blood was drawn into 8 mL Vacutainer CPT tubes at the end of each dosing period and processed as described below:

[0064] A. Preparation of Human WBC Pellet for Assay WBCs were prepared similarly to the procedure described in Example 1, except that no FBS / DMSO was added to the pellet before freezing. Preliminary data are summarized in the following table: B. Preparation of Human WBC Lysates for the Assay Add 0.6 mL of lysis buffer (26 mM citrate / 46 mM phosphate, pH 5.5) to the microfuge tube containing the WBC pellet; The tubes were vortexed until the cells were resuspended; Incubate the tubes at room temperature for approximately 15 minutes, but agitate the suspension by swirling for 2 minutes each time; · The tube was sonicated for 2 minutes and then vortexed for approximately 10 seconds; · Lysates were incubated on ice until cold and then pooled in pre-chilled polypropylene containers (on ice); · The vessel was vortexed and the pooled lysate was divided into pre-chilled, 0.100 mL aliquots into labeled, 0.5 mL, screw-cap polypropylene microcentrifuge tubes. The pooled lysate was mixed and aliquoted into 10-20 aliquots each by vortexing; Aliquots were stored at -80°C until use.

[0065] C. Human WBC Assay · Each tube containing lysate was thawed on ice, sonicated for 2 minutes, and then vortexed for 1 minute; · 50 μL of each standard, control, or clinical sample was added to the appropriate well of a black microplate (for standards, use 50 μL of WBC lysis buffer containing 0.5% BSA); · 50 μL of 117 mM GalNAc was added to all wells and the plate was incubated at ambient temperature for 5 minutes; · 50 μL of 5 mM 4-MU Gal substrate was added to all wells and the wells were mixed on a plate shaker for 30 seconds; The plate was covered and incubated at 37°C for approximately 1 hour; - 100 μL of 0.2 M NaOH / glycine buffer with a pH of 10.7 was added to each well to stop the reaction; The plate was read in a fluorescent plate reader as described in Example 1.

[0066] result Available data from the first 11 patients treated with DGJ for at least 12 weeks suggests that treatment with DGJ leads to an increase in the activity of the enzyme deficient in Fabry disease in 10 of the 11 patients (Figure 4). The patient with the L415P genotype showed no increase in the activity with DGJ treatment (week 6) (Figure 5). For the purpose of calculating the percentage of normal in the table, normal α-GAL levels were derived using the average α-GAL levels in leukocytes of 15 healthy volunteers from the multiple-dose Phase I study. The 11 patients represented 10 different genetic mutations and had baseline levels of α-GAL ranging from 0 to 30% of normal. These data show an average 2-fold increase in α-GAL, and up to 10- and 15-fold increases in some patients, measured in leukocytes. Activity remained elevated over 6-24 weeks and was counted when the dose was reduced to 25 mg bid (data not shown).

[0067] Essay Based on these results, it was believed that it would be possible to use T cells as described in Example 1 to screen candidate patients for suitability for chaperones using in vivo or in vitro assays, since the T cell assay demonstrated a significant increase in α-GAL activity in 10 of the 11 patients after 2 weeks of treatment with DGJ. In vivo screening may allow a more accurate assessment of the in vivo response to treatment with DGJ and other chaperones, since the in vitro assay does not allow a completely accurate prediction of the in vivo response due to systemic interactions and may be useful for determining appropriate doses. This significant increase in activity was seen after 2 weeks at a dose as low as 50 mg / day (25 mg bid) (although a different dosing regimen is contemplated in the present invention). These methods can be used for chaperone-based enhancement assays for other genetic disorders, such as cystic fibrosis, where protein maturation occurs in the ER, as well as other genetic disorders including glycosphingolipidoses, mucopolysaccharidoses, and other lysosomal storage diseases.

[0068] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Patents, patent applications, publications, product descriptions, GenBank accession numbers, and protocols are cited throughout this patent application, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0069] The present invention relates to the following: 1. A method for determining whether a patient having a deficiency in the activity of a protein will respond to treatment with a pharmacological chaperone specific for the protein, the method comprising: a. contacting a cell in or derived from a patient with a pharmacological chaperone specific for the protein; and b. comparing protein activity in cells not contacted with the specific pharmacological chaperone to protein activity in cells contacted with the specific pharmacological chaperone; Including, A sufficient increase in the protein activity in cells contacted with the specific pharmacological chaperone compared to the protein activity in cells not contacted with the specific pharmacological chaperone indicates that the individual will respond to treatment with the specific pharmacological chaperone. The method according to claim 1, 2. The method according to claim 1, wherein the deficiency in activity is caused by a missense mutation in the gene encoding the protein. 3. The method according to claim 1, wherein the protein is an enzyme. 4. The method according to claim 3, wherein the enzyme is a lysosomal enzyme. 5. The method of claim 4, wherein the patient has been diagnosed with a lysosomal storage disease. 6. The method according to 5, wherein the lysosomal enzyme is α-GAL and the lysosomal storage disease is Fabry disease. 7. The method according to claim 5, wherein the specific pharmacological chaperone is 1-deoxygalactonojirimycin. 8. The method of claim 7, wherein said cell is a white blood cell and said contacting with said specific pharmacological chaperone occurs in vivo. 9. The method of claim 8, wherein said leukocytes are T lymphocytes and said contacting with said specific pharmacological chaperone occurs in vitro. 10. The T lymphocytes, a. isolating leukocytes from a blood sample obtained from said patient; b. washing the leukocytes; and c. Establishing T lymphocyte-enriched cell cultures; 10. The method according to claim 9, wherein the method is obtained by 11. The method according to claim 10, wherein the T lymphocytes are cultured for about 1 to 3 days in the absence or presence of 1-deoxygalactonojirimycin as the specific pharmacological chaperone. 12. The method according to claim 11, wherein the culturing in the absence or presence of 1-deoxygalactonojirimycin is carried out for about 3 days. 13. The method of claim 12, wherein the activity of the α-GAL is measured using a fluorometric assay that quantifies the hydrolysis of a substrate in a lysate derived from the T lymphocytes. 14. The sufficient increase in activity in the lysate in the presence of the 1-deoxygalactonojirimycin to indicate whether the patient will respond is determined according to the following criteria: i) if the baseline activity is less than 1% of normal, the activity after culture or treatment with SPC should be at least 2% of normal; ii) if baseline activity is less than 1%-3% of normal, the activity after culture or treatment with SPC should be at least twice the baseline level; iii) if the baseline activity is less than 3%-10% of normal, the activity after culture or treatment with SPC should be at least 3% higher than the baseline level in a healthy individual; iv) if baseline activity is 10% or greater of normal, the activity after culture or treatment with SPC should be at least 1.3x the baseline level; 13. The method according to claim 12, wherein the measurement is performed according to the following procedure. 15. The method of claim 12, wherein a sufficient increase in the activity in the presence of the 1-deoxygalactonojirimycin to indicate whether the patient will respond is in the range of about 2-fold to 25-fold compared to the activity in the absence of the 1-deoxygalactonojirimycin. 16. The method of claim 15, wherein a sufficient increase in said activity in the presence of said 1-deoxygalactonojirimycin to indicate whether said patient will respond is at least about 20% greater than said activity in cells not cultured with said 1-deoxygalactonojirimycin. 17. The method of claim 8, wherein the patient receives daily doses of 1-deoxygalactonojirimycin for about two weeks. 18. The method according to claim 17, wherein the administration is oral administration. 19. The method of claim 17, wherein the 1-deoxygalactonojirimycin is administered at a dosage of about 50-500 mg / day. 20. The method of claim 19, wherein the dosage is in the range of about 100-250 mg / day. 21. The method of claim 20, wherein the dose is about 150 mg / day. 22. The method of claim 19, wherein the 1-deoxygalactonojirimycin is administered once per day. 23. The method of claim 17, further comprising the step of taking a blood sample and separating leukocytes at the end of the two week period. 24. The method according to claim 17, wherein the α-GAL activity is measured using a fluorometric assay that quantifies hydrolysis of a substrate in a lysate derived from the leukocytes. 25. The sufficient increase in activity in the lysate in the presence of the 1-deoxygalactonojirimycin to indicate whether the patient will respond is determined according to the following criteria: i) if the baseline activity is less than 1% of normal, the activity after culture or treatment with SPC should be at least 2% of normal; ii) if baseline activity is less than 1%-5% of normal, the activity after culture or treatment with SPC should be at least twice the baseline level; iii) if the baseline activity is less than 5%-10% of normal, the activity after culture or treatment with SPC should be at least 5% higher than the baseline level in a healthy individual; iv) if baseline activity is 10% or greater than normal, the activity after culture or treatment with SPC should be at least 1.5x the baseline level; 25. The method according to claim 24, wherein the measurement is performed according to the following procedure. 26.a. at least one T cell stimulating agent; b. specific pharmacological chaperones; c. a labeled substrate for the chaperone; d. GalNAc; and e. Instructions for performing the Protein Enhancement Assay A kit comprising: 27. The kit according to claim 26, wherein the T cell stimulatory agent is a mitogen. 28. The kit according to claim 27, wherein the mitogen is PHA. 29. The kit according to claim 26, wherein the stimulatory agent is a cytokine. 30. The kit according to claim 29, wherein the cytokine is IL-2. 31. The kit according to claim 26, wherein the pharmacological chaperone is 1-deoxygalactonojirimycin. 32. The kit according to claim 26, further comprising one or more of a blood collection tube, a centrifuge tube, and a cryotube. 33. The kit according to claim 26, wherein the protein is an enzyme. 34. The kit according to claim 33, wherein the enzyme is α-galactosidase A.

Claims

1. 1. A pharmaceutical composition for treating a patient with Fabry disease, comprising: comprising a therapeutically effective dose of 150 mg of 1-deoxygalactonojirimycin or a salt thereof; The pharmaceutical composition, wherein the pharmaceutical composition is administered to the patient every other day.

2. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered for at least two weeks.

3. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered for at least six weeks.

4. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered for at least 12 weeks.

5. 2. The pharmaceutical composition of claim 1, wherein the salt of 1-deoxygalactonojirimycin is migalastat hydrochloride.

6. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is administered orally.

7. 1. A pharmaceutical composition for enhancing α-GAL activity in a patient diagnosed with or suspected of having Fabry disease, comprising: comprising a therapeutically effective dose of 150 mg of 1-deoxygalactonojirimycin or a salt thereof; The pharmaceutical composition, wherein the pharmaceutical composition is administered to the patient every other day.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is administered for at least two weeks.

9. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is administered for at least six weeks.

10. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is administered for at least 12 weeks.

11. 8. The pharmaceutical composition of claim 7, wherein the salt of 1-deoxygalactonojirimycin is migalastat hydrochloride.

12. An oral solid formulation comprising 1-deoxygalactonojirimycin or a salt thereof and a pharma- ceutically acceptable carrier.

13. The oral solid formulation of claim 12, wherein the salt of 1-deoxygalactonojirimycin is migalastat hydrochloride.

14. The oral solid formulation of claim 12, which is a tablet.

15. The oral solid formulation of claim 12, which is a capsule.

16. 1. Use of 1-deoxygalactonojirimycin or a salt thereof for the manufacture of a medicament for treating a patient with Fabry disease, comprising: the medicament comprises a therapeutically effective dose of 150 mg of 1-deoxygalactonojirimycin or a salt thereof; The use wherein the medicament is administered to the patient every other day.

17. 17. The use according to claim 16, wherein the medicament is administered for at least two weeks.

18. 17. The use according to claim 16, wherein the medicament is administered for at least 6 weeks.

19. 17. The use according to claim 16, wherein the medicament is administered for at least 12 weeks.

20. 17. The use according to claim 16, wherein the salt of 1-deoxygalactonojirimycin is migalastat hydrochloride.

21. 17. The use according to claim 16, wherein the medicament is administered orally.

22. 1. Use of 1-deoxygalactonojirimycin or a salt thereof for the manufacture of a medicament for enhancing α-GAL activity in a patient diagnosed with or suspected of having Fabry disease, comprising: the medicament comprises a therapeutically effective dose of 150 mg of 1-deoxygalactonojirimycin or a salt thereof; The use wherein the medicament is administered to the patient every other day.

23. 23. The use according to claim 22, wherein the medicament is administered for at least two weeks.

24. 23. The use according to claim 22, wherein the medicament is administered for at least 6 weeks.

25. 23. The use according to claim 22, wherein the medicament is administered for at least 12 weeks.

26. 23. The use according to claim 22, wherein the salt of 1-deoxygalactonojirimycin is migalastat hydrochloride.

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