Methods of treating fabry disease in patients having mutation in gla gene

Abstract

To provide: methods of treating a patient diagnosed with or suspected of having Fabry disease; and methods of enhancing α-GalA in a patient diagnosed with or suspected of having Fabry disease.SOLUTION: There is provided a method comprising administering to a patient a therapeutically effective dose of a pharmacological chaperone for α-GalA, wherein the patient has a missense mutation in a nucleic acid sequence encoding α-GalA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Principles and embodiments of the present invention relate generally to the use of pharmacological chaperones for the treatment of Fabry disease, particularly in patients with mutations or variants in the alpha-galactosidase (GLA) gene. [Background technology]

[0002] Many human diseases result from mutations that cause changes in the amino acid sequence of proteins, reducing their stability and preventing their proper folding. Proteins generally fold within a specific region of the cell known as the endoplasmic reticulum, or ER. Cells have quality control mechanisms that ensure proteins fold into their correct three-dimensional shape and then move from the ER to their appropriate destination within the cell, a process generally referred to as protein trafficking. Misfolded proteins are often initially retained in the ER and then eliminated by the quality control mechanisms. In some cases, misfolded proteins can accumulate in the ER before being eliminated. Retention of misfolded proteins in the ER can interfere with their proper trafficking, resulting in reduced biological activity and impairing cellular function, ultimately leading to disease. In addition, accumulation of misfolded proteins in the ER can cause various stresses on the cell, which can also contribute to cellular dysfunction and disease.

[0003] Such mutations can lead to lysosomal storage disorders (LSDs), characterized by lysosomal enzyme deficiencies due to mutations in genes encoding the lysosomal enzymes. The resulting disease causes pathological accumulation of the enzyme's substrates, including lipids, carbohydrates, and polysaccharides. While many different mutational genotypes exist associated with each LSD, many of these mutations are missense mutations that can lead to the production of less stable enzymes. These less stable enzymes are sometimes prematurely degraded by ER-associated degradation pathways, resulting in enzyme deficiencies in lysosomes and pathological accumulation of substrates. Such mutant enzymes are sometimes referred to in the art as "folding mutants" or "conformational mutants."

[0004] Fabry disease is a rare genetic disorder caused by mutations in the GLA gene, which encodes the enzyme α-galactosidase A (α-Gal A). α-Gal A is required for glycosphingolipid metabolism. The mutations lead to the accumulation of the substrate globotriaosylceramide (GL-3) in various tissues and organs. Because the disease gene is encoded on the X chromosome, males with Fabry disease are hemizygous. Fabry disease is estimated to affect 1 in 40,000 and 1 in 60,000 men, with a lower incidence in women.

[0005] Several approaches to treating Fabry disease exist. One approved therapy for Fabry disease is enzyme replacement therapy (ERT), which typically involves intravenous infusion of a purified form of the corresponding wild-type protein. Two α-Gal A preparations are currently available for the treatment of Fabry disease: agalsidase alfa (Replagal®, Shire Human Genetic Therapies) and agalsidase beta (Fabrazyme®; Sanofi Genzyme Corporation). However, ERT has several drawbacks. One of the major complications of ERT is the rapid degradation of the injected protein, which leads to multiple, expensive, high-dose infusions. ERT also has several caveats, including the difficulty of large-scale production, purification, and storage of properly folded protein; the availability of glycosylated native protein; the development of anti-protein immune responses; and the inability of the protein to cross the blood-brain barrier and alleviate central nervous system pathology (i.e., low bioavailability). Additionally, replacement enzymes cannot penetrate the heart or kidney in sufficient quantities to reduce substrate accumulation in renal podocytes or cardiac myocytes, which is prominent in Fabry lesions.

[0006] Another therapeutic approach for certain enzyme deficiencies involves the use of small molecule inhibitors to reduce production of the natural substrate of the defective enzyme protein, thereby alleviating the pathology. This "substrate inhibition" approach has been described for approximately 40 classes of LSDs, including glycosphingolipid storage disorders in particular. The small molecule inhibitors proposed for use as therapy are specific for inhibiting enzymes involved in glycolipid synthesis, reducing the amount of cellular glycolipids that need to be degraded by the defective enzyme.

[0007] A third approach to treating Fabry disease is treatment with so-called pharmacological chaperones (PCs). Such PCs include small molecule inhibitors of α-Gal A, which can bind to α-Gal A and increase the stability of both the mutant enzyme and the corresponding wild-type enzyme. However, patients eligible for PC therapy must have an applicable mutation or variant that results in the production of an enzyme that is stabilized and has the potential to fold into a conformation that allows trafficking from the ER. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, even when Fabry disease is diagnosed by detection of deficient α-Gal A activity in plasma or peripheral white blood cells (WBCs), it is extremely difficult to predict whether a particular Fabry patient will respond to treatment with PC. Thus, there remains a need to identify new GLA mutations or variants that will be responsive to PC and to make available new treatment strategies for Fabry patients carrying these mutations or variants. [Means for solving the problem]

[0009] One aspect of the present invention relates to a method for treating a patient diagnosed with Fabry disease. The method comprises administering a therapeutically effective dose of a pharmacological chaperone for α-Gal A to the patient, wherein the patient has a missense mutation in the nucleic acid sequence encoding α-Gal A. In one or more embodiments, the mutation is D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, or R392T. In one or more embodiments, the mutation is N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, or N408Y. In one or more embodiments, the mutation is V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, or T412P. In various embodiments, these mutations are relative to SEQ ID NO:2. In one or more embodiments, the mutation is D33H relative to SEQ ID NO:2. In one or more embodiments, the mutation is G35A relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y88S relative to SEQ ID NO:2. In one or more embodiments, the mutation is T194A relative to SEQ ID NO:2. In one or more embodiments, the mutation is W204G relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y216S relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q250K relative to SEQ ID NO:2. In one or more embodiments, the mutation is R392T relative to SEQ ID NO:2. In one or more embodiments, the mutation is N53K relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q57R relative to SEQ ID NO:2. In one or more embodiments, the mutation is S62delinsLA relative to SEQ ID NO:2. In one or more embodiments, the mutation is M96V relative to SEQ ID NO:2. In one or more embodiments, the mutation is R112L relative to SEQ ID NO:2. In one or more embodiments, the mutation is D155E relative to SEQ ID NO:2.In one or more embodiments, the mutation is N228D relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q330P relative to SEQ ID NO:2. In one or more embodiments, the mutation is V339A relative to SEQ ID NO:2. In one or more embodiments, the mutation is K391E relative to SEQ ID NO:2. In one or more embodiments, the mutation is N408Y relative to SEQ ID NO:2. In one or more embodiments, the mutation is V22G relative to SEQ ID NO:2. In one or more embodiments, the mutation is N34H relative to SEQ ID NO:2. In one or more embodiments, the mutation is G80V relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q107R relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y152D relative to SEQ ID NO:2. In one or more embodiments, the mutation is A156S relative to SEQ ID NO:2. In one or more embodiments, the mutation is L189F relative to SEQ ID NO:2. In one or more embodiments, the mutation is W204L relative to SEQ ID NO:2. In one or more embodiments, the mutation is S238G relative to SEQ ID NO: 2. In one or more embodiments, the mutation is I239M relative to SEQ ID NO: 2. In one or more embodiments, the mutation is A257V relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P259Q relative to SEQ ID NO: 2. In one or more embodiments, the mutation is N320H relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P323T relative to SEQ ID NO: 2. In one or more embodiments, the mutation is E338V relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P380L relative to SEQ ID NO: 2. In one or more embodiments, the mutation is T412P relative to SEQ ID NO: 2. In some embodiments, the pharmaceutical chaperone comprises migalastat or a salt thereof. In one or more embodiments, the dose of migalastat or a salt thereof is about 100 mg to about 150 mg free base equivalent (FBE).In some embodiments, the salt of migalastat is migalastat hydrochloride. In one or more embodiments, the dose is about 150 mg of migalastat hydrochloride every other day, or an equivalent dose of migalastat or a salt thereof other than hydrochloride. In some embodiments, migalastat or a salt thereof is administered orally or by injection. These embodiments may be combined with each other or with other embodiments of the invention, such as with embodiments relating to methods of enhancing α-Gal A in patients diagnosed with or suspected of having Fabry disease, the use of a pharmacological chaperone for α-Gal A to manufacture a medicament for treating patients diagnosed with or suspected of having Fabry disease, or pharmacological chaperones for α-Gal A for use in treating patients diagnosed with Fabry disease, as well as applicable mutations, suitable PCs and doses, formulations, and their routes of administration.

[0010] Another aspect of the present invention relates to a method for enhancing a-Gal A in a patient diagnosed with or suspected of having Fabry disease. The method comprises administering a therapeutically effective dose of a pharmacological chaperone for a-Gal A to the patient, wherein the patient has a missense mutation in the nucleic acid sequence encoding a-Gal A. In one or more embodiments, the mutation is D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, or R392T. In one or more embodiments, the mutation is N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, or N408Y. In one or more embodiments, the mutation is V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, or T412P. In various embodiments, these mutations are relative to SEQ ID NO:2. In one or more embodiments, the mutation is D33H relative to SEQ ID NO:2. In one or more embodiments, the mutation is G35A relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y88S relative to SEQ ID NO:2. In one or more embodiments, the mutation is T194A relative to SEQ ID NO:2. In one or more embodiments, the mutation is W204G relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y216S relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q250K relative to SEQ ID NO:2. In one or more embodiments, the mutation is R392T relative to SEQ ID NO:2. In one or more embodiments, the mutation is N53K relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q57R relative to SEQ ID NO:2. In one or more embodiments, the mutation is S62delinsLA relative to SEQ ID NO:2. In one or more embodiments, the mutation is M96V relative to SEQ ID NO:2. In one or more embodiments, the mutation is R112L relative to SEQ ID NO:2.In one or more embodiments, the mutation is D155E relative to SEQ ID NO:2. In one or more embodiments, the mutation is N228D relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q330P relative to SEQ ID NO:2. In one or more embodiments, the mutation is V339A relative to SEQ ID NO:2. In one or more embodiments, the mutation is K391E relative to SEQ ID NO:2. In one or more embodiments, the mutation is N408Y relative to SEQ ID NO:2. In one or more embodiments, the mutation is V22G relative to SEQ ID NO:2. In one or more embodiments, the mutation is N34H relative to SEQ ID NO:2. In one or more embodiments, the mutation is G80V relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q107R relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y152D relative to SEQ ID NO:2. In one or more embodiments, the mutation is A156S relative to SEQ ID NO:2. In one or more embodiments, the mutation is L189F relative to SEQ ID NO:2. In one or more embodiments, the mutation is W204L relative to SEQ ID NO:2. In one or more embodiments, the mutation is S238G relative to SEQ ID NO:2. In one or more embodiments, the mutation is I239M relative to SEQ ID NO:2. In one or more embodiments, the mutation is A257V relative to SEQ ID NO:2. In one or more embodiments, the mutation is P259Q relative to SEQ ID NO:2. In one or more embodiments, the mutation is N320H relative to SEQ ID NO:2. In one or more embodiments, the mutation is P323T relative to SEQ ID NO:2. In one or more embodiments, the mutation is E338V relative to SEQ ID NO:2. In one or more embodiments, the mutation is P380L relative to SEQ ID NO:2. In one or more embodiments, the mutation is T412P relative to SEQ ID NO:2. In some embodiments, the pharmaceutical chaperone comprises migalastat or a salt thereof.In one or more embodiments, the dose of migalastat or a salt thereof is about 100 mg to about 150 mg FBE. In some embodiments, the migalastat salt is migalastat hydrochloride. In one or more embodiments, the dose is about 150 mg of migalastat hydrochloride every other day, or an equivalent dose of migalastat or a salt thereof other than hydrochloride. In some embodiments, migalastat or a salt thereof is administered orally or by injection. These embodiments may be combined with each other or with other embodiments of the invention, such as methods of treating patients with Fabry disease, the use of a pharmacological chaperone for α-Gal A to prepare a medicament for treating patients diagnosed with Fabry disease, or embodiments relating to a pharmacological chaperone for α-Gal A for use in treating patients diagnosed with Fabry disease, as well as applicable mutations, suitable PCs and doses, formulations, and their routes of administration.

[0011] Another aspect of the present invention relates to the use of a pharmacological chaperone for α-Gal A for the manufacture of a medicament for treating a patient diagnosed with Fabry disease, wherein the patient has a missense mutation in the nucleic acid sequence encoding α-Gal A. In one or more embodiments, the mutation is D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, or R392T. In one or more embodiments, the mutation is N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, or N408Y. In one or more embodiments, the mutation is V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, or T412P. In various embodiments, these mutations are relative to SEQ ID NO:2. In one or more embodiments, the mutation is D33H relative to SEQ ID NO:2. In one or more embodiments, the mutation is G35A relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y88S relative to SEQ ID NO:2. In one or more embodiments, the mutation is T194A relative to SEQ ID NO:2. In one or more embodiments, the mutation is W204G relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y216S relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q250K relative to SEQ ID NO:2. In one or more embodiments, the mutation is R392T relative to SEQ ID NO:2. In one or more embodiments, the mutation is N53K relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q57R relative to SEQ ID NO:2. In one or more embodiments, the mutation is S62delinsLA relative to SEQ ID NO:2. In one or more embodiments, the mutation is M96V relative to SEQ ID NO:2. In one or more embodiments, the mutation is R112L relative to SEQ ID NO:2. In one or more embodiments, the mutation is D155E relative to SEQ ID NO:2.In one or more embodiments, the mutation is N228D relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q330P relative to SEQ ID NO:2. In one or more embodiments, the mutation is V339A relative to SEQ ID NO:2. In one or more embodiments, the mutation is K391E relative to SEQ ID NO:2. In one or more embodiments, the mutation is N408Y relative to SEQ ID NO:2. In one or more embodiments, the mutation is V22G relative to SEQ ID NO:2. In one or more embodiments, the mutation is N34H relative to SEQ ID NO:2. In one or more embodiments, the mutation is G80V relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q107R relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y152D relative to SEQ ID NO:2. In one or more embodiments, the mutation is A156S relative to SEQ ID NO:2. In one or more embodiments, the mutation is L189F relative to SEQ ID NO:2. In one or more embodiments, the mutation is W204L relative to SEQ ID NO:2. In one or more embodiments, the mutation is S238G relative to SEQ ID NO: 2. In one or more embodiments, the mutation is I239M relative to SEQ ID NO: 2. In one or more embodiments, the mutation is A257V relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P259Q relative to SEQ ID NO: 2. In one or more embodiments, the mutation is N320H relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P323T relative to SEQ ID NO: 2. In one or more embodiments, the mutation is E338V relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P380L relative to SEQ ID NO: 2. In one or more embodiments, the mutation is T412P relative to SEQ ID NO: 2. In some embodiments, the pharmaceutical chaperone comprises migalastat or a salt thereof. In one or more embodiments, the dose of migalastat or a salt thereof is about 100 mg to about 150 mg FBE.In some embodiments, the salt of migalastat is migalastat hydrochloride. In one or more embodiments, the dose is about 150 mg of migalastat hydrochloride every other day, or an equivalent dose of migalastat or a salt thereof other than hydrochloride. In some embodiments, migalastat or a salt thereof is administered orally or by injection. These embodiments may be combined with each other or with other embodiments of the invention, such as with embodiments relating to methods of treating patients with Fabry disease, methods of enhancing α-Gal A in patients diagnosed with or suspected of having Fabry disease, or pharmacological chaperones to α-Gal A for use in treating patients diagnosed with Fabry disease, as well as applicable mutations, suitable PCs and doses, formulations, and their routes of administration.

[0012] Another aspect of the present invention relates to a pharmacological chaperone for α-Gal A for use in treating patients diagnosed with Fabry disease, wherein the patient has a missense mutation in the nucleic acid sequence encoding α-Gal A. In one or more embodiments, the mutation is D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, or R392T. In one or more embodiments, the mutation is N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, or N408Y. In one or more embodiments, the mutation is V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, or T412P. In various embodiments, these mutations are relative to SEQ ID NO:2. In one or more embodiments, the mutation is D33H relative to SEQ ID NO:2. In one or more embodiments, the mutation is G35A relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y88S relative to SEQ ID NO:2. In one or more embodiments, the mutation is T194A relative to SEQ ID NO:2. In one or more embodiments, the mutation is W204G relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y216S relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q250K relative to SEQ ID NO:2. In one or more embodiments, the mutation is R392T relative to SEQ ID NO:2. In one or more embodiments, the mutation is N53K relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q57R relative to SEQ ID NO:2. In one or more embodiments, the mutation is S62delinsLA relative to SEQ ID NO:2. In one or more embodiments, the mutation is M96V relative to SEQ ID NO:2. In one or more embodiments, the mutation is R112L relative to SEQ ID NO:2. In one or more embodiments, the mutation is D155E relative to SEQ ID NO:2.In one or more embodiments, the mutation is N228D relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q330P relative to SEQ ID NO:2. In one or more embodiments, the mutation is V339A relative to SEQ ID NO:2. In one or more embodiments, the mutation is K391E relative to SEQ ID NO:2. In one or more embodiments, the mutation is N408Y relative to SEQ ID NO:2. In one or more embodiments, the mutation is V22G relative to SEQ ID NO:2. In one or more embodiments, the mutation is N34H relative to SEQ ID NO:2. In one or more embodiments, the mutation is G80V relative to SEQ ID NO:2. In one or more embodiments, the mutation is Q107R relative to SEQ ID NO:2. In one or more embodiments, the mutation is Y152D relative to SEQ ID NO:2. In one or more embodiments, the mutation is A156S relative to SEQ ID NO:2. In one or more embodiments, the mutation is L189F relative to SEQ ID NO:2. In one or more embodiments, the mutation is W204L relative to SEQ ID NO:2. In one or more embodiments, the mutation is S238G relative to SEQ ID NO: 2. In one or more embodiments, the mutation is I239M relative to SEQ ID NO: 2. In one or more embodiments, the mutation is A257V relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P259Q relative to SEQ ID NO: 2. In one or more embodiments, the mutation is N320H relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P323T relative to SEQ ID NO: 2. In one or more embodiments, the mutation is E338V relative to SEQ ID NO: 2. In one or more embodiments, the mutation is P380L relative to SEQ ID NO: 2. In one or more embodiments, the mutation is T412P relative to SEQ ID NO: 2. In some embodiments, the pharmaceutical chaperone comprises migalastat or a salt thereof. In one or more embodiments, the dose of migalastat or a salt thereof is about 100 mg to about 150 mg FBE.In some embodiments, the salt of migalastat is migalastat hydrochloride. In one or more embodiments, the dose is about 150 mg of migalastat hydrochloride every other day, or an equivalent dose of migalastat or a salt thereof other than hydrochloride. In some embodiments, migalastat or a salt thereof is administered orally or by injection. These embodiments may be combined with each other or with other embodiments of the invention, such as methods of treating patients with Fabry disease, methods of enhancing α-Gal A in patients diagnosed with or suspected of having Fabry disease, or the use of a pharmacological chaperone for α-Gal A to manufacture a medicament for treating patients diagnosed with Fabry disease, as well as embodiments relating to applicable mutations, suitable PCs and doses, formulations, and their routes of administration.

[0013] Various embodiments are listed below. It will be understood that the embodiments listed below are not limited to those listed below and may be combined in other suitable combinations within the scope of the present invention. [Brief explanation of the drawings]

[0014] [Figure 1A] 1 shows the complete DNA sequence of the human wild-type GLA gene (SEQ ID NO: 1). [Figure 1B] 1 shows the complete DNA sequence of the human wild-type GLA gene (SEQ ID NO: 1). [Figure 1C] 1 shows the complete DNA sequence of the human wild-type GLA gene (SEQ ID NO: 1). [Figure 1D] 1 shows the complete DNA sequence of the human wild-type GLA gene (SEQ ID NO: 1). [Figure 1E] 1 shows the complete DNA sequence of the human wild-type GLA gene (SEQ ID NO: 1). [Figure 2] The wild-type α-Gal A protein (SEQ ID NO: 2) is shown. [Figure 3] 1 shows the nucleic acid sequence encoding the wild-type α-Gal A protein (SEQ ID NO: 3). DETAILED DESCRIPTION OF THE INVENTION

[0015] Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or method steps set forth in the following description. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0016] Various aspects of the present invention relate to the identification of novel GLA mutations in Fabry patients that will respond to treatment with pharmacological chaperones. Other aspects of the present invention relate to the treatment of these Fabry patients as well. For example, unexpectedly, low α-Gal A expression levels may be due to missense mutations D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, R392T, N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, N408Y, V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, or T412P. A activity was found to be increased upon exposure to pharmacological chaperones, and by extension, patients with these mutations would respond to treatment with pharmacological chaperones.

[0017] definition The terms used herein generally have their ordinary meaning in the art, within the context of this invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide further guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.

[0018] The term "Fabry disease" refers to an X-linked congenital abnormality of glycosphingolipid catabolism resulting from deficient lysosomal α-Gal A activity. This defect leads to the accumulation of the substrate globotriaosylceramide ("GL-3," also known as Gb3 or ceramide trihexoside) and related glycosphingolipids in vascular endothelial lysosomes in the heart, kidney, skin, and other tissues. Another substrate for this enzyme is plasma globotriaosylsphingosine ("plasma lyso-Gb3").

[0019] A "carrier" is a female in whom one X chromosome contains a defective α-Gal A gene and one X chromosome contains a normal gene, and in which there is X chromosome inactivation of the normal allele in one or more cell types. Carriers are often diagnosed with Fabry disease.

[0020] "Patient" refers to a subject who has been diagnosed with or is suspected of having a particular disease. The patient may be a human or an animal.

[0021] "Fabry patient" refers to an individual with mutant α-Gal A, as further defined below, who has been diagnosed with or is suspected of having Fabry disease. The hallmark markers for Fabry disease can occur with equal prevalence in hemizygous male and female carriers, although females are typically affected with less severe disease.

[0022] Human α-galactosidase A (α-Gal A) refers to the enzyme encoded by the human GLA gene. The complete DNA sequence of α-Gal A, including introns and exons, is available under GenBank accession number X14448.1 and is shown in Figures 1A-E (SEQ ID NO: 1). The human α-Gal A enzyme consists of 429 amino acids and is available under GenBank accession numbers X14448.1 and U78027.1, and is shown in SEQ ID NO: 2 and Figure 15. The nucleic acid sequence including only the coding region (i.e., exons) of SEQ ID NO: 1 is shown in Figure 3 (SEQ ID NO: 3).

[0023] The term "mutant protein" includes proteins that have a mutation in the gene encoding the protein that prevents the protein from achieving a stable conformation under conditions normally present in the ER. Failure to achieve a stable conformation results in significant amounts of the enzyme not being transported to lysosomes but rather being degraded. Such mutations are sometimes referred to as "conformational mutants." Such mutations include, but are not limited to, missense mutations and in-frame small deletions and insertions. As used herein, deletions are designated by the abbreviation "del" and insertions are designated by the abbreviation "ins." Thus, the nucleotide change "c.184_185insTAG" refers to the nucleotide sequence TAG between nucleotides 184 and 185, and the protein sequence change "S62delinsLA" refers to the deletion of amino acid S (serine) at position 62 and the insertion of amino acid sequence LA (leucine and alanine).

[0024] As used herein in one embodiment, the term "mutant α-Gal A" includes α-Gal A with a mutation in the gene encoding α-Gal A that prevents the enzyme from achieving a stable conformation under conditions normally present in the ER. The inability to achieve a stable conformation results in significant amounts of the enzyme not being transported to lysosomes but rather being degraded.

[0025] As used herein, the term "specific pharmacological chaperone" ("SPC") or "pharmacological chaperone" ("PC") 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) promoting the formation of a stable molecular conformation of the protein; (ii) directing trafficking of the protein from the ER to another cellular site, preferably the native cellular site, i.e., preventing ER-associated degradation of the protein; (iii) preventing aggregation of misfolded proteins; and / or (iv) restoring or enhancing at least some wild-type function and / or activity to a protein. For example, a compound that specifically binds to α-Gal A means that it binds to and exerts a chaperone effect on that enzyme and not on a general group of related or unrelated enzymes. More specifically, the term does not refer to endogenous chaperones such as BiP, or nonspecific agents, i.e., chemical chaperones, that demonstrate nonspecific chaperone activity for various proteins, such as glycerol, DMSO, or heavy water. In one or more embodiments of the present invention, the PC may be a reversible competitive inhibitor. In one or more embodiments, the PC is migalastat or a salt thereof. In another embodiment, the PC is migalastat free base (e.g., 123 mg of migalastat free base). In yet another embodiment, the PC is a salt of migalastat (e.g., 150 mg of migalastat HCl).

[0026] A "competitive inhibitor" of an enzyme can refer to a compound that is structurally similar to the chemical and molecular structure of the enzyme substrate and binds to the enzyme at approximately the same location as the substrate. Thus, the inhibitor competes for the same active site as the substrate molecule, thus increasing the Km. Competitive inhibition is usually reversible if enough substrate molecules are available to displace the inhibitor; i.e., competitive inhibitors can bind reversibly. Therefore, the amount of enzyme inhibition depends on the inhibitor concentration, the substrate concentration, and the relative affinities of the inhibitor and substrate for the active site.

[0027] As used herein, the term "specifically binds" refers to the interaction of a pharmacological chaperone with a protein, such as α-Gal A, specifically with amino acid residues in the protein that are directly involved in contact with the pharmacological chaperone. A pharmacological chaperone specifically binds to a target protein, e.g., α-Gal A, and exerts a chaperone effect on that protein, rather than on a general group of related or unrelated proteins. The amino acid residues in a protein that interact with a given pharmacological chaperone may or may not be within the "active site" of the protein. Specific binding can be assessed by routine binding assays or by structural studies, e.g., cocrystallization, NMR, etc. The active site of α-Gal A is the substrate-binding site.

[0028] "Deficient α-Gal A activity" refers to α-Gal A activity in cells from a patient that is below the normal range when compared (using the same method) with the activity of normal individuals who do not have or are not suspected of having Fabry disease or any other disease (particularly a hematological disorder).

[0029] As used herein, the terms "enhancing α-Gal A activity" or "increasing α-Gal A activity" refer to increasing the amount of α-Gal A in a stable conformation in cells contacted with an α-Gal A-specific pharmacological chaperone compared to the amount in cells (preferably of the same cell type, or the same cells, e.g., at an earlier stage) that have not been contacted with the α-Gal A-specific pharmacological chaperone. The terms also refer to increasing α-Gal A trafficking to lysosomes in cells contacted with an α-Gal A-specific pharmacological chaperone compared to trafficking of α-Gal A not contacted with a pharmacological chaperone specific for that protein. These terms refer to both wild-type and mutant α-Gal A. In one embodiment, the increase in α-Gal A amount in cells is measured by measuring the hydrolysis of an artificial substrate in lysates from cells treated with PC. Increased hydrolysis is an indication of increased α-Gal A activity.

[0030] The term "α-Gal A activity" refers to the normal physiological function of wild-type α-Gal A in a cell. For example, α-Gal A activity includes the hydrolysis of GL-3.

[0031] A "responder" is an individual diagnosed with or suspected of having a lysosomal storage disorder, e.g., Fabry disease, whose cells respond to contact with PC by exhibiting a sufficient increase in α-Gal A activity and / or alleviation of symptoms or enhancement of a surrogate marker, respectively. Non-limiting examples of enhanced Fabry surrogate markers include lyso-GB3 and those disclosed in U.S. Patent Application Publication No. 2010 / 0113517, hereby incorporated by reference in its entirety.

[0032] Non-limiting examples of improvements in surrogate markers of Fabry disease disclosed in U.S. Patent Application Publication No. 2010 / 0113517 include increased α-Gal A levels or activity in cells (e.g., fibroblasts) and tissues; decreased GL-3 accumulation; decreased plasma concentrations of homocysteine ​​and vascular cell adhesion molecule-1 (VCAM-1); decreased GL-3 accumulation in cardiomyocytes and valvular fibrocytes; decreased plasma lyso-Gb3; reduced cardiac hypertrophy (especially the left ventricle), valvular insufficiency, and arrhythmias; reduced proteinuria; decreased urinary concentrations of lipids such as CTH, lactosylceramide, and ceramide, and increased urinary concentrations of glucosylceramide and sphingomyelin; absence of lamellar inclusions (zebra bodies) in glomerular epithelial cells; improved renal function; reduced hypohidrosis; absence of angiokeratoma; and improvement in hearing abnormalities such as high-frequency sensorineural hearing loss, progressive hearing loss, sudden hearing loss, or tinnitus. Improvement of neurological symptoms includes prevention of transient ischemic attacks (TIA) or stroke; and reduction of neuropathic pain manifested as acroparesthesia (burning or tingling pain in the extremities). Another clinical marker that may determine Fabry disease is the prevalence of adverse cardiovascular symptoms. Common cardiac signs and symptoms in Fabry disease include left ventricular hypertrophy, valvular disease (especially mitral valve prolapse and / or regurgitation), premature coronary artery disease, angina, myocardial infarction, conduction abnormalities, arrhythmias, and congestive heart failure.

[0033] A dose that achieves one or more of the aforementioned responses is a "therapeutically effective dose."

[0034] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to humans. In some embodiments, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia. With respect to pharmaceutical carriers, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils. Water or aqueous solutions, saline solutions, and aqueous dextrose and glycerol solutions are preferably utilized as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition, or other editions.

[0035] As used herein, the term "isolated" means that the referenced material is removed from the environment in which it is normally found. Thus, isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of nucleic acid molecules, isolated nucleic acids include PCR products, mRNA bands on gels, cDNA, or restriction fragments. In another embodiment, isolated nucleic acids are preferably excised from the chromosome in which they are found, and more preferably, when found in a chromosome, they are no longer connected to non-regulatory regions, non-coding regions, or other genes located upstream or downstream of the gene contained in the isolated nucleic acid molecule. In yet another embodiment, isolated nucleic acids lack one or more introns. Isolated nucleic acids include sequences inserted into plasmids, cosmids, artificial chromosomes, etc. Thus, in a specific embodiment, recombinant nucleic acids are isolated nucleic acids. An isolated protein may be associated with other proteins or nucleic acids, or both, with the cell to which it is associated, or, if it is a membrane-bound protein, with the cell membrane. An isolated organelle, cell, or tissue is removed from the anatomical site in an organism in which it is found. An isolated material may, but need not, be purified.

[0036] The terms "about" and "approximately" are generally intended to refer to an acceptable degree of error for the measured quantity, given the nature or precision of the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, particularly in biological systems, the terms "about" and "approximately" can refer to values ​​within an order of magnitude of a given value, preferably within 10-fold or 5-fold, and more preferably within 2-fold. Numerical quantities provided herein are approximate unless otherwise specified, i.e., the terms "about" or "approximately" can be implied even when not explicitly stated.

[0037] The term "enzyme replacement therapy" or "ERT" refers to the introduction of a non-naturally occurring purified enzyme into an individual deficient in such an enzyme. The administered protein may be obtained from a natural source or by recombinant expression (as described in more detail below). The term also refers to the introduction of a purified enzyme in an individual who would otherwise require or benefit from the administration of the purified enzyme, e.g., an individual suffering from an enzyme deficiency. The introduced enzyme may be a purified recombinant enzyme made in vitro, or may be a protein purified from isolated tissue or body fluids, such as placenta or animal milk, or purified from a plant.

[0038] As used herein, the term "free base equivalent" or "FBE" refers to the amount of migalastat present in migalastat or a salt thereof. In other words, the term "FBE" refers to either the amount of migalastat free base or the equivalent amount of migalastat free base provided by a salt of migalastat. For example, due to the weight of the hydrochloride salt, only 150 mg of migalastat hydrochloride provides the same amount of migalastat as 123 mg of migalastat in free base form. Other salts are expected to have different conversion factors depending on the molecular weight of the salt.

[0039] The term "migalastat" includes migalastat free base or a pharmaceutically acceptable salt thereof (eg, migalastat HCl), unless specifically indicated to the contrary.

[0040] The terms "mutation" and "variant" (e.g., as in "applicable mutation or variant") refer to a change in the nucleotide sequence of a gene or chromosome. The two terms mentioned herein are typically used together—for example, as "mutation or variant"—to refer to a change in the nucleotide sequence mentioned in the perforation sequence. If only one of these two terms is mentioned for any reason, the missing term is included and should be understood as such. Furthermore, the terms "applicable mutation" and "applicable variant" refer to a mutation or variant that is applicable to PC therapy, for example, applicable to migalastat therapy. A particular type of applicable mutation or variant is a "HEK assay applicable mutation or variant," which is a mutation or variant that has been determined to be applicable to migalastat therapy according to the criteria in the in vitro HEK assay described herein.

[0041] Fabry disease Fabry disease is a rare, progressive, severe, X-linked lysosomal storage disorder. Mutations in the GLA gene cause a deficiency of the lysosomal enzyme α-Gal A, which is required for glycosphingolipid metabolism. Decreased α-Gal A activity, which begins in early childhood, leads to the accumulation of glycosphingolipids, including GL-3 and plasma lyso-Gb3, leading to the symptoms and fatal sequelae of Fabry disease, including pain, gastrointestinal symptoms, renal failure, cardiomyopathy, cerebrovascular events, and premature death. Early initiation of therapy and lifelong treatment offers the opportunity to slow disease progression and extend life expectancy.

[0042] Fabry disease encompasses a range of disease severity and age of onset, but is traditionally divided into two major phenotypes, "classic" and "late-onset." The classic phenotype is primarily considered to be in men with undetectable to low α-Gal A activity and early onset of renal, cardiac, and / or cerebrovascular symptoms. The late-onset phenotype is primarily considered to be in men with higher residual α-Gal A activity and later onset of these disease symptoms. Heterozygous female carriers typically express the late-onset phenotype, but may also exhibit the classic phenotype depending on the pattern of X-chromosome inactivation.

[0043] Over 1,000 GLA mutations that cause Fabry disease have been identified. Approximately 60% are missense mutations that result in a single amino acid substitution in the α-Gal A enzyme. Missense GLA mutations often result in the production of abnormally folded, unstable forms of α-Gal A, the majority of which are associated with the classic phenotype. Normal cellular quality control mechanisms in the endoplasmic reticulum prevent these abnormal proteins from trafficking to lysosomes, targeting them for premature degradation and removal. Many missense mutant forms are targets of migalastat, an α-Gal A-specific pharmacological chaperone.

[0044] The clinical manifestations of Fabry disease range in severity and roughly correlate with a patient's residual α-GAL levels. The majority of patients currently receiving treatment are referred to as patients with classic Fabry disease, most of whom are men. These patients experience disease in various organs, including the kidneys, heart, and brain. Disease symptoms first appear in adolescence and progress in severity until death, typically in the patient's 30s or 40s. Several recent studies suggest that there are many undiagnosed men and women with various Fabry disease symptoms, such as cardiac or renal dysfunction and stroke, that usually first appear in adulthood. This type of Fabry disease, referred to as late-onset Fabry disease, tends to have higher residual α-GAL levels than patients with classic Fabry disease. Late-onset Fabry disease patients typically experience their first disease symptoms in adulthood and often have disease symptoms localized to a single organ, such as left ventricular hypertrophy or progressive renal failure. In addition, late-onset Fabry disease can also present as stroke of unknown etiology.

[0045] Patients with Fabry disease have progressive renal dysfunction, and untreated individuals develop end-stage renal failure by their fourth decade. Deficiency of α-Gal A activity leads to the accumulation of GL-3 and related glycosphingolipids in many cell types, including those of the kidney. GL-3 accumulates in podocytes, distal tubules, and epithelial and tubular cells of the loop of Henle. Renal dysfunction can manifest as proteinuria and a reduced glomerular filtration rate.

[0046] Proper diagnosis of Fabry disease is challenging due to its rarity, multiorgan involvement, wide age range of onset, and heterogeneity. Misdiagnosis is frequent due to low awareness among medical professionals. The diagnosis of Fabry disease is most often confirmed after a patient presents with symptoms, based on reduced α-Gal A activity in plasma or peripheral white blood cells (WBCs) in conjunction with mutation analysis. Diagnosis is even more challenging in females, due to unreliable enzymatic identification of carrier females due to random X-chromosome inactivation in some carrier cells. For example, some obligate carriers (daughters of classically affected men) have α-Gal A enzyme activity ranging from normal to very low activity. Because carriers may have normal α-Gal A enzyme activity in white blood cells, only identification of the α-Gal A mutation by genetic testing provides accurate carrier identification and / or diagnosis.

[0047] Furthermore, as mentioned above, the age of onset, progression, and severity of Fabry disease depend, at least in part, on the rate of substrate accumulation, which correlates with intralysosomal enzyme activity. Thus, a complete lack of residual activity may correspond to rapid substrate accumulation and thus a more severe form of the disease (early onset and rapid progression). However, even small amounts of residual activity may be sufficient to degrade large amounts of substrate. This, in turn, would lead to a milder form of the disease with later onset and slower progression due to slowed substrate accumulation. Taking these factors into account, it is believed that even modest increases in enzyme activity may reduce the impact of a severe clinical phenotype. Data have estimated that for the majority of LSDs, just 1% to 6% of normal activity is sufficient to delay or prevent disease onset or produce a milder form of the disease. Thus, even small increases in activity can have a significant impact on substrate levels and, therefore, on the severity and rate of disease progression. Conversely, mutant lysosomal enzymes that show no response in vitro are predicted to be unresponsive in vivo.

[0048] In one or more embodiments, a mutation or variant of α-Gal A that is considered migalastat-applicable is defined as one that exhibits a ≥ 1.20-fold relative increase (+10 μM migalastat) over wild-type and a ≥ 3.0% absolute increase (+10 μM migalastat) when the mutant form of α-Gal A is expressed in HEK-293 cells according to a Good Laboratory Practice (GLP)-validated in vitro assay (GLP HEK or migalastat applicability assay) (referred to as the "HEK assay"). Such mutations or variants are also referred to herein as "HEK assay-applicable" mutations or variants.

[0049] Prior screening methods have been provided to determine enzyme enhancement before treatment begins. For example, an assay using HEK-293 cells has been utilized in clinical trials to predict whether a given mutation will respond to pharmacological chaperone (e.g., migalastat) treatment. In this assay, a cDNA construct is generated. The corresponding α-Gal A mutant form is transiently expressed in HEK-293 cells. The cells are then incubated with migalastat (17 nM to 1 mM) for 4 to 5 days. α-Gal A levels are then measured in cell lysates using a synthetic fluorogenic substrate (4-MU-α-Gal) or by Western blot. This has been performed for known disease-causing missense or small in-frame insertion / deletion mutations. Mutations previously identified as responsive to PC (e.g., migalastat) using these methods are listed in U.S. Patent No. 8,592,362 (hereby incorporated by reference in their entirety).

[0050] HEK assay applicable mutations include at least those mutations listed in a pharmacological reference table (e.g., those listed in the U.S. or international package insert for a migalastat product, such as GALAFOLD®). As used herein, a "pharmacological reference table" refers to any suitably accessible written or electronic record contained within the packaging of a migalastat product (e.g., GALAFOLD®) or within the package insert on a website accessible to a healthcare provider that conveys whether a particular mutation or variant is responsive to migalastat (e.g., GALAFOLD®) PC therapy, and is not necessarily limited to written records presented in tabular format. Thus, in one embodiment of the present invention, a "pharmacological reference table" refers to any convenient repository containing one or more applicable mutations or variants. In another embodiment, the "pharmacologic reference table" refers to an updated repository of applicable mutations or variants (i.e., D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, R392T, N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, N408Y, V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, or T412P), including the novel mutations or variants disclosed herein. Exemplary pharmacological reference tables for HEK assay applicable mutations can be found in the summaries of product characteristics and / or prescribing information for GALAFOLD® in the various countries in which it is approved for use, or at websites such as www.galafoldamenabilitytable.com or www.fabrygenevariantsearch.com, each of which is hereby incorporated herein in its entirety.

[0051] However, because only certain mutations are applicable to treatment with migalastat, there is a need to identify novel mutations and determine whether such mutations are applicable to migalastat therapy. As described in the Examples below, several novel mutations have been identified and determined to be applicable mutations to migalastat therapy. These mutations include D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, R392T, N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, N408Y, V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L and T412P.

[0052] Thus, in one or more embodiments, migalastat is used to treat Fabry disease and / or enhance a-Gal A activity in patients with an a-Gal A mutation selected from the group consisting of D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, and R392T. In one or more embodiments, migalastat is used to treat Fabry disease and / or enhance a-Gal A activity in patients with an a-Gal A mutation selected from the group consisting of N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, and N408Y. In one or more embodiments, migalastat is used to treat Fabry disease and / or enhance α-Gal A activity in patients with an α-Gal A mutation selected from the group consisting of V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, and T412P. In one or more embodiments, the patient has a D33H mutation. In one or more embodiments, the patient has a G35A mutation. In one or more embodiments, the patient has a Y88S mutation. In one or more embodiments, the patient has a T194A mutation. In one or more embodiments, the patient has a W204G mutation. In one or more embodiments, the patient has a Y216S mutation. In one or more embodiments, the patient has a Q250K mutation. In one or more embodiments, the patient has a mutation R392T. In one or more embodiments, the patient has a mutation N53K. In one or more embodiments, the patient has a mutation Q57R. In one or more embodiments, the patient has a mutation S62delinsLA. In one or more embodiments, the patient has a mutation M96V. In one or more embodiments, the patient has a mutation R112L. In one or more embodiments, the patient has a mutation D155E. In one or more embodiments, the patient has a mutation N228D.In one or more embodiments, the patient has a mutation Q330P. In one or more embodiments, the patient has a mutation V339A. In one or more embodiments, the patient has a mutation V339A. In one or more embodiments, the patient has a mutation K391E. In one or more embodiments, the patient has a mutation N408Y. In one or more embodiments, the patient has a mutation V22G. In one or more embodiments, the patient has a mutation N34H. In one or more embodiments, the patient has a mutation G80V. In one or more embodiments, the patient has a mutation Q107R. In one or more embodiments, the patient has a mutation Y152D. In one or more embodiments, the patient has a mutation A156S. In one or more embodiments, the patient has a mutation L189F. In one or more embodiments, the patient has a mutation W204L. In one or more embodiments, the patient has a mutation S238G. In one or more embodiments, the patient has a mutation I239M. In one or more embodiments, the patient has a mutation A257V. In one or more embodiments, the patient has a mutation P259Q. In one or more embodiments, the patient has the mutation N320H. In one or more embodiments, the patient has the mutation P323T. In one or more embodiments, the patient has the mutation E338V. In one or more embodiments, the patient has the mutation P380L. In one or more embodiments, the patient has the mutation T412P. In various embodiments, these α-Gal A mutations are relative to the amino acid sequence set forth in SEQ ID NO:2.

[0053] Exemplary nucleotide changes associated with these novel mutations are shown in Table 1 below.

[0054] TIFF2025165955000001.tif218170

[0055] Thus, in various embodiments, migalastat is used to treat Fabry disease and / or enhance α-Gal A activity in patients with α-Gal A mutations selected from the group consisting of c.97G>C, c.104G>C, c.263A>C, c.580A>G, c.610T>G, c.647A>C, c.748C>A, and c.1175G>C. In some embodiments, migalastat is used to treat Fabry disease and / or enhance α-Gal A activity in patients with a GLA mutation selected from the group consisting of c.159C>A c.159C>G, c.170A>G, c.184_185insTAG, c.286A>G, ​​c.335G>T, c.465T>c.465T>G, c.682A>G, c.989A>C, c.1016T>C, c.1171A>G, and c.1222A>T. In some embodiments, migalastat is used to treat Fabry disease and / or enhance α-Gal A activity in patients with a GLA mutation selected from the group consisting of c.65T>G, c.100A>C, c.239G>T, c.320A>G, c.454T>G, c.466G>T, c.567G>C or c.567G>T, c.611G>T, c.712A>G, c.717A>G, c.770C>T, c.776C>A, c.958A>C, c.967C>A, c.1013A>T, c.1139C>T, and c.1234A>C. In one or more embodiments, the patient has the GLA mutation c.97G>C. In one or more embodiments, the patient has the GLA mutation c.104G>C. In one or more embodiments, the patient has a GLA mutation c.263A>C. In one or more embodiments, the patient has a GLA mutation c.580A>G. In one or more embodiments, the patient has a GLA mutation c.610T>G. In one or more embodiments, the patient has a GLA mutation c.647A>C. In one or more embodiments, the patient has a GLA mutation c.748C>A. In one or more embodiments, the patient has a GLA mutation c.1175G>C. In one or more embodiments, the patient has a GLA mutation c.159C / G or c.159C>A.In one or more embodiments, the patient has a GLA mutation c.170A>G. In one or more embodiments, the patient has a GLA mutation c.184_185insTAG. In one or more embodiments, the patient has a GLA mutation c.286A>G. In one or more embodiments, the patient has a GLA mutation c.335G>T. In one or more embodiments, the patient has a GLA mutation c.465T>A or c.465T>G. In one or more embodiments, the patient has a GLA mutation c.682A>G. In one or more embodiments, the patient has a GLA mutation c.989A>C. In one or more embodiments, the patient has a GLA mutation c.1016T>C. In one or more embodiments, the patient has a GLA mutation c.1171A>G. In one or more embodiments, the patient has a GLA mutation c.1222A>T. In one or more embodiments, the patient has a GLA mutation c.65T>G. In one or more embodiments, the patient has a GLA mutation c.100A>C. In one or more embodiments, the patient has a GLA mutation c.239G>T. In one or more embodiments, the patient has a GLA mutation c.320A>G. In one or more embodiments, the patient has a GLA mutation c.454T>G. In one or more embodiments, the patient has a GLA mutation c.466G>T. In one or more embodiments, the patient has a GLA mutation c.567G>C or c.567G>T. In one or more embodiments, the patient has a GLA mutation c.611G>T. In one or more embodiments, the patient has a GLA mutation c.712A>G. In one or more embodiments, the patient has a GLA mutation c.717A>G. In one or more embodiments, the patient has a GLA mutation c.770C>T. In one or more embodiments, the patient has a GLA mutation c.776C>A. In one or more embodiments, the patient has a GLA mutation c.958A>C. In one or more embodiments, the patient has a GLA mutation c.967C>A. In one or more embodiments, the patient has the GLA mutation c.1013A>T. In one or more embodiments, the patient has the GLA mutation c.1139C>T.In one or more embodiments, the patient has the GLA mutation c.1234A>C. In various embodiments, these GLA mutations are relative to the amino acid sequence set forth in SEQ ID NO:3.

[0056] Furthermore, various embodiments of the present invention provide PCs for treating Fabry disease in patients with a mutation in the gene encoding α-Gal A, wherein the patient has been identified as having a missense mutation in human α-Gal A encoded by the nucleic acid sequence set forth in SEQ ID NO:1 and / or SEQ ID NO:3. Another aspect of the present invention relates to methods for treating patients diagnosed with Fabry disease. In one or more embodiments, the method comprises administering to the patient a therapeutically effective dose of α-Gal A PC. In another embodiment, the patient has a missense mutation in the nucleic acid sequence encoding α-Gal A. Another aspect of the present invention relates to methods for enhancing α-Gal A in patients diagnosed with or suspected of having Fabry disease. In one or more embodiments, the method comprises administering to the patient a therapeutically effective dose of α-Gal A PC, wherein the patient has a mutant α-Gal A encoded by a nucleic acid sequence having a missense mutation compared to SEQ ID NO:1 and / or SEQ ID NO:3. Details of these uses and methods, as well as additional embodiments, are provided below. Any of the embodiments relating to a method of treating a patient with Fabry disease, wherein the patient has been identified as having a missense mutation in human α-Gal A encoded by the nucleic acid sequence set forth in SEQ ID NO:1 and / or SEQ ID NO:3, a method of enhancing α-Gal A in a patient diagnosed with or suspected of having Fabry disease, the use of a pharmacological chaperone for α-Gal A for the manufacture of a medicament for the treatment of a patient diagnosed with Fabry disease, or the use of a pharmacological chaperone for α-Gal A for use in the treatment of a patient diagnosed with Fabry disease, can be combined with any of the other embodiments of the invention, for example, with embodiments relating to PCs and appropriate doses thereof.

[0057] In one or more embodiments, the patient may have other mutations in their GLA gene. For example, there may be mutations within intronic regions that may or may not affect the resulting α-Gal A enzyme. Thus, in one or more embodiments, the patient has a mutation encoded by a nucleic acid sequence having at least 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identity to SEQ ID NO:1. Furthermore, the patient may have one or more additional mutant α-Gal A within the coding region of the GLA gene. Thus, in one or more embodiments, the patient has a mutant α-Gal A encoded by a nucleic acid sequence having at least 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identity to SEQ ID NO:3. Additionally, in one or more embodiments, the patient has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 mutations compared to SEQ ID NO: 1 or SEQ ID NO: 3. It is further noted that some nucleic acid mutations in SEQ ID NO: 1 or SEQ ID NO: 3 may not result in amino acid changes to the resulting protein, as various nucleic acids are encoded by multiple nucleic acid sequences. Additionally, any of these embodiments may be combined with any of the other embodiments of the invention, such as those relating to applicable mutations, PCs, and their suitable dosages.

[0058] Pharmacological Chaperones Binding of small molecule inhibitors of LSD-related enzymes can increase the stability of both mutant and corresponding wild-type enzymes (see 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). Specifically, administration of small molecule derivatives of glucose and galactose, which are specific and selective competitive inhibitors of several target lysosomal enzymes, effectively increased the stability of the enzymes in cells in vitro, thereby increasing the trafficking of the enzymes to lysosomes. Therefore, increasing the amount of enzymes in lysosomes is expected to increase the hydrolysis of the enzyme substrates. The original theory behind this strategy was as follows: Because mutant enzyme proteins are unstable in the ER (Ishii et al., Biochem. Biophys. Res. Comm. 1996;220:812-815), they are delayed in their normal transport pathway (ER → Golgi apparatus → endosomes → lysosomes) and are prematurely degraded. Therefore, compounds that bind to and increase the stability of mutant enzymes could act as "chaperones" for the enzymes, increasing the amount that can exit the ER and be transported to lysosomes. In addition, because folding and trafficking of some wild-type proteins are incomplete, and in some cases up to 70% of some wild-type proteins are degraded before reaching their final cellular location, chaperones could be used to stabilize wild-type enzymes, increasing the amount of enzyme that can exit the ER and be transported to lysosomes.

[0059] In one or more embodiments, the pharmacological chaperone comprises migalastat or a salt thereof. The compound migalastat, also known as 1-deoxygalactonojirimycin (1-DGJ) or (2R,3S,4R,5S)-2-(hydroxymethyl)piperdine-3,4,5-triol, is a compound having the following chemical formula: TIFF2025165955000002.tif47161

[0060] As discussed herein, pharmaceutically acceptable salts of migalastat may also be used in the present invention. When a salt of migalastat is used, the dosage of the salt will be adjusted so that the patient receives a dose of migalastat equivalent to the amount that they would have received if migalastat free base had been used. An example of a pharmaceutically acceptable salt of migalastat is migalastat HCl: TIFF2025165955000003.tif46161

[0061] Migalastat is a low-molecular-weight iminosugar and an analog of the terminal galactose of GL-3. In vitro and in vivo pharmacological studies have demonstrated that migalastat acts as a pharmacological chaperone, binding with high affinity, selectivity, and reversibility to the active site of wild-type α-Gal A and specific mutant forms of α-Gal A. Binding of migalastat stabilizes these mutant forms of α-Gal A in the endoplasmic reticulum and promotes their proper trafficking to lysosomes, where its dissociation allows α-Gal A to reduce levels of GL-3 and other substrates.

[0062] In certain embodiments, the PC is migalastat or a salt thereof. In another embodiment, the PC comprises migalastat hydrochloride.

[0063] Any of these PCs for α-Gal A may be combined with any of the other embodiments of the invention, for example, with embodiments relating to methods of treating patients with Fabry disease, methods of increasing α-Gal A in patients diagnosed with or suspected of having Fabry disease, the use of a pharmacological chaperone for α-Gal A to manufacture a medicament for the treatment of patients diagnosed with Fabry disease, or a pharmacological chaperone for α-Gal A for use in treating patients diagnosed with Fabry disease, as well as with embodiments relating to suitable doses of the PC, applicable mutations, and to the treatment of Fabry patients with specific mutations in the nucleic acid sequence encoding α-Gal A.

[0064] Dosing, Formulation and Administration In one or more embodiments, a Fabry patient is administered migalastat or a salt thereof once every other day (also referred to as "QOD"). In various embodiments, the doses described herein relate to migalastat hydrochloride or an equivalent dose of migalastat or a salt thereof other than the hydrochloride salt. In some embodiments, these doses relate to the free base of migalastat. In alternative embodiments, these doses relate to a salt of migalastat. In further embodiments, the salt of migalastat is migalastat hydrochloride. The administration of migalastat or a salt of migalastat is referred to herein as "migalastat therapy."

[0065] An effective amount of migalastat or a salt thereof can range from about 100 mg FBE to about 150 mg FBE. Exemplary doses include about 100 mg FBE, about 105 mg FBE, about 110 mg FBE, about 115 mg FBE, about 120 mg FBE, about 123 mg FBE, about 125 mg FBE, about 130 mg FBE, about 135 mg FBE, about 140 mg FBE, about 145 mg FBE, or about 150 mg FBE.

[0066] It is again noted that 150 mg of migalastat hydrochloride is equivalent to 123 mg of the free base form of migalastat. Thus, in one or more embodiments, the dose is 150 mg of migalastat hydrochloride administered once every other day, or an equivalent dose of migalastat or a salt thereof other than the hydrochloride salt. As indicated above, this dose is referred to as 123 mg FBE of migalastat. In a further embodiment, the dose is 150 mg of migalastat hydrochloride administered once every other day. In other embodiments, the dose is 123 mg of migalastat free base administered once every other day.

[0067] In various embodiments, the effective amount is about 122 mg, about 128 mg, about 134 mg, about 140 mg, about 146 mg, about 150 mg, about 152 mg, about 159 mg, about 165 mg, about 171 mg, about 177 mg, or about 183 mg of migalastat hydrochloride.

[0068] Thus, in various embodiments, migalastat therapy comprises administering 150 mg of migalastat hydrochloride every other day, such as 123 mg FBE once every other day.

[0069] Administration of migalastat or a salt thereof can be over a period of time. In one or more embodiments, migalastat or a salt thereof is administered for a duration of at least 28 days, e.g., at least 30, 60, or 90 days, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 30, or 36 months, or at least 1, 2, 3, 4, or 5 years. In various embodiments, the migalastat therapy is long-term migalastat therapy for at least 6 months, e.g., at least 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 30, or 36 months, or at least 1, 2, 3, 4, or 5 years.

[0070] The administration of migalastat or a salt thereof according to the present invention may be in a formulation suitable for any route of administration, but is preferably administered in an oral dosage form such as a tablet, capsule, or solution. As an example, a patient is orally administered capsules each containing 150 mg of migalastat hydrochloride or an equivalent dose of migalastat or a salt thereof other than hydrochloride.

[0071] In some embodiments, the PC (e.g., migalastat or a salt thereof) is administered orally. In one or more embodiments, the PC (e.g., migalastat or a salt thereof) is administered by injection. The PC may be accompanied by a pharmaceutically acceptable carrier, which may depend on the method of administration.

[0072] In one or more embodiments, the PC (e.g., migalastat or a salt thereof) is administered as monotherapy and can be in a form suitable for any route of administration, such as orally in tablet or capsule or liquid form, or in a sterile aqueous solution for injection, etc. In other embodiments, the PC is provided as a lyophilized powder that is added to the replacement enzyme formulation during or immediately after reconstitution to prevent enzyme aggregation in vitro prior to administration.

[0073] When a PC (e.g., migastat or a salt thereof) is formulated for oral administration, tablets or capsules may be prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form, for example, of solutions, syrups, or suspensions, or may be prepared as a dry product for constitution with water or another suitable vehicle before use. Such liquid formulations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid). The formulations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents, as appropriate. Formulations for oral administration may be suitably formulated to give controlled release of the active chaperone compound.

[0074] Pharmaceutical formulations of PC (e.g., migastat or its salts) suitable for parenteral / injectable use generally include sterile aqueous solutions (where water soluble), or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, benzyl alcohol, sorbic acid, and the like. In many cases, it will be advisable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0075] Sterile injectable solutions are prepared by blending the required amount of purified enzyme (if present) and PC (e.g., migalastat or its salts) in an appropriate solvent with various other ingredients as enumerated above, as needed, followed by filtration or terminal sterilization. Generally, dispersions are prepared by blending the various sterilized active ingredients in a sterile vehicle containing the basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0076] The formulation may contain an excipient. Pharmaceutically acceptable excipients that may be included in the formulation include buffers, such as citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, amino acids, urea, alcohols, ascorbic acid, and phospholipids; proteins such as serum albumin, collagen, and gelatin; salts such as EDTA or EGTA, and sodium chloride; liposomes; polyvinylpyrollidone; sugars such as dextran, mannitol, sorbitol, and glycerol; propylene glycol and polyethylene glycol (e.g., PEG-4000, PEG-6000); glycerol; glycine or other amino acids; and lipids. Buffer systems used with the formulation include citrate, acetate, bicarbonate, and phosphate buffers. Phosphate buffers are a preferred embodiment.

[0077] The route of administration of the chaperone compound may be oral or parenteral, including intravenous, subcutaneous, intra-arterial, intraperitoneal, intraocular, intramuscular, buccal, rectal, intravaginal, intraorbital, intracerebral, intradermal, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intravesicular, intrapulmonary, intranasal, transmucosal, transdermal, or by inhalation.

[0078] Administration of the chaperone compound in the parenteral formulations described above may be by periodic injection of a bolus of the formulation, or by intravenous or intraperitoneal administration from an external (e.g., an IV bag) or internal (e.g., a bioerodible implant) reservoir.

[0079] The embodiments relating to pharmaceutical formulations and administration may be combined with any of the other embodiments of the invention, such as embodiments relating to methods of treating patients with Fabry disease, to the use of a pharmacological chaperone for α-Gal A to manufacture a medicament for the treatment of patients diagnosed with Fabry disease, or to a pharmacological chaperone for α-Gal A for use in the treatment of patients diagnosed with Fabry disease, as well as embodiments relating to applicable mutations, PCs and suitable dosages thereof.

[0080] In one or more embodiments, the PC (e.g., migalastat or a salt thereof) is administered in combination with ERT. ERT increases the amount of protein by exogenously introducing a wild-type or biologically functional enzyme via infusion. As discussed above, this therapy has been developed for many genetic disorders, including LSDs such as Fabry disease. After infusion, the exogenous enzyme is assumed to be taken up by tissues via nonspecific or receptor-specific mechanisms. Generally, uptake efficiency is not high, and the circulation time of exogenous proteins is short. In addition, exogenous proteins are unstable, subject to rapid intracellular degradation, and there is also the possibility of adverse immune reactions with subsequent treatment. In one or more embodiments, a chaperone is administered simultaneously with the replacement enzyme (e.g., replacement α-Gal A). In some embodiments, the chaperone is formulated with the replacement enzyme (e.g., replacement α-Gal A).

[0081] Throughout this specification, the phrases "one embodiment," "particular embodiment," "various embodiments," "one or more embodiments," or "an embodiment" mean that the particular feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearance of phrases such as "in one or more embodiments," "particular embodiment," "various embodiments," "in one embodiment," or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment of the invention. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0082] Although the invention herein has been described with reference to detailed embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made in the method and apparatus of the present invention without departing from the spirit and scope of the invention. Therefore, it is intended that the present invention cover such modifications and variations provided they come within the scope of the appended claims and their equivalents. [Example]

[0083] Effect of migalastat on α-Gal A mutations α-Gal A activity was measured in lysates prepared from HEK-293 cells transiently transfected with the indicated α-Gal A mutants and incubated for 5 days in the absence or presence of 10 μM migalastat. α-Gal A activity was expressed as nmol of free 4-MU liberated per mg of protein per hour (nmol / mg / hr). Baseline α-Gal A activity and α-Gal A activity after incubation with 10 μM migalastat were additionally expressed as a percentage of baseline wild-type α-Gal A activity (% WT). The wild-type α-Gal A activity used to calculate these percentages was the average activity measured in lysates from wild-type transfected cells incubated in the absence of migalastat and measured in parallel.

[0084] The α-Gal A activity results for the novel mutations D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, and R392T tested are shown in Table 2 below:

[0085] TIFF2025165955000004.tif91170

[0086] The results of α-Gal A activity testing for the novel mutations N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, and N408Y are shown in Table 3 below:

[0087] TIFF2025165955000005.tif77170

[0088] The α-Gal A activity results for the novel mutations V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, and T412P tested are shown in Table 4 below:

[0089] TIFF2025165955000006.tif103170

[0090] In Tables 2-4, values ​​were calculated for the mean ± standard error of the mean (SEM). nmol / mg / hr refers to "nmol of free 4-MU released per mg of protein per hour." WT refers to "wild type." NC refers to "not calculable." N / A refers to "not applicable."

[0091] Baseline and 10 μM migalastat α-Gal A activity: Migalastat differences in α-Gal A activity between lysates incubated in the absence and presence of 10 μM migalastat were determined using a one-tailed Mann-Whitney U test; increases at 10 μM migalastat with p<0.05 were considered statistically significant. "BLD" indicates that the mean α-Gal A activity was below the limit of detection (<142 nmol / mg / hr).

[0092] Baseline α-Gal A activity (% WT) = (α-Gal A activity in mutant-transfected cell lysate without migalastat ÷ α-Gal A activity in lysate from wild-type-transfected cells without migalastat). * 100.

[0093] 10 μM migalastat α-Gal A activity (% WT) = (α-Gal A activity in mutant-transfected cell lysate incubated with migalastat ÷ α-Gal A activity in wild-type-transfected cell lysate without migalastat). * 100.

[0094] Absolute increase (% WT) = 10 μM migalastat α-Gal A activity (% WT) - baseline α-Gal A activity (% WT).

[0095] Relative increase is the 10 μM migalastat α-Gal A activity in mutant-transfected cell lysates divided by the baseline α-Gal A activity in mutant-transfected cell lysates incubated without migalastat.

[0096] As can be clearly seen from Table 2, the novel α-Gal A mutations D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, and R392T demonstrated in vitro responses to incubation with migalastat that met compliance criteria. Therefore, patients with these mutations are predicted to be treatable with the migalastat therapy described herein.

[0097] As can be clearly seen from Table 3, the novel α-Gal A mutations D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, and R392T demonstrated in vitro responses to incubation with migalastat that met compliance criteria. Thus, patients with these mutations are predicted to be treatable with the migalastat therapy described herein.

[0098] As can be clearly seen from Table 4, the novel α-Gal A mutations V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, and T412P demonstrated in vitro responses to incubation with migalastat that met compliance criteria. Thus, patients with these mutations are predicted to be treatable with the migalastat therapy described herein.

[0099] The patents and scientific literature referred to herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, articles and scientific literature cited herein are hereby incorporated by reference.

[0100] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, workers skilled in the art will understand that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. 1. A method for treating Fabry disease in a human patient in need thereof, comprising administering to said patient a therapeutically effective dose of migalastat or a salt thereof, wherein said patient has an α-galactosidase A mutation selected from the group consisting of: D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, and R392T.

2. 10. The method of claim 1, wherein the migalastat or a salt thereof is administered to the patient every other day.

3. 10. The method of claim 1, wherein the patient is administered about 100 to about 150 mg free base equivalent of the migalastat or salt thereof every other day.

4. 10. The method of claim 1, wherein the patient is administered about 123 mg free base equivalent of the migalastat or salt thereof every other day.

5. 10. The method of claim 1, wherein the patient is administered about 123 mg of migalastat free base every other day.

6. 10. The method of claim 1, wherein the patient is administered about 150 mg of migalastat hydrochloride every other day.

7. The method of any one of claims 1 to 6, wherein the migalastat or a salt thereof is administered orally.

8. The method according to any one of claims 1 to 6, wherein the migalastat or a salt thereof is administered by injection.

9. The method according to any one of claims 1 to 8, wherein the migalastat or a salt thereof enhances α-galactosidase A activity.

10. The method of any one of claims 1 to 9, wherein the patient is male.

11. The method of any one of claims 1 to 9, wherein the patient is female.

12. The method of any one of claims 1 to 11, wherein the mutation is D33H.

13. The method of any one of claims 1 to 11, wherein the mutation is G35A.

14. The method of any one of claims 1 to 11, wherein the mutation is Y88S.

15. The method of any one of claims 1 to 11, wherein the mutation is T194A.

16. The method of any one of claims 1 to 11, wherein the mutation is W204G.

17. The method of any one of claims 1 to 11, wherein the mutation is Y216S.

18. The method of any one of claims 1 to 11, wherein the mutation is Q250K.

19. The method of any one of claims 1 to 11, wherein the mutation is R392T.

20. The method of any one of claims 1 to 19, wherein the mutation is disclosed in a pharmacological reference table.

21. 21. The method of claim 20, wherein the pharmacological reference table is provided in the package insert for a migalastat product approved for treating Fabry disease.

22. 21. The method of claim 20, wherein the pharmacological reference table is provided in the package insert for GALAFOLD®.

23. 21. The method of claim 20, wherein the pharmacological reference table is provided on a website.

24. 24. The method of claim 23, wherein the website is one or more of www.galafoldamenabilitytable.com or www.fabrygenevariantsearch.com.

25. 1. A method of enhancing α-galactosidase A in a patient diagnosed with or suspected of having Fabry disease, comprising administering to said patient a therapeutically effective dose of migalastat or a salt thereof, wherein said patient has an α-galactosidase A mutation selected from the group consisting of: D33H, G35A, Y88S, T194A, W204G, Y216S, Q250K, and R392T.

26. 26. The method of claim 25, wherein the migalastat or a salt thereof is administered to the patient every other day.

27. 26. The method of claim 25, wherein the patient is administered about 100 to about 150 mg free base equivalent of the migalastat or salt thereof every other day.

28. 26. The method of claim 25, wherein the patient is administered about 123 mg free base equivalent of the migalastat or salt thereof every other day.

29. 26. The method of claim 25, wherein the patient is administered about 123 mg of migalastat free base every other day.

30. 26. The method of claim 25, wherein the patient is administered about 150 mg of migalastat hydrochloride every other day.

31. The method of any one of claims 25 to 30, wherein the migalastat or a salt thereof is administered orally.

32. The method of any one of claims 25 to 30, wherein the migalastat or a salt thereof is administered by injection.

33. The method of any one of claims 25 to 32, wherein the patient is male.

34. The method of any one of claims 25 to 32, wherein the patient is female.

35. 35. The method of any one of claims 25 to 34, wherein the mutation is D33H.

36. 35. The method of any one of claims 25 to 34, wherein the mutation is G35A.

37. 35. The method of any one of claims 25 to 34, wherein the mutation is Y88S.

38. The method of any one of claims 25 to 34, wherein the mutation is T194A.

39. 35. The method of any one of claims 25 to 34, wherein the mutation is W204G.

40. 35. The method of any one of claims 25 to 34, wherein the mutation is Y216S.

41. 35. The method of any one of claims 25 to 34, wherein the mutation is Q250K.

42. The method of any one of claims 25 to 34, wherein the mutation is R392T.

43. The method of any one of claims 25 to 42, wherein the mutation is disclosed in a pharmacological reference table.

44. 44. The method of claim 43, wherein the pharmacological reference table is provided in the package insert for a migalastat product approved for treating Fabry disease.

45. 44. The method of claim 43, wherein the pharmacological reference table is provided in the package insert for GALAFOLD®.

46. 44. The method of claim 43, wherein the pharmacological reference table is provided on a website.

47. 47. The method of claim 46, wherein the website is one or more of www.galafoldamenabilitytable.com or www.fabrygenevariantsearch.com.

48. 1. A method for treating Fabry disease in a human patient in need thereof, comprising administering to said patient a therapeutically effective dose of migalastat or a salt thereof, wherein said patient has an α-galactosidase A mutation selected from the group consisting of: N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, and N408Y.

49. 49. The method of claim 48, wherein the migalastat or a salt thereof is administered to the patient every other day.

50. 49. The method of claim 48, wherein the patient is administered about 100 to about 150 mg free base equivalent of the migalastat or salt thereof every other day.

51. 49. The method of claim 48, wherein the patient is administered about 123 mg free base equivalent of the migalastat or salt thereof every other day.

52. 49. The method of claim 48, wherein the patient is administered about 123 mg of migalastat free base every other day.

53. 49. The method of claim 48, wherein the patient is administered about 150 mg of migalastat hydrochloride every other day.

54. 54. The method of any one of claims 48 to 53, wherein the migalastat or a salt thereof is administered orally.

55. 54. The method of any one of claims 48 to 53, wherein the migalastat or a salt thereof is administered by injection.

56. The method according to any one of claims 48 to 55, wherein the migalastat or a salt thereof enhances α-galactosidase A activity.

57. 57. The method of any one of claims 48 to 56, wherein the patient is male.

58. 57. The method of any one of claims 48 to 56, wherein the patient is female.

59. 59. The method of any one of claims 48 to 58, wherein the mutation is N53K.

60. 59. The method of any one of claims 48 to 58, wherein the mutation is Q57R.

61. 59. The method of any one of claims 48 to 58, wherein the mutation is S62delinsLA.

62. 59. The method of any one of claims 48 to 58, wherein the mutation is M96V.

63. 59. The method of any one of claims 48 to 58, wherein the mutation is R112L.

64. 59. The method of any one of claims 48 to 58, wherein the mutation is D155E.

65. 59. The method of any one of claims 48 to 58, wherein the mutation is N228D.

66. 59. The method of any one of claims 48 to 58, wherein the mutation is Q330P.

67. 59. The method of any one of claims 48 to 58, wherein the mutation is V339A.

68. 59. The method of any one of claims 48 to 58, wherein the mutation is K391E.

69. 59. The method of any one of claims 48 to 58, wherein the mutation is N408Y.

70. 70. The method of any one of claims 48 to 69, wherein the mutation is disclosed in a pharmacological reference table.

71. 71. The method of claim 70, wherein the pharmacological reference table is provided in the package insert for a migalastat product approved for treating Fabry disease.

72. 71. The method of claim 70, wherein the pharmacological reference table is provided in the package insert for GALAFOLD®.

73. 71. The method of claim 70, wherein the pharmacological reference table is provided on a website.

74. 74. The method of claim 73, wherein the website is one or more of www.galafoldamenabilitytable.com or www.fabrygenevariantsearch.com.

75. 1. A method of enhancing α-galactosidase A in a patient diagnosed with or suspected of having Fabry disease, comprising administering to said patient a therapeutically effective dose of migalastat or a salt thereof, wherein said patient has an α-galactosidase A mutation selected from the group consisting of: N53K, Q57R, S62delinsLA, M96V, R112L, D155E, N228D, Q330P, V339A, K391E, and N408Y.

76. 76. The method of claim 75, wherein the migalastat or a salt thereof is administered to the patient every other day.

77. 76. The method of claim 75, wherein said patient is administered about 100 to about 150 mg free base equivalent of said migalastat or salt thereof every other day.

78. 76. The method of claim 75, wherein the patient is administered about 123 mg free base equivalent of the migalastat or salt thereof every other day.

79. 76. The method of claim 75, wherein the patient is administered about 123 mg of migalastat free base every other day.

80. 76. The method of claim 75, wherein the patient is administered about 150 mg of migalastat hydrochloride every other day.

81. 81. The method of any one of claims 75 to 80, wherein the migalastat or a salt thereof is administered orally.

82. 81. The method of any one of claims 75 to 80, wherein the migalastat or a salt thereof is administered by injection.

83. 83. The method of any one of claims 75 to 82, wherein the patient is male.

84. 83. The method of any one of claims 75 to 82, wherein the patient is female.

85. 85. The method of any one of claims 75 to 84, wherein the mutation is N53K.

86. 85. The method of any one of claims 75 to 84, wherein the mutation is Q57R.

87. 85. The method of any one of claims 75 to 84, wherein the mutation is S62delinsLA.

88. 85. The method of any one of claims 75 to 84, wherein the mutation is M96V.

89. 85. The method of any one of claims 75 to 84, wherein the mutation is R112L.

90. 85. The method of any one of claims 75 to 84, wherein the mutation is D155E.

91. 85. The method of any one of claims 75 to 84, wherein the mutation is N228D.

92. 85. The method of any one of claims 75 to 84, wherein the mutation is Q330P.

93. 85. The method of any one of claims 75 to 84, wherein the mutation is V339A.

94. 85. The method of any one of claims 75 to 84, wherein the mutation is K391E.

95. 85. The method of any one of claims 75 to 84, wherein the mutation is N408Y.

96. 96. The method of any one of claims 75 to 95, wherein the mutation is disclosed in a pharmacological reference table.

97. 97. The method of claim 96, wherein the pharmacological reference table is provided in the package insert for a migalastat product approved for treating Fabry disease.

98. 97. The method of claim 96, wherein the pharmacological reference table is provided in the package insert for GALAFOLD®.

99. 97. The method of claim 96, wherein the pharmacological reference table is provided on a website.

100. 100. The method of claim 99, wherein the website is one or more of www.galafoldamenabilitytable.com or www.fabrygenevariantsearch.com.

101. 1. A method for treating Fabry disease in a human patient in need thereof, comprising administering to said patient a therapeutically effective dose of migalastat or a salt thereof, wherein said patient has an α-galactosidase A mutation selected from the group consisting of: V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, and T412P.

102. 102. The method of claim 101, wherein the migalastat or a salt thereof is administered to the patient every other day.

103. 102. The method of claim 101, wherein said patient is administered about 100 to about 150 mg free base equivalent of said migalastat or salt thereof every other day.

104. 102. The method of claim 101, wherein the patient is administered about 123 mg free base equivalent of migalastat or a salt thereof every other day.

105. 102. The method of claim 101, wherein the patient is administered about 123 mg of migalastat free base every other day.

106. 102. The method of claim 101, wherein the patient is administered about 150 mg of migalastat hydrochloride every other day.

107. 107. The method of any one of claims 101 to 106, wherein the migalastat or a salt thereof is administered orally.

108. The method of any one of claims 101 to 106, wherein the migalastat or a salt thereof is administered by injection.

109. The method of any one of claims 101 to 108, wherein the migalastat or a salt thereof enhances α-galactosidase A activity.

110. 110. The method of any one of claims 101 to 109, wherein the patient is male.

111. 110. The method of any one of claims 101 to 109, wherein the patient is female.

112. 112. The method of any one of claims 101 to 111, wherein the mutation is V22G.

113. 112. The method of any one of claims 101 to 111, wherein the mutation is N34H.

114. 112. The method of any one of claims 101 to 111, wherein the mutation is G80V.

115. 112. The method of any one of claims 101 to 111, wherein the mutation is Q107R.

116. 112. The method of any one of claims 101 to 111, wherein the mutation is Y152D.

117. 112. The method of any one of claims 101 to 111, wherein the mutation is A156S.

118. 112. The method of any one of claims 101 to 111, wherein the mutation is L189F.

119. 112. The method of any one of claims 101 to 111, wherein the mutation is W204L.

120. 112. The method of any one of claims 101 to 111, wherein the mutation is S238G.

121. 112. The method of any one of claims 101 to 111, wherein the mutation is I239M.

122. 112. The method of any one of claims 101 to 111, wherein the mutation is A257V.

123. 112. The method of any one of claims 101 to 111, wherein the mutation is P259Q.

124. 112. The method of any one of claims 101 to 111, wherein the mutation is N320H.

125. 112. The method of any one of claims 101 to 111, wherein the mutation is P323T.

126. 112. The method of any one of claims 101 to 111, wherein the mutation is E338V.

127. 112. The method of any one of claims 101 to 111, wherein the mutation is P380L.

128. 112. The method of any one of claims 101 to 111, wherein the mutation is T412P.

129. 129. The method of any one of claims 101 to 128, wherein the mutation is disclosed in a pharmacological reference table.

130. 130. The method of claim 129, wherein the pharmacological reference table is provided in the package insert for a migalastat product approved for treating Fabry disease.

131. 130. The method of claim 129, wherein the pharmacological reference table is provided in the package insert for GALAFOLD®.

132. 130. The method of claim 129, wherein the pharmacological reference table is provided on a website.

133. 133. The method of claim 132, wherein the website is one or more of www.galafoldamenabilitytable.com or www.fabrygenevariantsearch.com.

134. 1. A method of enhancing α-galactosidase A in a patient diagnosed with or suspected of having Fabry disease, comprising administering to said patient a therapeutically effective dose of migalastat or a salt thereof, wherein said patient has an α-galactosidase A mutation selected from the group consisting of: V22G, N34H, G80V, Q107R, Y152D, A156S, L189F, W204L, S238G, I239M, A257V, P259Q, N320H, P323T, E338V, P380L, and T412P.

135. 135. The method of claim 134, wherein the migalastat or a salt thereof is administered to the patient every other day.

136. 135. The method of claim 134, wherein said patient is administered about 100 to about 150 mg free base equivalent of said migalastat or salt thereof every other day.

137. 135. The method of claim 134, wherein the patient is administered about 123 mg free base equivalent of the migalastat or salt thereof every other day.

138. 135. The method of claim 134, wherein the patient is administered about 123 mg of migalastat free base every other day.

139. 135. The method of claim 134, wherein the patient is administered about 150 mg of migalastat hydrochloride every other day.

140. 140. The method of any one of claims 134 to 139, wherein the migalastat or a salt thereof is administered orally.

141. 140. The method of any one of claims 134 to 139, wherein the migalastat or a salt thereof is administered by injection.

142. 142. The method of any one of claims 134 to 141, wherein the patient is male.

143. 142. The method of any one of claims 134 to 141, wherein the patient is female.

144. 144. The method of any one of claims 134 to 143, wherein the mutation is V22G.

145. 144. The method of any one of claims 134 to 143, wherein the mutation is N34H.

146. 144. The method of any one of claims 134 to 143, wherein the mutation is G80V.

147. 144. The method of any one of claims 134 to 143, wherein the mutation is Q107R.

148. 144. The method of any one of claims 134 to 143, wherein the mutation is Y152D.

149. 144. The method of any one of claims 134 to 143, wherein the mutation is A156S.

150. 144. The method of any one of claims 134 to 143, wherein the mutation is L189F.

151. 144. The method of any one of claims 134 to 143, wherein the mutation is W204L.

152. 144. The method of any one of claims 134 to 143, wherein the mutation is S238G.

153. 144. The method of any one of claims 134 to 143, wherein the mutation is I239M.

154. 144. The method of any one of claims 134 to 143, wherein the mutation is A257V.

155. 144. The method of any one of claims 134 to 143, wherein the mutation is P259Q.

156. 144. The method of any one of claims 134 to 143, wherein the mutation is N320H.

157. 144. The method of any one of claims 134 to 143, wherein the mutation is P323T.

158. 144. The method of any one of claims 134 to 143, wherein the mutation is E338V.

159. 144. The method of any one of claims 134 to 143, wherein the mutation is P380L.

160. 144. The method of any one of claims 134 to 143, wherein the mutation is T412P.

161. 161. The method of any one of claims 134 to 160, wherein the mutation is disclosed in a pharmacological reference table.

162. The method of claim 161, wherein the pharmacological reference table is provided in the package insert for a migalastat product approved for treating Fabry disease.

163. 162. The method of claim 161, wherein the pharmacological reference table is provided in the package insert for GALAFOLD®.

164. 162. The method of claim 161, wherein the pharmacological reference table is provided on a website.

165. 165. The method of claim 164, wherein the website is one or more of www.galafoldamenabilitytable.com or www.fabrygenevariantsearch.com.

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