Medication regimens for treating Fabry disease

Abstract

A method for treating Fabry disease is provided. [Solution] Provided are dosing regimens and administration schedules for using 1-deoxygalactonojirimycin and enzyme replacement therapy for the treatment of Fabry disease. In certain embodiments, the present application provides dosing regimens and administration schedules for using migalastat hydrochloride and agalsidase (e.g., agalsidase alfa or agalsidase beta) for the treatment of Fabry disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 451,798, filed March 11, 2011; U.S. Provisional Patent Application No. 61 / 578,201, filed December 20, 2011, and U.S. Provisional Patent Application No. 61 / 596,165, filed February 7, 2012, the disclosures of which are hereby incorporated by reference in their entireties.

[0002] The present application provides dosing regimens and administration schedules for using 1-deoxygalactonojirimycin and enzyme replacement therapy for the treatment of Fabry disease. [Background technology]

[0003] Fabry disease is a progressive, X-linked, congenital disorder of glycospingolipid metabolism caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) as a result of mutations in the α-Gal A gene (GLA). Despite being an X-linked disorder, females may develop clinical symptoms to varying degrees. Fabry disease is rare, with an estimated incidence ranging from 1 in 40,000 males to 1 in 117,000 in the general population. Furthermore, a later-onset phenotypic variant of Fabry disease exists, which may be underdiagnosed because it does not exhibit classic signs and symptoms. This, along with newborn screening tests for Fabry disease, suggests that the actual incidence of Fabry disease may be higher than current estimates.

[0004] Clinical manifestations of the disease may correlate with residual α-Gal A levels. Untreated, Fabry patients have a reduced life expectancy, with death usually occurring within the first decade of life after the third or fourth decade due to vascular disease affecting the kidneys, heart, and / or central nervous system. The enzyme deficiency causes the intracellular accumulation of the substrate globotriaosylceramide (GL-3) in vascular endothelial and visceral tissues throughout the body. Gradual decline in renal function and worsening azotemia due to glycospingolipid deposition usually occurs between the ages of 20 and 40, but can occur as early as the teenage years. Renal involvement is observed in both hemizygous (male) and heterozygous (female) patients.

[0005] Cardiac disease occurs in most men and many women. Early cardiac findings include left ventricular enlargement, valvular complications, and conduction abnormalities. Mitral regurgitation is the most common valvular lesion, typically presenting in childhood or adolescence. Cerebrovascular symptoms are primarily due to multifocal small vessel complications and can include thrombosis, transient ischemic attacks, basilar artery ischemia and aneurysms, seizures, hemiplegia, hemisensory loss, aphasia, labyrinthine disorders, or cerebral hemorrhage. The average age at onset of cerebrovascular symptoms is 33.8 years. With advancing age, personality changes and psychotic behavior may become apparent.

[0006] The current approved treatment for Fabry disease is enzyme replacement therapy ("ERT"). Two α-Gal A products are currently available for the treatment of Fabry disease: agalsidase alfa (Replagal®, Shire Human Genetic Therapies) and agalsidase beta (Fabrazyme®; Genzyme Corporation). These two forms of ERT are intended to replace a patient's insufficient α-Gal A activity by administering a recombinant form of the enzyme intravenously. While ERT is effective in many situations, this treatment also has limitations. ERT has not been demonstrated to reduce stroke risk, it causes a slow myocardial response, and there is limited GL-3 excretion from some cell types in the kidney. Some patients develop an immune response to ERT.

[0007] 1-Deoxygalactonojirimycin and its salt, 1-deoxygalactonojirimycin hydrochloride (also known by its United States Adoptive Name (USAN) migalastat hydrochloride) act as pharmacological chaperones for mutant α-Gal A by selectively binding to the enzyme, thereby increasing its stability and assisting the enzyme in folding into its correct three-dimensional shape. This stabilization of α-Gal A allows the cellular quality control machinery to recognize the enzyme as correctly folded, thereby increasing its transport to lysosomes, allowing lysosomes to fulfill their intended biological function of metabolizing GL-3. As a result of restoring correct transport of α-Gal A from the ER to lysosomes, migalastat hydrochloride also reduces the accumulation of misfolded proteins in the ER, thereby alleviating cellular stress and some of the inflammatory responses that may contribute to Fabry disease. Multiple in vitro and in vivo preclinical studies, as well as clinical trials, of migalastat hydrochloride have been conducted. Migalastat hydrochloride has been shown to increase the amount of intracellular α-Gal A protein and enhance the transport of the mutant enzyme to lysosomes. Summary of the Invention [Means for solving the problem]

[0008] This application provides dosing regimens and administration schedules for using 1-deoxygalactonojirimycin and enzyme replacement therapy for the treatment of Fabry disease. In certain embodiments, this application provides dosing regimens and administration schedules for using migalastat hydrochloride and agalsidase (e.g., agalsidase alfa or agalsidase beta) for the treatment of Fabry disease.

[0009] In one embodiment, the method comprises administering to a patient in need thereof about 50 mg to about 600 mg of 1-deoxygalactonojirimycin and an effective amount of α-Gal A enzyme replacement therapy. 1-Deoxygalactonojirimycin can be administered before, after, or simultaneously with α-Gal A enzyme replacement therapy. In one embodiment, the patient fasts for a period beginning about 0.5 to about 4 hours prior to and ending about 0.5 to about 4 hours after administration of 1-deoxygalactonojirimycin. In a further embodiment, the patient fasts for at least about 2 hours prior to and at least about 2 hours after administration of 1-deoxygalactonojirimycin.

[0010] In another embodiment, 1-deoxygalactonojirimycin is administered simultaneously with or about 4 hours before (T=-4 hours to T=0 hours) the administration of α-Gal A enzyme replacement therapy. In a further embodiment, 1-deoxygalactonojirimycin is administered about 2 hours before the administration of α-Gal A enzyme replacement therapy.

[0011] In a particular embodiment, the 1-deoxygalactonojirimycin is migalastat hydrochloride. In one embodiment, the α-Gal A enzyme replacement therapy is agalsidase alfa or agalsidase beta.

[0012] In one embodiment, 1-deoxygalactonojirimycin is administered as an adjunct to α-Gal A enzyme replacement therapy. In another embodiment, 1-deoxygalactonojirimycin and α-Gal A enzyme replacement therapy are administered as combination therapy.

[0013] In a specific embodiment, the amount of 1-deoxygalactonojirimycin administered by the above-described method is from about 150 mg to about 450 mg. In one embodiment, the amount of 1-deoxygalactonojirimycin administered is selected from 150 mg, 300 mg, and 450 mg.

[0014] In particular embodiments, 1-deoxygalactonojirimycin is administered immediately prior to or simultaneously with the administration of α-Gal A enzyme replacement therapy. In alternative embodiments, a second dose of 1-deoxygalactonojirimycin is administered within 4 hours of the administration of α-Gal A enzyme replacement therapy.

[0015] In certain embodiments, 1-deoxygalactonojirimycin is administered every 1 to 4 weeks to a patient who is also receiving α-Gal A enzyme replacement therapy. In further embodiments, 1-deoxygalactonojirimycin is administered every 12 to 16 days to a patient who is also receiving α-Gal A enzyme replacement therapy. In further embodiments, 1-deoxygalactonojirimycin is administered every 14 days to a patient who is also receiving α-Gal A enzyme replacement therapy. In certain embodiments, α-Gal A enzyme replacement therapy is administered every 14 days as a combination or adjunctive therapy to a patient who is also receiving 1-deoxygalactonojirimycin.

[0016] The present application also provides 1-deoxygalactonojirimycin for use in treating Fabry disease, wherein the treatment comprises administering to a human subject in need thereof about 50 mg to about 600 mg of 1-deoxygalactonojirimycin and an effective amount of α-Gal A enzyme replacement therapy.

[0017] The present application also provides a use of 1-deoxygalactonojirimycin in the preparation of a medicament for treating Fabry disease, wherein the treatment comprises administering to a human subject in need thereof about 50 mg to about 600 mg of 1-deoxygalactonojirimycin and an effective amount of α-Gal A enzyme replacement therapy.

[0018] The present application also provides a kit for treating Fabry disease in a subject, the kit comprising about 50 mg to about 600 mg of 1-deoxygalactonojirimycin and an effective amount of α-Gal A enzyme replacement therapy. In certain embodiments, the amount of 1-deoxygalactonojirimycin in the kit is selected from 150 mg, 300 mg, and about 450 mg. [Brief explanation of the drawings]

[0019] [Figure 1] 1 shows a composite profile of plasma α-Gal A activity in patients treated with 0.5 mg / kg or 1.0 mg / kg agalsidase beta alone (Period 1) or in combination with 150 mg migalastat (Period 2) during Periods 1 and 2. [Figure 2] 1 shows the increase in plasma α-Gal A activity AUC in all patients after co-administration with migalastat. [Figure 3] The partial AUC for each sampling time point is shown, which demonstrated the potent increase in plasma α-Gal A activity with co-administration of 0.5 mg / kg or 1.0 mg / kg agalsidase beta with 150 mg migalastat. [Figure 4A-B] 1 shows increased skin α-Gal A activity in two patients following co-administration of 0.5 mg / kg agalsidase beta and 150 mg migalastat. [Figure 5A-B] Figure 5A shows an increase in skin α-Gal A activity in two patients after co-administration of 0.5 mg / kg agalsidase beta with 150 mg migalastat. Figure 5B shows an increase in skin α-Gal A activity after co-administration of 0.5 mg / kg agalsidase beta with 150 mg migalastat in a patient who received a 40-minute long ERT infusion during period 2. [Figure 6A-B] 1 shows increased skin α-Gal A activity in two patients following co-administration of 1.0 mg / kg agalsidase beta with 150 mg migalastat. [Figure 7A-B] 1 shows increased PBMC α-Gal A activity in two patients following co-administration of 0.5 mg / kg agalsidase beta with 150 mg migalastat. [Figure 8A-B] 1 shows increased PBMC α-Gal A activity in two patients following co-administration of 0.5 mg / kg agalsidase beta with 150 mg migalastat. [Figure 9A-B] 1 shows increased PBMC α-Gal A activity in two patients following co-administration of 1.0 mg / kg agalsidase beta with 150 mg migalastat. [Figure 10] 1 shows a table summarizing the increase in α-Gal A activity in plasma, skin, and PBMCs following co-administration of 0.5 mg / kg or 1.0 mg / kg agalsidase beta with 150 mg migalastat. [Figure 11] The genotypes of each test subject in Examples 2 and 3 are shown. [Figure 12] Plasma AUC α-Gal A activity for treatment with 1.0 mg / kg agalsidase beta and treatment with 1.0 mg / kg agalsidase beta in combination with 150 mg migalastat HCl is shown (inset shows mean and standard deviation). [Figure 13] Plasma AUC α-Gal A activity for treatment with 0.5 mg / kg agalsidase beta and treatment with 0.5 mg / kg agalsidase beta in combination with 150 mg migalastat HCl is shown (inset shows mean and standard deviation). [Figure 14] Skin α-Gal A activity on day 2 after treatment with agalsidase beta alone or in combination with 150 mg migalastat HCl is shown (baseline-subtracted ratios to agalsidase beta alone are noted). [Figure 15] Skin α-Gal A activity (baseline-subtracted ratio to agalsidase beta alone) on day 7 after treatment with agalsidase beta alone or in combination with 150 mg migalastat HCl. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present application provides dosing regimens and administration schedules for using 1-deoxygalactonojirimycin and agalsidase for the treatment of Fabry disease.

[0021] definition "Fabry disease" refers to classic Fabry disease, late-onset Fabry disease, and females who are hemizygous for a mutation in the gene encoding α-galactosidase A (α-Gal A). The term "Fabry disease," as used herein, further includes any condition in a subject that exhibits lower-than-normal endogenous α-Gal A activity.

[0022] The term "AUC" refers to a mathematical calculation that assesses the body's total exposure to a given drug over time. In a graph plotting blood concentration after dosing, the drug concentration variable is on the y-axis and time is on the x-axis. The area between the drug concentration curve and the x-axis for a specified time interval is the AUC. AUC is used to guide dosing schedules and to compare the bioavailability of various drugs.

[0023] The term "Cmax" refers to the maximum plasma concentration achieved after dosing.

[0024] The terms "therapeutically effective dose" and "effective amount" refer to that amount of a particular pharmaceutical compound or composition sufficient to result in a beneficial therapeutic response. A beneficial therapeutic response can be any response that a user (e.g., a clinician) may recognize as an effective response to the treatment, including the symptoms and surrogate clinical markers described above. Thus, a therapeutic response can generally be the amelioration of one or more symptoms of a disease or disorder, e.g., Fabry disease, e.g., symptoms known in the art for that disease or disorder, e.g., Fabry disease.

[0025] Non-limiting examples of surrogate marker improvement in Fabry disease include increased α-GAL levels or activity in cells (e.g., fibroblasts) and tissues; decreased GL-3 accumulation as measured by changes in renal interstitial capillary biopsies using histology; decreased urinary GL-3 levels; assessment of renal function (including glomerular filtration rate (GFR) and 24-hour urinary protein); decreased plasma levels of homocysteine ​​and vascular cell adhesion molecule-1 (VCAM-1); decreased GL-3 accumulation in cardiomyocytes and valvular fibroblasts; decreased cardiac hypertrophy (particularly left ventricular hypertrophy), valvular insufficiency, and arrhythmias; remission of proteinuria; decreased urinary levels of lipids such as CTH, lactosylceramide, and ceramide, and increased urinary levels of glucosylceramide and sphingomyelin (Fuller et al., Clinical Chemistry. 2005;51:688-694); absence of lamellar inclusion bodies (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 in neurological symptoms includes prevention of transient ischemic attack (TIA) or stroke; and relief of neuropathic pain manifested as acroparaesthesia (burning or tingling pain in the extremities).

[0026] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerated and typically do not produce adverse reactions when administered to humans. Preferably, 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 Pharmacopoeia or other generally recognized pharmacopeia. 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 saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences," E.W. Martin, 18th Edition, or other editions (hereby incorporated by reference in their entirety).

[0027] "1-Deoxygalactonojirimycin" (DGJ) refers to (2R,3S,4R,5S)-2-(hydroxymethyl)piperdine-3,4,5-triol. As used herein, references to "1-deoxygalactonojirimycin" or "DGJ" throughout include both its free base form and any pharmaceutically acceptable salt forms. The hydrochloride salt of DGJ is known as migalastat hydrochloride.

[0028] The term "adjunct" or "adjunct therapy" refers to any additional substance, treatment, or procedure used to improve the efficacy, safety, or otherwise facilitate or enhance the performance of the primary substance, treatment, or procedure.

[0029] The term "combination therapy" refers to any therapy that provides enhanced results compared to the effects of each therapy when administered individually. The individual therapies in the combination therapy may be administered simultaneously or sequentially.

[0030] Enhancement may include any improvement in the effectiveness of various therapies that may result in advantageous results compared to those achieved when the therapies are administered alone. Enhancement of effectiveness and the determination of enhanced effectiveness may be measured by various parameters, including but not limited to: temporal parameters (e.g., length of treatment, recovery time, long-term effects of treatment, or reversibility of treatment); biological parameters (e.g., cell number, cell volume, cell composition, tissue volume, tissue size, tissue composition); spatial parameters (e.g., tissue strength, tissue size, or tissue accessibility), and physiological parameters (e.g., body contouring, pain, discomfort, recovery time, or visible scars). Enhancement of effectiveness may include synergistic enhancement, where the enhancement of effectiveness is greater than the additive effect of each therapy when administered alone. Enhancement of effectiveness may include additive enhancement, where the enhancement of effectiveness is substantially equal to the additive effect of each therapy when administered alone. Enhancement of effectiveness may include less than synergistic enhancement, where the enhancement of effectiveness is less than the additive effect of each therapy when administered alone, but still better than the effect of each therapy when administered alone.

[0031] The terms "about" and "approximately" are generally intended to mean 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" may refer to an average value that is within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical values ​​provided herein are approximate unless otherwise specified.

[0032] Formulation and Administration 1-Deoxygalactonojirimycin can be administered as a free base or in the form of a pharmacologically acceptable salt, including 1-deoxygalactonojirimycin hydrochloride (also known as migastat hydrochloride). It can be administered in a form suitable for any route of administration, for example, orally in tablet, capsule, or liquid form, or in a sterile aqueous solution for injection. It can be administered orally in the form of a tablet, capsule, ovule, elixir, solution or suspension, gel, syrup, mouthwash, or dry powder to be constituted with water or another suitable vehicle before use, optionally containing flavoring and coloring agents, for immediate-release, delayed-release, modified-release, sustained-release, pulsed-release, or controlled-release applications. Solid compositions, such as tablets, capsules, lozenges, troches, pills, boli, powders, pastes, granules, bullets, or premixed formulations, can also be used. Solid and liquid compositions for oral use can be prepared according to methods well known in the art. Such compositions can also contain one or more pharmaceutically acceptable carriers and excipients, which can be in solid or liquid form.When compound is formulated for oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized 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 can also be coated by methods well known in the art.

[0033] Pharmaceutically acceptable excipients also include, but are not limited to, microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium hydrogen phosphate, and glycine, disintegrants such as starch (preferably corn starch, potato starch, or tapioca starch), sodium starch glycolate, croscarmellose sodium, and certain complex silicates, and granulation binders such as polyvinylpyrolidone, hydroxypropyl ethylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included.

[0034] In a specific embodiment, migalastat hydrochloride is formulated in a white hard gelatin capsule with magnesium stearate and pregelatinized starch. In another embodiment, the solid dosage form comprises about 75-80% migalastat hydrochloride, about 0.1-2% magnesium stearate, and about 20-25% pregelatinized starch. In another specific embodiment, the capsule comprises about 76.5% migalastat hydrochloride, about 0.5% magnesium stearate, and about 23% pregelatinized starch.

[0035] enzyme replacement therapy The current approved treatment for Fabry disease is enzyme replacement therapy. Two products are currently available for the treatment of Fabry disease and are marketed worldwide: agalsidase alfa (Replagal®, Shire Human Genetic Therapies) and agalsidase beta (Fabrazyme®; Genzyme Corporation). These two forms of ERT are intended to compensate for patients' insufficient α-Gal A activity by administering a recombinant form of the enzyme intravenously. ERT has been demonstrated to reduce GL-3 deposition in the renal capillary endothelium and certain other cell types. While ERT is effective in many conditions, this treatment also has limitations. ERT has not been demonstrated to reduce stroke risk, myocardial response is slow, and GL-3 excretion from some renal cell types is limited. Some patients develop an immune response to ERT.

[0036] The recommended dosage of agalsidase alfa is 0.2 mg / kg body weight administered as an intravenous infusion every 2 weeks. A 10-week study was conducted in ERT-naive adult male Fabry patients to evaluate the pharmacokinetics and pharmacodynamics of agalsidase alfa. The mean half-life after administration of agalsidase alfa at doses ranging from 0.1 to 0.4 mg / kg was 56 to 76 minutes, with no significant relationship between dose and half-life, clearance, or volume of distribution. AUC was linearly proportional to dose across this dose range. Plasma GL-3 levels decreased by approximately 50% in all dose groups; this decrease was independent of dose and dosing frequency. Two of 18 patients became IgG positive during the study. No IgE antibodies were detected in any patient during the study.

[0037] The recommended dosage of agalsidase beta is 1 mg / kg body weight administered as an intravenous infusion every 2 weeks. The manufacturer of agalsidase beta has announced a drug shortage of this only ERT approved for Fabry disease in the United States. As a result, agalsidase beta is currently in limited supply, and patients typically receive reduced doses of the enzyme and / or longer dosing intervals (i.e., longer than 2 weeks between doses). Agalsidase beta exhibits nonlinear pharmacokinetics, with a disproportionate increase in exposure (AUC) and a decrease in clearance with increasing dose. Increasing the dose from 0.3 mg / kg to 1 mg / kg and from 1 mg / kg to 3 mg / kg resulted in approximately 6-fold and 8-fold increases in AUC, respectively. The elimination half-life of agalsidase beta in adult patients following doses ranging from 0.3 mg / kg to 3 mg / kg was dose-dependent and ranged from 45 to 100 minutes.

[0038] In clinical trials, 79% of adult patients and 69% of pediatric patients treated with agalsidase developed IgG antibodies to agalsidase beta; the majority of patients who developed IgG antibodies developed within 3 months of exposure. Men, particularly those with low residual α-Gal A levels, were more likely to develop IgG antibodies than men or women with high residual levels. IgG seroconversion in pediatric patients was associated with a prolonged half-life of agalsidase. However, in adult patients, one study observed identical agalsidase pharmacokinetic properties before and after seroconversion; in another study, patients with the highest IgG titers experienced decreases in maximum agalsidase concentrations and AUC values ​​of up to 26% of baseline. The presence of IgG antibodies to agalsidase has been reported to reduce enzyme activity.

[0039] Migalastat hydrochloride stabilizes wild-type α-Gal A both in vitro and in vivo. In vitro, binding of migalastat hydrochloride to rhα-Gal A has been demonstrated to significantly increase the stability of rhα-Gal A at neutral pH in a time- and concentration-dependent manner, as measured by thermal denaturation and activity. In neutral pH buffer, rhα-Gal A exhibited a half-life of approximately 3 hours and a decline in activity; when co-incubated with migalastat hydrochloride, the half-life for the decline of rhα-Gal A activity increased to approximately 40 hours.

[0040] In rats, oral administration of 3 mg / kg migalastat hydrochloride, followed 30 minutes later by injection of 10 mg / kg agalsidase beta, increased the plasma half-life of rhα-Gal A by 2.6-fold and increased plasma α-Gal A levels by 2.5-fold and 1.5-fold at 60 and 240 minutes, respectively. In GLA-deficient mice, oral administration of 30, 100, or 300 mg / kg migalastat hydrochloride 30 minutes before and 2 hours after injection of rhα-Gal A dose-dependently increased tissue α-Gal A levels and dose-dependently decreased GL-3 levels in the skin, heart, kidney, and plasma compared with rhα-Gal A alone.

[0041] Migalastat hydrochloride has been shown to stabilize agalsidase alfa both in vitro and in vivo. The effect of migalastat hydrochloride on the physical stability of agalsidase alfa was assessed using an in vitro thermal denaturation assay. Using this assay, agalsidase alfa exhibited a melting temperature (Tm) of approximately 51°C at pH 7.4. However, when 10 μM migalastat hydrochloride was included in the denaturation reaction, the Tm of agalsidase alfa increased substantially to 59°C. As expected for a lysosomal enzyme, agalsidase alfa was more stable at low pH (Tm of 58°C at pH 5.2) and exhibited further resistance to thermal denaturation in the presence of 10 μM migalastat hydrochloride (Tm of 68°C). These data indicate that the binding of migalastat hydrochloride confers a high degree of physical stability to agalsidase alfa.

[0042] The effect of migalastat hydrochloride on the clearance rate of agalsidase alfa from the blood of male Sprague-Dawley rats was also examined. Animals received either vehicle (water) or a single oral gavage dose of 1, 3, 10, or 30 mg / kg migalastat hydrochloride, followed 30 minutes later by a bolus tail vein injection of 0.2 mg / kg agalsidase alfa. Blood was collected at the appropriate time points, and plasma α-Gal A activity was measured. In the absence of migalastat hydrochloride, α-Gal A activity declined rapidly; pretreatment with migalastat hydrochloride dose-dependently increased the half-life of agalsidase alfa (as measured by α-Gal A activity) by approximately 2- and 3-fold, respectively, and increased plasma α-Gal A levels by approximately 2.5- and 1.5-fold at 60 and 240 minutes, respectively. The effects of migalastat hydrochloride on agalsidase alfa both in vitro and in vivo are comparable to those observed with migalastat hydrochloride on agalsidase beta.

[0043] A pilot study has been conducted in GLA-deficient mice to evaluate the safety of co-administered migalastat hydrochloride and Fabrazyme®. Migalastat hydrochloride was administered at doses of 3 and 30 mg / kg three times a week for four weeks in combination with Fabrazyme®, administered intravenously once a week at a dose of 1 mg / kg. There appeared to be no directly drug-related changes in survival, clinical status, or hematology and clinical chemistry parameters observed in male GLA-deficient mice co-administered migalastat hydrochloride and Fabrazyme®. [Example]

[0044] Example 1: Dosage regimen for the treatment of Fabry disease using migalastat hydrochloride and agalsidase One objective of this study was to evaluate the safety, efficacy, and pharmacodynamics of a dosing regimen involving the co-administration of migalastat hydrochloride and agalsidase in patients with Fabry disease.

[0045] Another objective of this study was to evaluate the effect of 150 mg and 450 mg doses of migalastat hydrochloride on the distribution of α-Gal A. This can be assessed by measuring the distribution of agalsidase in the skin after dosing with agalsidase alone and agalsidase in combination with migalastat hydrochloride by measuring α-Gal A and protein levels 24 hours and 7 days after dosing.

[0046] Other metrics that can be evaluated are: Urinary GL-3 excretion before and 14 days after each agalsidase dose; Skin GL-3 after administration of agalsidase alone and agalsidase in combination with migalastat hydrochloride at 24 hours and 7 days post-dose; -α-Gal A enzyme levels in WBCs measured before the start of agalsidase infusion and at 2, 4, 24, 7, and 14 days after administration; · Antibody titer (IgG) before starting agalsidase infusion; Plasma globotriaosylsphingosine (lyso-GB3) concentrations and urinary excretion of lyso-GB3 before and 14 days after each dose of agalsidase.

[0047] Measurements of α-Gal A enzyme levels in plasma, WBC, and skin were all performed with and without Con A capture, and protein levels were measured by Western blot.

[0048] Study Design: This is a Phase 2, two-stage, open-label study evaluating the safety and efficacy of coadministration of migalastat hydrochloride with agalsidase. The study will be conducted in male subjects aged 18 to 65 years who have been receiving a stable dose (0.3-1.0 mg / kg) of agalsidase beta (Fabrazyme®) or agalsidase alfa (Replagal®) at least 1 month prior to study enrollment. Approximately 18 subjects will be enrolled.

[0049] This open-label study consists of two stages. Stage 1 consists of screening and a three-period study evaluating the effect of 150 mg migalastat hydrochloride on the pharmacokinetics and safety of agalsidase and the effect of agalsidase on the pharmacokinetics and safety of 150 mg migalastat hydrochloride. Stage 2 consists of screening and a two-period study evaluating the effect of 450 mg migalastat hydrochloride on the pharmacokinetics and safety of agalsidase. Stage 2 will not evaluate the effect of agalsidase on the pharmacokinetics and safety of the 450 mg dose of migalastat hydrochloride. The plasma exposure of migalastat hydrochloride when administered with agalsidase alone will be characterized to ensure that sufficient migalastat hydrochloride plasma concentrations are achieved.

[0050] Each subject will receive each of the following treatments in the order listed below. Stage 1 will consist of the following periods: Period 1: agalsidase alone as an intravenous infusion; Period 2: 150 mg oral dose of migalastat hydrochloride, followed 2 hours later by the initiation of intravenous agalsidase infusion; Period 3: 150 mg oral dose of migalastat hydrochloride.

[0051] The dose of agalsidase administered during Period 1 and Period 2 is the same. Agalsidase alfa is administered as a 40-minute intravenous infusion, and agalsidase beta is administered as a 2-hour intravenous infusion.

[0052] For Period 1, prior to the next scheduled agalsidase infusion, subjects will undergo the following assessments: adverse event assessment, concomitant medications, physical examination, weight, vital signs, 12-lead ECG, clinical laboratory tests (serum chemistry, hematology, and urinalysis), skin biopsy (punch biopsy for measurement of α-Gal A enzyme levels; if sufficient sample is available, skin GL-3 will also be measured).

[0053] On the morning of Day 1, urine is collected for measurement of urinary GL-3 and lyso-GB3, followed by the subject's current dose of agalsidase, administered as an infusion using an infusion pump. Blood samples for pharmacokinetic and pharmacodynamic analyses are collected immediately prior to the start of the agalsidase infusion and for 24 hours after the start of the agalsidase infusion. Plasma and WBC α-Gal A enzyme levels, plasma lyso-GB3, and plasma antibody titers are measured from the collected blood samples at the time points summarized in Table 2 for agalsidase beta and Table 4 for agalsidase alfa. At the end of the agalsidase infusion, immediately after the post-infusion blood sample is drawn, a 12-lead ECG is performed.

[0054] On day 2, 24 hours after the start of the previous day's infusion, punch skin biopsies are taken and α-Gal A enzyme levels are measured from the biopsies; if sufficient sample is available, skin GL-3 is also measured. After the final pharmacokinetic sample is taken, the following assessments are performed: adverse event assessment, concomitant medications, physical examination, weight, vital signs, and clinical laboratory tests (serum chemistry, hematology, and urinalysis).

[0055] On Day 7, subjects undergo the following evaluations: physical examination, vital signs, concomitant medications, and adverse event assessment. Skin biopsies are taken and α-Gal A enzyme levels are measured from the biopsies; if sufficient sample is available, skin GL-3 is also measured. Blood samples are also taken for measurement of WBC α-Gal A and plasma enzyme levels. On Day 14, urine samples are taken for measurement of urinary GL-3 and lyso-GB3 excretion. Blood samples are also taken for measurement of WBC α-Gal A and plasma enzyme levels, and vital signs are assessed.

[0056] For Period 2, prior to the next scheduled agalsidase infusion, subjects will undergo the following assessments: adverse event assessment, concomitant medications, physical examination, weight, vital signs, 12-lead ECG, clinical laboratory tests (serum chemistry, hematology, and urinalysis).

[0057] On the morning of Day 1, urine is collected for measurement of urinary GL-3 and lyso-GB3, followed by administration of a 150 mg oral dose of migalastat hydrochloride two hours before the scheduled agalsidase infusion. Subjects fast for at least two hours before and two hours after administration of migalastat hydrochloride. In Period 2, each subject receives the same agalsidase dose administered in Period 1 as an infusion using an infusion pump. The agalsidase infusion begins two hours after administration of the migalastat hydrochloride dose.

[0058] Blood samples for pharmacokinetic and pharmacodynamic analyses are collected prior to and 1 hour after administration of migalastat hydrochloride. Additional blood samples are collected immediately prior to and over 24 hours after the start of the agalsidase infusion. Plasma and WBC α-Gal A enzyme levels, plasma lyso-GB3, and plasma antibody titers are measured from the collected blood samples at the time points summarized in Table 2 for agalsidase beta and Table 4 for agalsidase alfa. At the end of the agalsidase infusion, immediately after the post-infusion blood sample is collected, a 12-lead ECG is performed.

[0059] On day 2, 24 hours after the start of the previous day's infusion, punch skin biopsies are taken and α-Gal A enzyme levels are measured from the biopsies; if sufficient sample is available, skin GL-3 is also measured. After the final pharmacokinetic sample is taken, the following assessments are performed: adverse event assessment, concomitant medications, physical examination, weight, vital signs, and clinical laboratory tests (serum chemistry, hematology, and urinalysis).

[0060] On Day 7, subjects will undergo the following evaluations: physical examination, vital signs, concomitant medications, and adverse event assessment. Skin biopsies will be taken to measure α-Gal A enzyme levels; if sufficient sample is available, skin GL-3 will also be measured. Blood samples will also be taken to measure WBC α-Gal A and plasma enzyme levels.

[0061] On day 14, urine samples are collected for measurement of urinary GL-3 and lyso-GB3 excretion. Blood samples are also collected for measurement of α-Gal A in WBC and plasma enzyme levels, and vital signs are assessed.

[0062] After completing all assessments after Period 2, subjects enter Period 3. All subjects receive their next agalsidase infusion on Day 1 according to their usual dosing schedule. On Day 6, all subjects undergo the following assessments: adverse event assessment, concomitant medications, physical examination, weight, vital signs, 12-lead ECG, and clinical laboratory tests (serum chemistry, hematology, and urinalysis).

[0063] On Day 7, a 150 mg oral dose of migalastat hydrochloride is administered. Subjects fast for at least 2 hours before and 2 hours after administration of migalastat hydrochloride. Blood samples are collected before and over 24 hours after administration of migalastat hydrochloride. Migalastat hydrochloride concentrations are measured in all plasma samples (see Table 2 for agalsidase beta and Table 4 for subjects receiving agalsidase alfa for sample collection time points).

[0064] On Day 8, after collection of the final pharmacokinetic sample, the following assessments will be performed: adverse event assessment, concomitant medications, physical examination, vital signs, and laboratory tests (serum chemistry, hematology, and urinalysis).

[0065] Follow-up for Period 3 will be by telephone 28 days after Period 3. The following assessments will be performed: concomitant medications and adverse events.

[0066] For Stage 2, each subject will receive each of the following treatments in the order listed below: Period 1: agalsidase alone as infusion; Period 2: 450 mg oral dose of migalastat hydrochloride, starting 2 hours later; intravenous infusion of agalsidase.

[0067] The dose of agalsidase administered in periods 1 and 2 is the same. Agalsidase alfa is administered as a 40-minute intravenous infusion, and agalsidase beta is administered as a 2-hour intravenous infusion.

[0068] For Period 1, subjects who meet all eligibility criteria will undergo the following assessments prior to their next scheduled agalsidase infusion: adverse event assessment, concomitant medications, physical examination, weight, vital signs, 12-lead ECG, clinical laboratory tests (serum chemistry, hematology, and urinalysis), skin biopsy (punch biopsy for measurement of α-Gal A enzyme levels; if sufficient sample is available, skin GL-3 will also be measured).

[0069] On the morning of Day 1, urine is collected for measurement of urinary GL-3 and lyso-GB3, followed by the subject's current dose of agalsidase, administered as an infusion using an infusion pump. Blood samples for pharmacokinetic and pharmacodynamic analyses are collected immediately prior to the start of the agalsidase infusion and for 24 hours after the start of the agalsidase infusion. Plasma and WBC α-Gal A enzyme levels, plasma lyso-GB3, and plasma antibody titers are measured from the collected blood samples at the time points summarized in Table 3 for agalsidase beta and Table 5 for agalsidase alfa. At the end of the agalsidase infusion, immediately after the post-infusion blood sample is drawn, a 12-lead ECG is performed.

[0070] On day 2, 24 hours after the start of the previous day's infusion, punch skin biopsies are taken and α-Gal A enzyme levels are measured from the biopsies; if sufficient sample is available, skin GL-3 is also measured. After the final pharmacokinetic sample is taken, the following assessments are performed: adverse event assessment, concomitant medications, physical examination, weight, vital signs, and clinical laboratory tests (serum chemistry, hematology, and urinalysis).

[0071] On Day 7, subjects will undergo the following assessments: vital signs, concomitant medications, and adverse event assessment. Skin biopsies will be taken to measure α-Gal A enzyme levels; if sufficient sample is available, skin GL-3 will also be measured. Blood samples will also be taken to measure WBC α-Gal A and plasma enzyme levels.

[0072] On day 14, urine samples are collected for measurement of urinary GL-3 and lyso-GB3 excretion. Blood samples are also collected for measurement of α-Gal A in WBC and plasma enzyme levels, and vital signs are assessed.

[0073] For Period 2, subjects will undergo the following assessments prior to their next scheduled agalsidase infusion: adverse event assessment, concomitant medications, physical examination, weight, vital signs, 12-lead ECG, clinical laboratory tests (serum chemistry, hematology, and urinalysis).

[0074] On the morning of Day 1, urine is collected for measurement of urinary GL-3 and lyso-GB3, followed by administration of a 450 mg oral dose of migalastat hydrochloride two hours before the scheduled agalsidase infusion. Subjects fast for at least two hours before and two hours after administration of migalastat hydrochloride. In Period 2, each subject receives the same agalsidase dose administered in Period 1 as an infusion using an infusion pump. The agalsidase infusion begins two hours after administration of the migalastat hydrochloride dose.

[0075] Blood samples for pharmacokinetic and pharmacodynamic analyses are collected prior to the administration of the migalastat hydrochloride dose and 1 hour after administration of migalastat hydrochloride. Additional blood samples are collected immediately prior to the start of the agalsidase infusion and over a 24-hour period following the start of the agalsidase infusion. Plasma and WBC α-Gal A enzyme levels, plasma lyso-GB3, and plasma antibody titers are measured from the collected blood samples at the time points summarized in Table 3 for agalsidase beta and Table 5 for agalsidase alfa. At the end of the agalsidase infusion, immediately after the post-infusion blood sample is collected, a 12-lead ECG is performed.

[0076] On day 2, 24 hours after the start of the previous day's infusion, punch skin biopsies are taken and α-Gal A enzyme levels are measured from the biopsies; if sufficient sample is available, skin GL-3 is also measured. After the final pharmacokinetic sample is taken, the following assessments are performed: adverse event assessment, concomitant medications, physical examination, weight, vital signs, and clinical laboratory tests (serum chemistry, hematology, and urinalysis).

[0077] On Day 7, subjects will undergo the following assessments: vital signs, concomitant medications, and adverse event assessment. Skin biopsies will be taken and α-Gal A enzyme levels will be measured from the biopsies. Blood samples will also be taken for measurement of α-Gal A in WBC and plasma enzyme levels.

[0078] On day 14, urine collection for measurement of urinary GL-3 and lyso-GB3 excretion is performed. Blood samples for measurement of α-Gal A in WBC and plasma enzyme levels are also collected, and vital signs are assessed.

[0079] Follow-up is 28 days after Period 2. The following assessments will be performed: concomitant medications and adverse events.

[0080] Assessment and Sampling Schedule. Table 1 shows the assessment schedule for Stages 1 and 2. Sampling time points and analytes for co-administration of migalastat hydrochloride with Fabrazyme® are shown in Tables 2 and 3. Sampling time points and analytes for co-administration of migalastat hydrochloride with Replagal® are shown in Tables 4 and 5.

[0081] [Table 1]

[0082] [Table 2]

[0083] [Table 3]

[0084] [Table 4]

[0085] [Table 5]

[0086] Pharmacokinetics of migalastat HCl and rhα-Gal A. Migalastat HCl concentrations in blood samples are measured in plasma using a validated LC-MS / MS assay. Plasma α-Gal A levels are measured with and without Con A using a validated assay that measures enzyme activity using 4-MUG. α-Gal A protein levels are measured by Western blotting using an anti-human Gal A antibody.

[0087] Skin α-Gal A Enzyme Levels. Skin biopsy samples are tested for α-Gal A enzyme levels. Skin biopsies are performed using a "punch" device. A strip is removed at each visit. Skin α-Gal A levels are measured using a validated assay that measures enzyme activity using 4-MUG with and without Con A. α-Gal A protein levels are measured by Western blotting using an anti-human Gal A antibody.

[0088] WBC α-Gal A levels. WBC α-Gal A levels are measured in blood samples using a validated assay that measures enzyme activity using 4-MUG with and without Con A. α-Gal A protein levels are measured by Western blotting using an anti-human Gal A antibody.

[0089] Plasma Lyso-GB3. Plasma lyso-GB3 measurements will be piloted to obtain data in patients receiving ERT alone and ERT co-administered with migalastat hydrochloride. Plasma lyso-GB3 concentrations will be measured using a validated assay.

[0090] Urinary GL-3 and Lyso-GB3. On Days 1 and 14 of Periods 1 and 2, a first-morning urine sample is collected from each subject for analysis of urinary GL-3 and lyso-GB3 excretion. Subjects collect urine on the mornings of Days 1 and 14. Urinary GL-3 and lyso-GB3 are expressed as a function of urinary creatinine concentration.

[0091] Antibody titer. Blood samples are collected and the IgG antibody titer of each blood sample is measured.

[0092] Safety parameters: Safety parameters will be assessed by reviewing changes in physical examination findings, vital signs, ECG changes over time, laboratory tests, and adverse events.

[0093] Vital signs, weight, and height. Temperature and respiration will be measured at screening and admission. To monitor safety, temperature, respiration, sitting blood pressure, and heart rate will be measured on days 2, 7, and 14, prior to and approximately 1, 2, 3, 4, and 6 hours after administration of agalsidase (Period 1) or migalastat hydrochloride (Periods 2 and 3). If vital sign monitoring overlaps with blood draws, blood draws will take priority and vital signs will be adjusted accordingly.

[0094] ECG Monitoring. ECG monitoring is performed with a standard 12-lead ECG.

[0095] Laboratory Tests. Blood samples and urine samples for laboratory tests (hematology, serum chemistry) will be collected at each visit and analyzed at a central laboratory. Hematology tests include total hemoglobin, hematocrit, red blood cells, platelets, and differential white blood cell count. Coagulation (screening only) includes INR and aPTT. · Serum chemistry includes measurements of AST, ALT, alkaline phosphatase, total bilirubin, creatinine, urea, glucose, calcium, sodium, potassium, magnesium, total protein, albumin, bicarbonate, LDH, blood urea nitrogen, chloride, and phosphate. Serum creatinine measurements are performed using reagents calibrated for the Isotope Dilution Mass Spectrometry (IDMS) reference method. Urinalysis includes microscopic examination of color, appearance, specific gravity, pH, protein, glucose, ketones, blood, leukocyte esterase, nitrites, bilirubin, urobilinogen, and sediment.

[0096] Pharmacokinetic parameters. AUC 0-t , AUC 無限大 , C max , t max , k el Non-compartmental pharmacokinetic parameters of elimination half-life and elimination time are calculated. Pharmacokinetic parameters are summarized by processing using descriptive statistics. AUC of each compound alone versus each compound in combination. 0-t ratio, AUC 無限大 The ratio will be calculated. The pharmacokinetic and pharmacodynamic data for subjects receiving agalsidase alfa and agalsidase beta will be analyzed separately.

[0097] Statistical Analysis. Provide descriptive statistics (N, mean, standard deviation, and coefficient of variation, standard error, median, minimum, and maximum) as appropriate. Individual (per subject) AUC ratios and C max The effect of the compound relative to the co-administered compound is assessed by calculating the ratio as follows:

number

[0098] AUC ratio and C max Ratios will be expressed as the mean of the individual ratios and 90% confidence intervals around the mean. Pharmacokinetic and pharmacodynamic data for subjects receiving agalsidase alfa and agalsidase beta will be analyzed separately. Results will be provided in tabular and graphical formats as appropriate. All subjects receiving study drug and with sufficient data to generate reliable pharmacokinetic parameters will be included in the safety and pharmacokinetic analyses.

[0099] Example 2: Dosage regimen for the treatment of Fabry disease using migalastat hydrochloride and agalsidase Migalastat HCl is a pharmacological chaperone for α-galactosidase A (α-Gal A) that increases the stability and correct folding of this enzyme. Migalastat may act by stabilizing the enzyme and preventing α-Gal A inactivation under blood pH / temperature conditions. The objectives of this study were to characterize the effects of 150 mg and 450 mg migalastat administered 2 hours before agalsidase administration on the safety and plasma pharmacokinetics of agalsidase in subjects with Fabry disease. The objectives of this study were also to characterize the effects of 150 mg and 450 mg migalastat on the plasma, skin, and PBMC pharmacokinetics of agalsidase in patients with Fabry disease; to characterize the effect of plasma agalsidase on the plasma pharmacokinetics of migalastat; to evaluate urinary GL-3 and plasma and urinary lyso-GB3 levels before and 14 days after agalsidase infusion; and to evaluate antibody titers before agalsidase infusion.

[0100] Methods. This study was conducted according to the methods described in Example 1. Specifically, this was an open-label, single-dose, non-randomized, fixed-sequence, two-stage study. Stage 1 consisted of three periods: (1) Enzyme replacement monotherapy (ERT) with agalsidase beta 0.5 mg / kg or 1.0 mg / kg (approximately 2-hour infusion) or agalsidase alfa 0.2 mg / kg (approximately 40-minute infusion); (2) ERT + 150 mg migalastat oral tablet (single dose) coadministration, with migalastat administered 2 hours before ERT; and (3) 150 mg migalastat monotherapy.

[0101] Stage 2 consisted of two periods in the same order as Stage 1 (i.e., Periods (1) and (2)), except that 3 x 150 mg migalastat oral tablets (i.e., 450 mg migalastat) were co-administered during Period 2. Stage 2 consisted of the following two periods: (1) agalsidase beta 0.5 mg / kg or 1.0 mg / kg (approximately 2-hour infusion) or agalsidase alfa 0.2 mg / kg (approximately 40-minute infusion) enzyme replacement monotherapy (ERT); and (2) Simultaneous administration of ERT + 450 mg migalastat oral tablets. Migalastat was administered 2 hours before ERT.

[0102] Treatment periods were separated by a washout period of at least 14 days.

[0103] α-Gal A catalyzes the first step in the degradation of the substrate GL-3 in vivo. α-Gal A also acts on other substrates, such as the artificial low-molecular-weight florescent substrate 4-MUG, through the same α-linkage. α-Gal A activity toward 4-MUG was measured in vitro from plasma, skin, and PBMC samples after dissociation of migalastat at serial dilutions.

[0104] Six patients with plasma, skin, and PBMC α-galactosidase A activity from Periods 1 and 2 of Stage 1 were evaluated. In Period 1, four patients received 0.5 mg / kg agalsidase beta ERT and two patients received 1.0 mg / kg agalsidase beta ERT; in Period 2, 150 mg migalastat was coadministered 2 hours before the start of the same dose of ERT. For both periods, the duration of the infusion was 2 hours, with one exception: one patient receiving 0.5 mg / kg agalsidase beta had an uneven infusion; this patient received a 2 hour 40 minute infusion in Period 2 but only a 2 hour infusion in Period 1.

[0105] result: Coadministration of migalastat increases plasma α-Gal-A activity The following mean increases in plasma α-Gal A activity AUC (area under the curve) were observed for co-administration with migalastat (Period 2) compared to ERT alone (Period 1): Mean increase in 0.5mg / kg agalsidase beta infusion: 0.5 mg / kg agalsidase beta: 3.0 times (N=4) Individual patient increases: 2.0-fold, 2.2-fold, 3.4-fold, and 4.2-fold Average increase: 3.3x, excluding patients with uneven infusion times (2.0x) Mean increase in 1.0 mg / kg agalsidase beta infusion: 1.9 times (N=2) for 1.0 mg / kg agalsidase beta Increase in individual patients: 1.6 and 2.2 times

[0106] The plasma α-Gal A activity composite profiles for six patients in Periods 1 and 2 are shown in Figure 1. The increase in plasma α-Gal A activity AUC for all patients after coadministration with migalastat is shown in Figure 2. Figure 3 shows the partial AUCs at each sampling time point, demonstrating the increase in plasma α-Gal A activity following coadministration of 0.5 mg / kg or 1.0 mg / kg agalsidase beta with 150 mg migalastat.

[0107] Increase in skin α-Gal-A activity by migalastat The following mean increases in skin α-Gal A activity were observed for co-administration with migalastat (Period 2) compared to ERT alone (Period 1): Mean increase in 0.5mg / kg agalsidase beta infusion: 2.6-fold increase for 0.5 mg / kg agalsidase beta on day 2 (N=3, one patient's sample was lost) Individual patient increases: 2.8-fold, 3.9-fold, and 1.1-fold Skin activity remained unchanged from day 7 of period 1 Mean increase in 1.0 mg / kg agalsidase beta infusion: 1.9-fold and 1.5-fold for 1.0 mg / kg agalsidase beta on days 2 and 7, respectively (N=2) Individual patient increases: 1.6-fold and 2.1-fold on day 2, 1.7-fold and 1.2-fold on day 7

[0108] The increase in skin α-Gal A activity after co-administration of 0.5 mg / kg or 1.0 mg / kg agalsidase beta with 150 mg migalastat is shown in Figures 4 to 6. Figure 5A shows the increase in skin α-Gal A activity after co-administration of 0.5 mg / kg agalsidase beta with 150 mg migalastat in patients who received a 40-minute long ERT infusion during period 2 compared to other patients.

[0109] Increase in PBMC α-Gal-A activity by migalastat The following mean increases in PBMC α-Gal A activity were observed for co-administration with migalastat (Period 2) compared to ERT alone (Period 1): Mean increase in 0.5mg / kg agalsidase beta infusion: 2.3-fold, 2.0-fold, and 2.2-fold for 0.5 mg / kg agalsidase beta on days 2, 7, and 14, respectively (N=4) Increase range for individual patients: 1.4-3.1 times, 1.4-2.3 times, and 1.7-2.8 times on days 2, 7, and 14, respectively Skin activity remained unchanged from day 7 of period 1 Mean increase in 1.0 mg / kg agalsidase beta infusion: 1.8-fold, 4.8-fold, and 3.5-fold for 1.0 mg / kg agalsidase beta on days 2, 7, and 14, respectively (N=2) Individual patient increases: 1.1-fold and 2.5-fold, 3.6-fold and 6.0-fold, and 1.7-fold and 5.4-fold on days 2, 7, and 14, respectively

[0110] The increase in PBMC α-Gal A activity following co-administration of 0.5 mg / kg or 1.0 mg / kg agalsidase beta with 150 mg migalastat is shown in Figures 7-9.

[0111] Conclusion: The interaction of 150 mg migalastat with 0.5 mg / kg and 1.0 mg / kg agalsidase beta resulted in increased α-Gal A activity for: Plasma α-Gal A AUC in all patients (N=6) Skin α-Gal A on day 2 in all patients (N=5), but on day 7 in only 3 of 5 patients PBMC α-Gal A on days 2, 7, and 14 in all patients (N=6)

[0112] The interaction of 150 mg migalastat with 0.5 mg / kg or 1.0 mg / kg agalsidase beta resulted in a 2- to 4-fold increase in α-galactosidase A activity AUC compared with agalsidase beta alone, a 1.1- to 3.9-fold increase in skin α-galactosidase A activity on day 2, and a 1.1- to 6.0-fold increase in PBMC α-galactosidase A activity on days 2, 7, and 14. On day 7, α-galactosidase A activity increased in the skin of four patients after coadministration.

[0113] The 150 mg migalastat dose increased the enzyme activity of agalsidase beta at a half dose (0.5 mg / kg) better than at the full dose (1.0 mg / kg) in plasma, skin, and PBMCs up to 24 hours post-dose; however, the reverse was true in skin and PBMCs (1.0 mg / kg > 0.5 mg / kg) on ​​days 7 and 14 post-dose. A table summarizing the results is shown in Figure 10.

[0114] With agalsidase beta alone, all patients had increased PBMC α-Gal A activity compared to baseline at all time points; however, two patients had decreased skin α-Gal A activity on day 2 after the 0.5 mg / kg infusion compared to baseline.

[0115] Example 3: Dosage regimen for the treatment of Fabry disease using migalastat hydrochloride and agalsidase The following example is an updated version of the study described in Example 2. This example includes additional subjects, for a total of 7 subjects.

[0116] The purpose of this example is to evaluate the safety and PK of two doses of migalastat HCl (150 mg and 450 mg) co-administered with ERT (agalsidase) in men diagnosed with Fabry disease.

[0117] Methods: This is an ongoing, open-label, non-randomized, two-stage, fixed-sequence study. Stage 1 consists of three periods. Period 1: Intravenous infusion of ERT alone Period 2: Oral administration of migalastat HCl (150 mg), followed 2 hours later by intravenous infusion of ERT (same dose as in Period 1). Period 3: Oral administration of migalastat HCl 150 mg alone. Eligible patients: Men, aged 18-65 years, with Fabry disease. Inclusion Criteria: Body Mass Index (BMI) 18-35. Treatment with agalsidase has been initiated at least one month prior to administration. · Estimated creatinine clearance (CLcr) ≥ 50 mL / min at screening. Exclusion criteria: - Documented transient ischemic attack, ischemic stroke, or unstable myocardial infarction within the 3 months prior to screening. Clinically significant unstable heart disease. Sensitivity to or concomitant therapy with iminosugars (e.g., miglustat, miglitol).

[0118] Subjects will receive their current dose and regimen of agalsidase beta alone (0.5 or 1.0 mg / kg over approximately 2 hours) during one infusion, followed by 150 mg of migalastat HCl administered orally 2 hours before agalsidase beta during the next infusion.

[0119] Five of the current seven subjects received a dose of 0.5 mg / kg every two weeks, and two of the seven subjects received a dose of 1.0 mg / kg every four weeks.

[0120] Stage 2 will test a 450 mg dose of migalastat HCl.

[0121] Stages 1 and 2 are repeated in unique subjects with an ERT infusion of agalsidase alfa (0.2 mg / kg over approximately 40 minutes).

[0122] sample: Serial blood samples were collected up to 24 hours post-dose for plasma α-Gal A activity and protein levels for each period. Blood samples for α-Gal A activity in peripheral blood mononuclear cells (PBMCs) were collected pre-dose and on days 1, 2, 7, and 14 of each period. Punch biopsies for skin α-Gal A activity were collected pre-dose in Period 1 and on days 2 and 7 between Periods 1 and 2. Plasma α-Gal A activity PK parameters included C max , T max , AUC 0-t , AUC 0-inf , and t 1 / 2Pharmacokinetic parameters will be calculated using standard non-compartmental procedures (WINNONLIN version 5.0 or higher).

[0123] α-Gal A activity in plasma, skin, and PBMC lysates was measured by a fluorogenic enzyme assay using 4-methylumbelliferyl-α-D-galactopyranoside (4-MUG). α-Gal A activity against 4MUG was measured in vitro after dissociation of migalastat by serial dilution.

[0124] Western blot analysis of α-Gal A protein was performed on plasma samples using anti-human α-Gal A antibody. A rhα-Gal A (agalsidase) standard curve was run to calculate the appropriate α-Gal protein concentration in each sample.

[0125] Safety parameters included adverse events (AEs), vital signs, clinical tests (hematology, serum chemistry, and urinalysis), electrocardiogram (ECG), physical examination, and concomitant medication use.

[0126] Results: Preliminary results are available for Stage 1, Period 1 and Period 2.

[0127] Patient Disposition and Demographics: Seven patients with plasma, skin, and PBMC α-Gal A activity from Stage 1 Period 1 and Period 2 were evaluated. In period 1, all seven patients received agalsidase beta alone; in period 2, all patients received 150 mg migalastat HCl concomitantly 2 hours before the initiation of agalsidase beta. Two patients (identified as Subjects A and B) received an intravenous infusion of agalsidase beta 1.0 mg / kg over a 2-hour duration. Five patients (identified as Subjects C, D, E, F, and G) received an intravenous infusion of agalsidase beta 0.5 mg / kg over a 2-hour duration, with one exception: Subject E received a 2-hour 40-minute infusion during Period 2, but a 2-hour infusion during Period 1. All subjects were men with Fabry disease, aged 44 to 61 years, with a body mass index (BMI) of 20.9 to 29.1 kg / m 2 The range of CI was 1.0 to 1.2, and the estimated CLcr ranged from 54 to 88 mL / min. The genotypes of each subject are shown in FIG.

[0128] Safety: Twelve adverse events (AEs) have been reported to date, one of which was serious. The serious AE was a transient ischemic attack (TIA) that occurred after the screening visit but was less severe than required hospitalization before dosing and was deemed unrelated to study drug by the investigator. The TIA resolved without sequelae. All other AEs were mild in severity, all were deemed unrelated to study drug, and most resolved without treatment. Three AEs in three different subjects: atrial premature beats, atrial flutter, and leg edema, are ongoing and all are unrelated to study drug.

[0129] Plasma α-Gal A activity: The plasma AUC (area under the curve) of the activity-time profiles of α-Gal A activity (mean and standard deviation shown in inset) for treatment with 1.0 mg / kg agalsidase beta alone and treatment with 1.0 mg / kg agalsidase beta in combination with 150 mg migalastat HCl are shown in Figure 12 for subjects A and B. Treatment with 1.0 mg / kg agalsidase beta in combination with 150 mg migalastat HCl resulted in a 2.2-fold and 1.6-fold increase in α-Gal A activity for subjects A and B, respectively, compared to treatment with 1.0 mg / kg agalsidase beta monotherapy.

[0130] The plasma AUC (area under the curve) of the activity-time profile of α-Gal A activity (mean and standard deviation shown in inset) for treatment with 0.5 mg / kg agalsidase beta alone and treatment with 0.5 mg / kg agalsidase beta in combination with 150 mg migalastat HCl is shown for subjects C through G in Figure 13. Combination treatment with 0.5 mg / kg agalsidase beta and 150 mg migalastat HCl resulted in a 2.0- to 4.2-fold increase in α-Gal A activity compared to treatment with 0.5 mg / kg agalsidase beta monotherapy.

[0131] Plasma α-Gal A activity increased at all time points in most subjects with coadministration compared with enzyme replacement therapy (ERT) alone, with one exception. Subject E received a 40-minute longer, uneven infusion during period 2, which resulted in a relative decrease in enzyme activity during the infusion period. However, at all time points after peak activity in Subject E, activity increased compared with ERT alone. In addition, consistent with Eng et al., Am. J. Hum. Genet. 68:711-722 (2001), exposure increased nonlinearly with α-Gal A activity.

[0132] Coadministration of migalastat HCl with ERT (Period 2) compared with ERT alone (Period 1) resulted in an increase in plasma α-Gal A activity AUC in all subjects, as shown in Figures 12 and 13. The mean increase in plasma α-Gal A activity AUC was 3.0-fold with 0.5 mg / kg agalsidase beta. Excluding the patient with an uneven infusion time of 2.0-fold, the mean increase was 3.2-fold. The mean increase in plasma α-Gal A activity AUC was 1.9-fold with 1.0 mg / kg agalsidase beta.

[0133] Skin α-Gal A activity Figure 14 shows that treating subjects with 0.5 mg / kg or 1.0 mg / kg agalsidase beta in combination with 150 mg migalastat HCl increased α-Gal A activity in skin samples on day 2 compared to enzyme monotherapy treatment alone. Figure 15 shows α-Gal A activity in skin samples on day 7 after monotherapy and after combination therapy with agalsidase beta and 150 mg migalastat HCl. The following mean increases in skin α-Gal A activity were observed: 2.6-fold and 1.4-fold (N=5) on days 2 and 7, respectively, for 0.5 mg / kg agalsidase beta, and 1.9-fold and 1.5-fold (N=2) on days 2 and 7, respectively, for 1.0 mg / kg agalsidase beta.

[0134] PMBC α-Gal A activity The following mean increases in PBMC α-Gal A activity were observed: 2.4-fold, 1.9-fold, and 2.1-fold (N=5) for 0.5 mg / kg agalsidase beta on days 2, 7, and 14, respectively, and 1.8-fold, 4.8-fold, and 3.5-fold for 1.0 mg / kg (N=2) agalsidase beta on days 2, 7, and 14, respectively.

[0135] Western blot analysis In five of seven subjects, no changes in plasma α-Gal A protein were observed by Western blot analysis of the Period 2 (coadministration) / Period 1 (ERT alone) AUC ratio, but two subjects (Subject C, who received 0.5 mg / kg agalsidase beta, and Subject G, who received 1.0 mg / kg agalsidase beta) experienced a 20% increase in protein amount after coadministration compared to ERT alone (data not shown).

[0136] conclusion The interaction of 150 mg migalastat with 0.5 mg / kg and 1.0 mg / kg agalsidase beta resulted in increased α-Gal A activity in plasma, skin, and PBMCs. Coadministration of 150 mg migalastat HCl with agalsidase beta was generally safe and well tolerated.

[0137] The present application is not to be limited in scope by the specific embodiments described herein. Instead, various modifications of the present application, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be encompassed by the appended claims.

[0138] Furthermore, it should be understood that all values ​​are approximate and are provided for illustrative purposes.

[0139] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. A method for treating Fabry disease in a subject, comprising administering to a patient in need thereof about 50 mg to about 600 mg of 1-deoxygalactonojirimycin and an effective amount of α-Gal A enzyme replacement therapy.

2. 10. The method of claim 1, wherein the amount of 1-deoxygalactonojirimycin administered is from about 150 mg to about 450 mg.

3. 10. The method of claim 1, wherein the amount of 1-deoxygalactonojirimycin administered is selected from about 150 mg, about 300 mg, and about 450 mg.

4. 10. The method of claim 1, wherein the patient fasts for a period beginning about 0.5 to about 4 hours prior to administration of 1-deoxygalactonojirimycin and ending about 0.5 to about 4 hours after administration.

5. 5. The method of claim 4, wherein the patient fasts for at least about 2 hours before and at least about 2 hours after administration of 1-deoxygalactonojirimycin.

6. 10. The method of claim 1, wherein the 1-deoxygalactonojirimycin is administered simultaneously with or about 4 hours before the administration of the α-Gal A enzyme replacement therapy.

7. 7. The method of claim 6, wherein the 1-deoxygalactonojirimycin is administered about 2 hours before the administration of the α-Gal A enzyme replacement therapy.

8. 2. The method of claim 1, wherein the 1-deoxygalactonojirimycin is migalastat hydrochloride.

9. 10. The method of claim 1, wherein the α-Gal A enzyme replacement therapy is selected from agalsidase alpha and agalsidase beta.

10. 10. The method of claim 1, wherein the 1-deoxygalactonojirimycin is administered as an adjunct to the α-Gal A enzyme replacement therapy.

11. 10. The method of claim 1, wherein the 1-deoxygalactonojirimycin and α-Gal A enzyme replacement therapy are administered as a combination therapy.

12. 7. The method of claim 6, wherein a second dose of 1-deoxygalactonojirimycin is administered between about 4 hours and the administration of the α-Gal A enzyme replacement therapy.

13. 8. The method of claim 7, wherein the 1-deoxygalactonojirimycin and α-Gal A enzyme replacement therapy is administered every 1 to 4 weeks.

14. 14. The method of claim 13, wherein the 1-deoxygalactonojirimycin and α-Gal A enzyme replacement therapy is administered every two weeks.

15. A kit for treating Fabry disease in a subject, the kit comprising about 50 mg to about 600 mg of 1-deoxygalactonojirimycin and an effective amount of α-Gal A enzyme replacement therapy.

16. 16. The kit of claim 15, wherein the amount of 1-deoxygalactonojirimycin is selected from about 150 mg, about 300 mg, and about 450 mg.

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