Augmented acid alpha-glucosidase for treatment of pompe disease

Abstract

To provide recombinant human acid α-glucosidase for use in improved enzyme replacement therapy for treating Pompe disease.SOLUTION: Recombinant acid α-glucosidase for use in treating Pompe disease in combination with miglustat is provided, the recombinant acid α-glucosidase being expressed in Chinese hamster ovary (CHO) cells and comprising N-glycan units having one or two mannose-6-phosphate residues, the content of the N-glycan units having one or two mannose-6-phosphate residues being increased compared to the content of N-glycan units having one or two mannose-6-phosphate residues in alglucosidase alfa.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides methods for treating Pompe disease comprising administering to an individual a combination of acid α-glucosidase and its pharmacological chaperone. More specifically, the present invention provides methods for treating Pompe disease comprising administering to an individual a combination of recombinant human acid α-glucosidase and miglustat. [Background technology]

[0002] Pompe disease, also known as acid maltase deficiency or type II glycogen storage disease, is one of several lysosomal storage disorders. Lysosomal storage disorders are a group of autosomal recessive disorders characterized by the accumulation of cellular glycosphingolipids, glycogen, or mucopolysaccharides in intracellular compartments called lysosomes. Individuals with these disorders carry mutated genes encoding enzymes that are defective in catalyzing the hydrolysis of one or more of these substances, which accumulate in the lysosomes. Other examples of lysosomal disorders include Gaucher disease, G M1 -Includes gangliosidoses, fucosidoses, mucopolysaccharidoses, Hurler syndrome, Niemann-Pick A and B disease, and Fabry disease. Pompe disease is also classified as a neuromuscular or metabolic myopathy.

[0003] Pompe disease, estimated to occur in approximately 1 in 40,000 births, is caused by mutations in the GAA gene, which encodes the enzyme lysosomal α-glucosidase (EC:3.2.1.20), commonly known as acid α-glucosidase. Acid α-glucosidase is involved in the metabolism of glycogen, a branched polysaccharide that is the primary storage form of glucose in animals, by catalyzing its hydrolysis to glucose within the lysosomes. Individuals with Pompe disease produce a defective acid α-glucosidase that is inactive or has reduced activity. Therefore, glycogen breakdown occurs slowly or not at all, and glycogen accumulates in lysosomes in various tissues, particularly in striated muscle, resulting in a wide range of clinical symptoms, including progressive muscle weakness and respiratory failure. Tissues such as cardiac and skeletal muscle are particularly affected.

[0004] Pompe disease varies greatly depending on the degree of enzyme deficiency, the severity, and the age at which it begins. More than 500 different mutations in the GAA gene have been identified, many of which cause disease symptoms of varying severity. The disease is broadly classified into early-onset or infantile-onset and later-onset forms. Earlier onset and lower enzyme activity are generally associated with a more severe clinical course. Pediatric Pompe disease is the most severe and results from complete or near-complete acid α-glucosidase deficiency, manifesting as severe loss of muscle tone, weakness, liver and cardiac enlargement, and cardiomyopathy. The tongue may be enlarged and protruding, and swallowing may be difficult. Most children die of respiratory or cardiac complications by the age of 2 years. Late-onset Pompe disease can present at any age above 12 months and is characterized by the absence of cardiac involvement and a more favorable short-term prognosis. Symptoms are associated with progressive skeletal muscle dysfunction, with generalized muscle weakness in the trunk, proximal limbs, and diaphragm, and respiratory muscle wasting. Adult patients may not have significant symptoms or mobility limitations. Prognosis generally depends on the extent of respiratory muscle involvement. Most subjects with Pompe disease experience gradual progression to wheelchair use and physical debilitation requiring assisted ventilation, often with early death due to respiratory failure.

[0005] Recent treatment options for Pompe disease include enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA). Conventional rhGAA products are known as alglucosidase alfa, Myozyme®, or Lumizyme®; Genzyme, Inc. ERT is a chronic treatment required for the patient's lifetime and involves administering replacement enzymes via intravenous infusion. The replacement enzyme is then transported through the circulation and enters intracellular lysosomes, where it acts to break down accumulated glycogen, compensating for the deficient activity of the endogenous defective mutant enzyme and thus alleviating disease symptoms. In patients with infantile-onset Pompe disease, treatment with alglucosidase alfa has been shown to significantly improve survival compared with historical controls, and in late-onset Pompe disease, alglucosidase alfa has been shown to have modest, but statistically significant, effects on the 6-minute walk test (6MWT) and forced vital capacity (FVC) compared with placebo.

[0006] However, most subjects' condition remains stable or continues to worsen while undergoing treatment with alglucosidase alfa. The reasons for the apparently suboptimal effects of ERT with alglucosidase alfa are unclear but may be due in part to the progressive nature of the underlying muscle lesion or the inadequate tissue targeting of current ERTs. For example, the infused enzyme is not stable at neutral pH, including that of plasma (approximately pH 7.4), and may be irreversibly inactivated in the circulation. Furthermore, infused alglucosidase alfa exhibits poor uptake in critical disease-related muscle, possibly due to insufficient glycosylation with mannose-6-phosphate (M6P) residues. Such residues bind to the cation-independent mannose-6-phosphate receptor (CIMPR) on the cell surface, allowing the enzyme to enter cells and lysosomes. Therefore, to ensure sufficient amounts of active enzyme reach the lysosomes, high doses of the enzyme may be required for effective treatment, making therapy costly and time-consuming.

[0007] Furthermore, the development of anti-recombinant human acid α-glucosidase neutralizing antibodies often occurs in Pompe disease patients due to repeated exposure to treatment. Such immune responses can significantly reduce a patient's tolerance to treatment. The U.S. product label for alglucosidase alfa contains a black box warning containing information about the potential risk of hypersensitivity reactions. Life-threatening anaphylactic reactions, including anaphylactic shock, have been observed in subjects treated with alglucosidase alfa.

[0008] To address these shortcomings, next-generation ERTs are being developed.In one strategy, recombinant enzymes can be co-administered in combination with pharmacological chaperones that can induce or stabilize the enzyme's proper conformation and prevent or reduce the enzyme's degradation and / or unfolding into its inactive form in vitro (e.g., during storage before administration) or in vivo.Such strategies are described in International Publication Nos. 2004 / 069190, 2006 / 125141, 2013 / 166249 and 2014 / 014938.

[0009] The results of a clinical trial of coadministration of alglucosidase alfa with miglustat in patients with Pompe disease have been described. In a clinical trial conducted in 13 subjects with Pompe disease (3 cases of early onset (infancy) and 10 cases of late onset) at four treatment centers in Italy, 20–40 mg / kg of alglucosidase alfa was administered alone, followed by four doses of 80 mg miglustat. The results of this study showed a 6.8-fold increase in acid α-glucosidase activity exposure (measured by the pharmacokinetic parameter AUC (area under the concentration vs. time curve)) with coadministration compared with alglucosidase alone (Parenti, G., G. Andria, et al. (2015). “Lysosomal Storage Diseases: From Pathophysiology to Therapy.” Annu. Rev. Med. 66(1):471–486). Additionally, a study conducted at the University of Florida evaluated the plasma pharmacokinetics (PK) of miglustat when coadministered with intravenous infusion of alglucosidase alfa in subjects with Pompe disease (Doerfler, PA, J.S. Kelley, et al. (2014). “Pharmacological chaperones prevent the precipitation of rhGAA by anti-GAA antibodies during enzyme replacement therapy.” Mol. Genet. Metab. 111(2):S38).

[0010] However, there is a need for further improvements in enzyme replacement therapy for the treatment of Pompe disease.For example, new recombinant human acid α-glucosidase enzymes are desirable, which may have one or more advantages over currently used enzymes, including but not limited to improved tissue uptake, improved enzyme activity, improved stability or reduced immunogenicity. Summary of the Invention

[0011] The present invention provides a method of treating Pompe disease in a patient in need thereof, comprising administering to the patient miglustat in combination with recombinant human acid α-glucosidase (rhGAA), the recombinant human acid α-glucosidase being expressed in Chinese hamster ovary (CHO) cells and comprising an increased content of N-glycan units having one or two mannose-6-phosphate residues compared to the content of N-glycan units having one or two mannose-6-phosphate residues in alglucosidase alfa. In at least one embodiment, the recombinant human acid α-glucosidase is administered intravenously at a dose of about 20 mg / kg, and miglustat is administered orally at a dose of about 260 mg.

[0012] In another aspect, the present invention provides a combination of miglustat and recombinant human acid α-glucosidase as defined herein for the treatment of Pompe disease in a patient in need thereof.

[0013] In another aspect, the present invention provides the use of a combination of miglustat and recombinant human acid α-glucosidase as defined herein in the preparation of a medicament for the treatment of Pompe disease in a patient in need thereof.

[0014] Another aspect of the present invention provides a kit for combination treatment of Pompe disease in a patient in need thereof, the kit comprising a pharmaceutically acceptable dosage form comprising miglustat, a pharmaceutically acceptable dosage form comprising recombinant human acid alpha-glucosidase as defined herein, and instructions for administering the pharmaceutically acceptable dosage form comprising miglustat and the pharmaceutically acceptable dosage form comprising recombinant acid alpha-glucosidase to a patient in need thereof.

[0015] Further features of the present invention will become apparent from the following specification and accompanying drawings. [Brief explanation of the drawings]

[0016] [Figure 1]1 is a graph showing the percentage of unfolded ATB200 protein versus temperature at various pH values, and in the presence and absence of miglustat. [Figure 2A] Figures 2A and 2B show the results of CIMPR affinity chromatography of Lumizyme® and Myozyme®, respectively. The dashed line indicates the M6P elution gradient. Elution with M6P resulted in the displacement of GAA molecules bound via M6P-containing glycans by CIMPR. As shown in Figure 2A, 78% of the GAA activity in Lumizyme® eluted before the addition of M6P. Figure 2B shows that 73% of the GAA activity in Myozyme® eluted before the addition of M6P. Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme® eluted with M6P. These figures demonstrate that the majority of rhGAA in these two conventional rhGAA products lacks the M6P-bearing glycans necessary for cellular uptake and lysosomal targeting. [Figure 2B] Figures 2A and 2B show the results of CIMPR affinity chromatography of Lumizyme® and Myozyme®, respectively. The dashed line indicates the M6P elution gradient. Elution with M6P resulted in the displacement of GAA molecules bound via M6P-containing glycans by CIMPR. As shown in Figure 2A, 78% of the GAA activity in Lumizyme® eluted before the addition of M6P. Figure 2B shows that 73% of the GAA activity in Myozyme® eluted before the addition of M6P. Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme® eluted with M6P. These figures demonstrate that the majority of rhGAA in these two conventional rhGAA products lacks the M6P-bearing glycans necessary for cellular uptake and lysosomal targeting. [Figure 3] 1 shows a DNA construct for transforming CHO cells with DNA encoding rhGAA. CHO cells were transformed with a DNA construct encoding rhGAA. [Figure 4A]The results of CIMPR affinity chromatography of Myozym (registered trademark) and ATB200 rhGAA are shown in Figure 4B. As is clear from Figure 4B, approximately 70% of the rhGAA in ATB200 rhGAA contained M6P. [Figure 4B] The results of CIMPR affinity chromatography of Myozym (registered trademark) and ATB200 rhGAA are shown in Figure 4B. As is clear from Figure 4B, approximately 70% of the rhGAA in ATB200 rhGAA contained M6P. [Figure 5A] 1 shows the results of CIMPR affinity chromatography of ATB200 rhGAA with and without capture on an anion exchange (AEX) column. [Figure 5B] 1 shows the results of CIMPR affinity chromatography of ATB200 rhGAA with and without capture on an anion exchange (AEX) column. [Figure 6] 1 shows the Polywax elution profiles of Lumizyme® and ATB200 rhGAA. [Figure 7] 1 shows an overview of the N-glycan structures of Lumizyme® compared to three different preparations of ATB200 rhGAA identified as BP-rhGAA, ATB200-1 and ATB200-2. [Figure 8A] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 8B] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 8C] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 8D] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 8E] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 8F] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 8G]1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 8H] 1 shows the results of site-specific N-glycosylation analysis of ATB200 rhGAA. [Figure 9A] FIG. 9A compares the CIMPR binding affinity of ATB200 rhGAA (left trace) and Lumizyme (right trace). [Figure 9B] FIG. 9B compares the Bis-M6P content of Lumizyme® and ATB200 rhGAA. [Figure 10A] FIG. 10A compares ATB200 rhGAA activity (left trace) with Lumizyme® rhGAA activity (right trace) in normal fibroblasts at various GAA concentrations. [Figure 10B] FIG. 10B compares ATB200 rhGAA activity (left trace) with Lumizyme® rhGAA activity (right trace) in fibroblasts from a subject with Pompe disease at various GAA concentrations. [Figure 10C] FIG. 10C compares (K uptake) of fibroblasts from healthy subjects and subjects with Pompe disease. [Figure 11] 1 is a graph showing the goodness of fit of a population pharmacokinetic (PK) model of ATB200. [Figure 12] 1 is a graph showing the dose-normalized plasma concentration-time profiles of miglustat and duvoglustat. [Figure 13A] FIG. 13A is a graph showing the goodness of fit of the population PK model of duvoglustat in plasma. [Figure 13B] FIG. 13B is a graph showing the goodness of fit of the population PK model of duvoglustat in muscle tissue. [Figure 14] 1 is a graph showing the goodness of fit of the population PK model for miglustat. [Figure 15] 1 is a graph showing the predicted concentration-time profile resulting from a 4-hour infusion of a single 20 mg / kg intravenous (IV) dose of ATB200 in humans. [Figure 16A]FIG. 16A is a graph showing the amount of glycogen versus dose of recombinant human acid α-glucosidase in mouse myocardium after contact with vehicle (negative control), 20 mg / kg alglucosidase alfa (Lumizyme®), or 5, 10, or 20 mg / kg ATB200. [Figure 16B] Figure 16B is a graph showing the amount of glycogen versus dose of recombinant human acid α-glucosidase in mouse quadriceps muscle after contact with vehicle (negative control), 20 mg / kg alglucosidase alfa (Lumizyme®), or 5, 10, or 20 mg / kg ATB200. [Figure 16C] Figure 16C is a graph showing the amount of glycogen versus dose of recombinant human acid α-glucosidase in mouse triceps muscle after contact with vehicle (negative control), 20 mg / kg alglucosidase alfa (Lumizyme®), or 5, 10, or 20 mg / kg ATB200. [Figure 17] 1 is a graph plotting the ratio of the AUC value of miglustat to the AUC value of ATB200 versus the ratio of the glycogen level in mice treated with varying doses of miglustat in the presence of ATB200 to the glycogen level in mice treated with ATB200 alone. [Figure 18] 1 is a graph showing predicted concentration-time profiles of miglustat in plasma following repeated administration of 466 mg, 270 mg, and 233 mg doses of miglustat. [Figure 19] 1 is a graph showing predicted concentration-time profiles of miglustat in tissue lysosomes following repeated administration of 466 mg, 270 mg, and 233 mg doses of miglustat. [Figure 20] A series of photomicrographs of heart, septum, and soleus muscles from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing levels of lysosome-associated membrane protein 1 (LAMP1). [Figure 21] A series of photomicrographs of heart and soleus muscles from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing glycogen levels by staining with periodic acid-Schiff reagent (PAS). [Figure 22] Series of photomicrographs (1000x) of quadriceps muscles from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, stained with methylene blue to show vacuoles (indicated by arrows). [Figure 23] A series of photomicrographs (400x) of quadriceps muscles from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing levels of the autophagy markers microtubule-associated protein type 1A / 1B light chain 3 phosphatidylethanolamine conjugate (LC3A II) and p62, the insulin-dependent glucose transporter GLUT4, and the insulin-independent glucose transporter GLUT1. [Figure 24A] 1 is a graph showing the concentration-time profile of GAA activity in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, or 20 mg / kg ATB200 and 130 or 260 mg miglustat. [Figure 24B] 1 is a graph showing the concentration-time profile of GAA activity in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, or 20 mg / kg ATB200 and 130 or 260 mg miglustat. [Figure 24C] 1 is a graph showing the concentration-time profile of GAA activity in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, or 20 mg / kg ATB200 and 130 or 260 mg miglustat. [Figure 24D]1 is a graph showing the concentration-time profile of GAA activity in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, or 20 mg / kg ATB200 and 130 or 260 mg miglustat. [Figure 25A] 1 is a graph showing the concentration-time profile of GAA total protein in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 25B] 1 is a graph showing the concentration-time profile of GAA total protein in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 25C] 1 is a graph showing the concentration-time profile of GAA total protein in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 25D] 1 is a graph showing the concentration-time profile of GAA total protein in plasma in human subjects after administration of 5, 10 or 20 mg / kg ATB200, 20 mg / kg ATB200 and 130 mg miglustat, or 20 mg / kg ATB200 and 260 mg miglustat. [Figure 26] 1 is a graph showing the concentration-time profile of miglustat in plasma in human subjects after administration of 130 mg or 260 mg of miglustat. [Figure 27] 1 is a series of immunofluorescence micrographs of GAA and LAMP1 levels in wild-type and Pompe fibroblasts. [Figure 28]1 is a series of photomicrographs of muscle fibers from wild-type and Gaa knockout mice showing dystrophin, α- and β-dystroglycan, and dysferlin levels. [Figure 29A] A series of photomicrographs (200x) of rectus femoris (RF) and vastus lateralis / vastus medialis (VL / VM) muscle fibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing LMAP1 IHC signal. [Figure 29B] A series of photomicrographs (200x) of rectus femoris (RF) and vastus lateralis / vastus medialis (VL / VM) muscle fibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing LMAP1 IHC signal. [Figure 30A] A series of photomicrographs (200x) of RF and VL / VM muscle fibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing LC3 II IHC signal. [Figure 30B] A series of photomicrographs (200x) of RF and VL / VM muscle fibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing LC3 II IHC signal. [Figure 31A] A series of photomicrographs (200x) of RF and VL / VM muscle fibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing dysferlin IHC signal. [Figure 31B] A series of photomicrographs (200x) of RF and VL / VM muscle fibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing dysferlin IHC signal. [Figure 32A] 1 is a graph showing glycogen levels in quadriceps, triceps, gastrocnemius and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 32B] 1 is a graph showing glycogen levels in quadriceps, triceps, gastrocnemius and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 32C] 1 is a graph showing glycogen levels in quadriceps, triceps, gastrocnemius and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 32D] 1 is a graph showing glycogen levels in quadriceps, triceps, gastrocnemius and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 33A] 1 is a graph showing wirehand and grip strength muscle data from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence of miglustat. [Figure 33B] 1 is a graph showing wirehand and grip strength muscle data from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence of miglustat. [Figure 34A] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 34B]1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 34C] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 34D] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 34E] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 34F] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 34G] 1 is a graph showing glycogen levels in quadriceps, triceps and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200 in the presence and absence of miglustat. [Figure 35] 1 is a series of photomicrographs of VL / VM myofibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing LAMP1, LC3, and dysferlin IHC signals. [Figure 36]1 is a graph showing the concentration-time profile of GAA activity in plasma in Gaa knockout mice after administration of two batches of ATB200 having different sialic acid contents. [Figure 37A] 1 is a graph showing glycogen levels in quadriceps, triceps, gastrocnemius and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200. [Figure 37B] 1 is a graph showing glycogen levels in quadriceps, triceps, gastrocnemius and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200. [Figure 37C] 1 is a graph showing glycogen levels in quadriceps, triceps, gastrocnemius and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200. [Figure 37D] 1 is a graph showing glycogen levels in quadriceps, triceps, gastrocnemius and heart cells from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa and ATB200. [Figure 38] 1 is a graph showing alanine aminotransferase (ALT) levels in human patients after administration of increasing doses of ATB200 (5, 10 and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 39] 1 is a graph showing aspartate aminotransferase (AST) levels in human patients after administration of increasing doses of ATB200 (5, 10 and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 40] FIG. 1 is a graph showing creatine phosphokinase (CPK) levels in human patients after administration of increasing doses of ATB200 (5, 10 and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 41] 1 is a graph showing mean ALT, AST, and CPK levels in human patients after administration of increasing doses of ATB200 (5, 10, and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). [Figure 42] A series of photomicrographs (100x and 200x) of vastus lateralis (VL) muscle fibers from wild-type and Gaa knockout mice treated with vehicle, alglucosidase alfa, and ATB200 in the presence and absence of miglustat, showing dystrophin signals. DETAILED DESCRIPTION OF THE INVENTION

[0017] definition The terms used herein generally have their ordinary meanings in the art, with respect to this invention and in the specific context in which each term is used. Certain terms are explained below or elsewhere in this specification to provide additional guidance to those skilled in the art.

[0018] In this specification, for literal explanation or necessary implication, unless the context otherwise requires, the word "comprises" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., to specify the presence of stated features, and do not exclude the presence or addition of further features in various embodiments of the invention.

[0019] As used herein, the term "Pompe disease," also known as acid maltase deficiency, glycogenic storage disease type II (GSDII), and glycogenic disease type II, is intended to refer to a genetic lysosomal storage disorder characterized by mutations in the GAA gene, which encodes the human acid α-glucosidase enzyme. The term includes, but is not limited to, early- and late-onset forms of Pompe disease, including, but not limited to, infantile, juvenile, and adult-onset forms.

[0020] As used herein, the term "acid α-glucosidase" refers to a lysosomal enzyme that hydrolyzes the α-1,4 bond between the D-glucose units of glycogen, maltose, and isomaltose. Alternative names include, but are not limited to, lysosomal α-glucosidase (EC: 3.2.1.20); glucoamylase; 1,4-α-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase; and exo-1,4-α-glucosidase. Human acid α-glucosidase is encoded by the GAA gene (National Center for Biotechnology Information (NCBI) Gene ID 2548), which is mapped to the long arm of chromosome 17 (location 17q25.2-q25.3). More than 500 mutations have now been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations that result in misfolding or misprocessing of the acid α-glucosidase enzyme include T1064C (Leu355Pro) and C2104T (Arg702Cys). Additionally, GAA mutations that affect enzyme maturation and processing include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE at amino acid residues 516-521 is required for activity of the acid α-glucosidase protein. As used herein, the abbreviation "GAA" is intended to refer to the acid α-glucosidase enzyme, and the italicized abbreviation "GAA" is intended to refer to the human gene encoding the human acid α-glucosidase enzyme. The italicized abbreviation "Gaa" is intended to refer to non-human genes encoding non-human acid α-glucosidase enzymes, including, but not limited to, rat or mouse genes, and the abbreviation "Gaa" is intended to refer to non-human acid α-glucosidase enzymes. Thus, the abbreviation "rhGAA" is intended to refer to recombinant human acid alpha-glucosidase enzyme.

[0021] As used herein, the term "alglucosidase alfa" is intended to refer to recombinant human acid alpha-glucosidase identified as [199-arginine, 223-histidine] prepro-alpha-glucosidase (human); Chemical Abstracts Registry Number 420794-05-0. Alglucosidase alfa has been approved for commercial sale in the United States by Genzyme as Lumizyme® and Myozyme® on October 1, 2014.

[0022] As used herein, the term "ATB200" is intended to refer to the recombinant human acid α-glucosidase described in co-pending patent application PCT / US2015 / 053252, the disclosure of which is incorporated herein by reference.

[0023] As used herein, the term "glycan" refers to a polysaccharide chain covalently bonded to an amino acid residue on a protein or polypeptide. As used herein, the term "N-glycan" or "N-linked glycan" refers to a polysaccharide chain covalently bonded to the nitrogen atom of an amino acid residue on a protein or polypeptide. For example, an N-glycan may be covalently bonded to the side chain nitrogen atom of an asparagine residue. A glycan may contain one or several monosaccharide units, and the monosaccharide units may be covalently bonded to form a linear or branched chain. In at least one embodiment, the N-glycan unit bound to ATB200 may contain one or more monosaccharide units independently selected from N-acetylglucosamine, mannose, galactose, or sialic acid. The N-glycan units on a protein may be determined by any suitable analytical technique, such as mass spectrometry. In some embodiments, N-glycan units can be determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using instruments such as a Thermo Scientific Orbitrap Velos Pro™ mass spectrometer, a Thermo Scientific Orbitrap Fusion Lumos Tribid™ mass spectrometer, or a Waters Xevo® G2-XS QTof mass spectrometer.

[0024] As used herein, the term "high mannose N-glycan" is intended to refer to an N-glycan having one to six or more mannose units. In at least one embodiment, a high mannose N-glycan unit contains a bis(N-acetylglucosamine) chain attached to an asparagine residue, which may be further attached to a branched polymannose chain. When used interchangeably herein, the term "M6P" or "mannose-6-phosphate" is intended to refer to a mannose unit that is phosphorylated at the 6-position, i.e., has a phosphate group attached to the hydroxyl group at the 6-position. In at least one embodiment, one or more mannose units of one or more N-glycan units are phosphorylated at the 6-position to form a mannose-6-phosphate unit. In at least one embodiment, the term "M6P" or "mannose-6-phosphate" refers to both a mannose phosphodiester having an N-acetylglucosamine (GlcNAc) as a "cap" on the phosphate group, and a mannose unit with an exposed phosphate group that lacks a GlcNAc cap. In at least one embodiment, the N-glycans of the protein can have multiple M6P groups, with at least one M6P group having a GlcNAc cap and at least one other M6P group lacking a GlcNAc cap.

[0025] As used herein, the term "complex N-glycan" is intended to refer to an N-glycan containing one or more galactose and / or sialic acid units. In at least one embodiment, the complex N-glycan may be a high-mannose N-glycan in which one or more mannose units are further attached to one or more monosaccharide units independently selected from N-acetylglucosamine, galactose, and sialic acid.

[0026] As used herein, the compound miglustat, also known as N-butyl-1-deoxynojirimycin or NB-DNJ or (2R,3R,4R,5S)-1-butyl-2-(hydroxymethyl)piperidine-3,4,5-triol, has the following chemical formula: JPEG2025163019000001.jpg29154

[0027] One formulation of miglustat is marketed under the trade name Zavesca® as a monotherapy for type 1 Gaucher disease.

[0028] As described below, pharmaceutically acceptable salts of miglustat may also be used in the present invention. When a salt of miglustat is used, the dose of the salt is adjusted so that the dose of miglustat received by the patient is equivalent to the dose they would receive if the free base of miglustat were used.

[0029] As used herein, the compound duvoglustat, also known as 1-deoxynojirimycin or DNJ or (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol, has the following chemical formula: JPEG2025163019000002.jpg31154

[0030] As used herein, the term "pharmacological chaperone" or sometimes simply the term "chaperone" is intended to refer to something that specifically binds to acid α-glucosidase and has one or more of the following effects: • Promotes the formation of stable molecular conformations of proteins; • Facilitating proper transport of proteins from the endoplasmic reticulum to another cellular location, preferably their native cellular location, to prevent endoplasmic reticulum-associated degradation of the protein; • Preventing aggregation of structurally unstable or misfolded proteins; • Restoring and / or improving wild-type function, stability and / or activity of at least a portion of a protein; and / or • Improve the phenotype or function of cells that possess acid α-glucosidase.

[0031] Thus, a pharmacological chaperone for acid α-glucosidase is a molecule that binds to acid α-glucosidase and ensures its proper folding, transport, non-aggregation, and activity. As used herein, the term includes, but is not limited to, active site-specific chaperones (ASSCs), inhibitors or antagonists, and agonists that bind to the active site of the enzyme. In at least one embodiment, the pharmacological chaperone may be an inhibitor or antagonist of acid α-glucosidase. As used herein, the term "antagonist" is intended to refer to any molecule that binds to acid α-glucosidase and partially or completely blocks, inhibits, reduces, or neutralizes the activity of acid α-glucosidase. In at least one embodiment, the pharmacological chaperone is miglustat. Another non-limiting pharmacological chaperone for acid α-glucosidase is duvoglustat.

[0032] As used herein, the term "active site" is intended to refer to a region of a protein that is associated with and necessary for the protein's specific biological activity. In at least one embodiment, the active site may be a site that binds to a substrate or other binding partner and contributes amino acid residues directly involved in the formation and cleavage of chemical bonds. Active sites in the present invention may include the catalytic site of an enzyme, the antigen-binding site of an antibody, the ligand-binding domain of a receptor, the binding domain of a regulator, or the receptor-binding domain of a secreted protein. Active sites may also include transactivation, protein-protein interaction, or DNA-binding domains of transcription factors and regulators.

[0033] As used herein, the term "AUC" refers to a mathematical calculation for evaluating the total exposure of the body to a given drug over time.In a graph plotting how the blood concentration of a drug administered to a subject changes with time after administration, the drug concentration variable is on the y-axis, and time is on the x-axis.The area between the drug concentration curve for a given time interval and the x-axis is AUC ("area under the curve").AUC is used as a guide for dosing schedules and to compare the bioavailability of different drugs in the body.

[0034] As used herein, the term "C 最大値 " is intended to refer to the maximum plasma concentration of a drug achieved after administration to a subject.

[0035] As used herein, the term "volume of distribution" or "V" is intended to refer to the theoretical volume required to contain the total amount of drug administered at the same concentration as observed in plasma, and represents the extent to which a drug is distributed in body tissues rather than in plasma. A higher value of V indicates a greater degree of tissue distribution. "Central volume of distribution" or "Vc" is intended to refer to the volume of distribution in blood and tissues highly perfused by blood. "Peripheral volume of distribution" or "V2" is intended to refer to the volume of distribution in peripheral tissues.

[0036] As used interchangeably herein, the terms "clearance," "systemic clearance," or "CL" are intended to refer to the volume of plasma from which an administered drug is completely removed per unit time. "Peripheral clearance" is intended to refer to the volume of peripheral tissue from which an administered drug is cleared per unit time.

[0037] As used herein, "therapeutically effective dose" and "effective amount" are intended to refer to the amount of acid α-glucosidase and / or miglustat and / or a combination thereof that results in a therapeutic response in a subject. A therapeutic response can be any response that a user (e.g., a clinician) recognizes as an effective response to treatment, including any surrogate clinical marker or symptom described herein and known in the art. Thus, in at least one embodiment, a therapeutic response can be an improvement or suppression of one or more symptoms or markers of Pompe disease, such as those known in the art. Symptoms or markers of Pompe disease include, but are not limited to, cardiomyopathy, cardiac hypertrophy, progressive muscle weakness, especially in the trunk or lower limbs, severe hypotension, macroglossia (and sometimes tongue protrusion), difficulty swallowing, sucking, and / or feeding, respiratory failure, hepatomegaly (moderate), facial muscle relaxation, areflexia, exercise intolerance, exertional dyspnea, orthopnea, sleep apnea, morning headache, somnolence, lordosis and / or scoliosis, decreased deep tendon reflexes, lower back pain, and failure to achieve developmental motor milestones. It should be noted that the concentration of miglustat that has an inhibitory effect on acid α-glucosidase may constitute an "effective amount" for purposes of the present invention due to dilution (and resulting changes in binding due to shifts in equilibrium), bioavailability, and metabolism of miglustat upon in vivo administration.

[0038] As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of a non-natural purified enzyme into an individual with a deficiency of such an enzyme. The administered protein can be obtained from a natural source or by recombinant expression. The term also refers to the introduction of a purified enzyme into an individual who otherwise requires or would benefit from the administration of the purified enzyme. In at least one embodiment, such an individual suffers from an enzyme deficiency. The introduced enzyme may be a purified recombinant enzyme produced in vitro, or a protein purified from an isolated tissue or fluid, such as the placenta or milk of an animal, or from a plant.

[0039] As used herein, the term "combination therapy" is intended to refer to any therapy in which two or more individual therapies are administered simultaneously or sequentially. In at least one embodiment, the results of the combination therapy are enhanced compared to the effects of each therapy when administered individually. Enhancement can include any improvement in the effects of various therapies that can result in advantageous results compared to the results achieved by the therapies when administered alone. Enhanced effects or results can include synergistic enhancement, where the enhanced effect is greater than the additive effect of each therapy when administered alone; additive enhancement, where the enhanced effect is substantially equal to the additive effect of each therapy when administered alone; or subsynergistic, where the enhanced effect is less than the additive effect of each therapy when administered alone, but superior to the effect of each therapy when administered alone. Enhanced effects can be measured by any means known in the art that can measure treatment effects or results.

[0040] As used herein, the term "pharmaceutically acceptable" is intended to refer to molecular entities and compositions that are physiologically tolerable 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 government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, particularly humans.

[0041] As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Suitable pharmaceutical carriers are known in the art and, at least in one embodiment, are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other editions.

[0042] As used herein, the term "subject" or "patient" is intended to refer to a human or non-human animal. In at least one embodiment, the subject is a mammal. In at least one embodiment, the subject is a human.

[0043] As used herein, the term "anti-drug antibody" is intended to refer to an antibody that specifically binds to a drug administered to a subject and is produced by the subject as at least part of a humoral immune response to the administration of the drug to the subject. In at least one embodiment, the drug is a therapeutic protein drug product. The presence of anti-drug antibodies in a subject can result in immune responses ranging from mild to severe, including, but not limited to, life-threatening immune responses, including, but not limited to, anaphylaxis, cytokine release syndrome, and cross-reactive neutralization of endogenous proteins that mediate critical functions. Additionally or alternatively, the presence of anti-drug antibodies in a subject can reduce the effectiveness of the drug.

[0044] As used herein, the term "neutralizing antibody" is intended to refer to an anti-drug antibody that acts to neutralize the function of a drug. In at least one embodiment, a therapeutic protein drug product is a counterpart of an endogenous protein whose expression is reduced or absent in a subject. In at least one embodiment, a neutralizing antibody can act to neutralize the function of an endogenous protein.

[0045] As used herein, the terms "about" and "approximately" are intended to refer to an acceptable degree of error for the measured quantity, given the nature or precision of the measurement. For example, the degree of error can be indicated by the number of significant figures provided for a measurement, as understood in the art, and includes, but is not limited to, a variation of ±1 of the most accurate significant figure reported for 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, preferably within 5-fold, and more preferably within 2-fold of a given value. Numerical values ​​described herein are approximate unless otherwise specified, and the terms "about" or "approximately" mean that they can be inferred unless expressly stated.

[0046] The term "simultaneously" as used herein is intended to mean simultaneously or within a reasonably short time before or after, as understood by those skilled in the art.For example, when two treatments are administered simultaneously, one treatment can be administered before or after the other treatment to allow the time required for preparation of the later of the two treatments.Therefore, "simultaneous administration" of two treatments includes, but is not limited to, one treatment following the other treatment by 20 minutes or less, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute or less.

[0047] The term "pharmaceutically acceptable salt," as used herein, is intended to mean a salt that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., and is generally water- or oil-soluble or dispersible, and effective for its intended use, in accordance with a reasonable benefit / risk ratio. This term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. Lists of suitable salts can be found, for example, in S. M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19, which is incorporated herein by reference.

[0048] The term "pharmaceutically acceptable acid addition salts," as used herein, refers to those salts with inorganic acids, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and the like, which retain the biological effectiveness and properties of the free base and which are not biologically or otherwise undesirable, as well as acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphate, hemisulfonic acid, hemisulfonic acid, hexafluorophosphate ... "Salts" are intended to mean salts formed with organic acids, including, but not limited to, xanthic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, undecanoic acid, and the like.

[0049] The term "pharmaceutically acceptable base addition salts," as used herein, is intended to mean salts formed with inorganic bases, including, but not limited to, hydroxides, carbonates, or bicarbonates of ammonia, or ammonium or metal cations such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like, which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable. Salts derived from pharmaceutically acceptable organic non-toxic bases include those derived from primary, secondary, and tertiary amines, quaternary amine compounds, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrazine ... The salts of amine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, polyamine resins, and the like.

[0050] The present invention provides a method for treating Pompe disease in a patient in need thereof, comprising administering to the patient miglustat or a pharmaceutically acceptable salt thereof in combination with recombinant human acid α-glucosidase, wherein the recombinant human acid α-glucosidase is expressed in Chinese hamster ovary (CHO) cells and contains an increased content of N-glycan units having one or two mannose-6-phosphate residues compared to the content of N-glycan units having one or two mannose-6-phosphate residues in alglucosidase alfa. In at least one embodiment, the recombinant human acid α-glucosidase has low levels of complex glycans with terminal galactose. In another aspect, the present invention provides a combination of miglustat and recombinant human acid α-glucosidase for the treatment of Pompe disease in a patient in need thereof.

[0051] In at least one embodiment, miglustat is administered orally. In at least one embodiment, miglustat is administered at an oral dose of about 200 mg to about 600 mg, or at an oral dose of about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg. In at least one embodiment, miglustat is administered at an oral dose of about 233 mg to about 400 mg. In at least one embodiment, miglustat is administered at an oral dose of about 250 to about 270 mg, or at an oral dose of about 250 mg, about 255 mg, about 260 mg, about 265 mg, or about 270 mg. In at least one embodiment, miglustat is administered as an oral dose of about 260 mg.

[0052] Those skilled in the art will appreciate that for an adult patient weighing an average of about 70 kg, an oral dose of miglustat in the range of about 200 mg to 600 mg, or any smaller range, may be appropriate. For patients weighing significantly less than about 70 kg, including, but not limited to, infants, children, or underweight adults, a lower dose may be deemed appropriate by a physician. Thus, in at least one embodiment, miglustat is administered at an oral dose of about 50 mg to about 200 mg, or at oral doses of about 50 mg, about 75 mg, about 100 mg, 125 mg, about 150 mg, about 175 mg, or about 200 mg. In at least one embodiment, miglustat is administered at an oral dose of about 65 mg to about 195 mg, or at oral doses of about 65 mg, about 130 mg, or about 195 mg.

[0053] In at least one embodiment, miglustat is administered in a pharmaceutically acceptable dosage form suitable for oral administration, including, but not limited to, tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or dry powders for reconstitution with water or other suitable solvents prior to use, optionally with flavoring and coloring, for immediate-release, delayed-release, modified-release, sustained-release, pulsed-release, or controlled-release applications. Solid compositions such as tablets, capsules, lozenges, troches, pills, boluses, powders, pastes, granules, bullets, dragees, or premixed preparations can also be used. In at least one embodiment, miglustat is administered as a tablet. In at least one embodiment, miglustat is administered as a capsule. In at least one embodiment, the dosage form contains about 50 mg to about 300 mg of miglustat. In at least one embodiment, the dosage form contains about 65 mg of miglustat. In at least one embodiment, the dosage form contains about 130 mg of miglustat. In at least one embodiment, the dosage form contains about 260 mg of miglustat.When the dosage form contains about 65 mg of miglustat, it is contemplated that miglustat can be administered as four dosage forms or a total dosage of 260 mg of miglustat.However, for patients with body weight significantly lower than the average adult weight of 70 kg, including but not limited to infants, children, or underweight adults, miglustat can be administered as one dosage form (a total dosage of 65 mg of miglustat), two dosage forms (a total dosage of 130 mg of miglustat), or three dosage forms (a total dosage of 195 mg of miglustat).

[0054] 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. Tablets or capsules can be prepared by conventional means using pharmaceutically acceptable excipients, including, but not limited to, binders, fillers, lubricants, disintegrants, or wetting agents. Suitable pharmaceutically acceptable excipients are known in the art and include, but are not limited to, pregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl ethylcellulose (HPEC), hydroxypropyl cellulose (HPC), sucrose, gelatin, gum arabic, lactose, microcrystalline cellulose, calcium hydrogen phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato, or tapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine croscarmellose sodium, and silicate complex. Tablets may be coated by methods well known in the art. In at least one embodiment, miglustat is administered in the formulation marketed as Zavesca® (Actelion Pharmaceuticals).

[0055] In at least one embodiment, the recombinant human acid α-glucosidase is expressed in Chinese hamster ovary (CHO) cells and comprises an increased content of N-glycan units having one or more mannose-6-phosphate residues compared to the content of N-glycan units having one or more mannose-6-phosphate residues of alglucosidase alfa. In at least one embodiment, the acid α-glucosidase is a recombinant human acid α-glucosidase, referred to herein as ATB200, as described in co-pending International Patent Application PCT / US2015 / 053252. ATB200 has a high affinity (K DIt binds to the cation-independent mannose-6-phosphate receptor (CIMPR) at ~2–4 nM and is expressed in Pompe fibroblasts and skeletal myoblasts (K 取り込み ATB200 has been characterized in vivo and has been shown to be efficiently internalized by alglucosidase alfa (t 1 / 2 Its apparent plasma half-life (t ) is shorter than 60 min. 1 / 2 It was shown that the solubility of the solubility is approximately 45 minutes.

[0056] In at least one embodiment, the recombinant human acid α-glucosidase is an enzyme having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2 (or encoded by SEQ ID NO:2), SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. JPEG2025163019000003.jpg152154JPEG2025163019000004.jpg209154JPEG2025163019000005.jpg202154JPEG2025163019000006.jpg199154 JPEG2025163019000007.jpg204154JPEG2025163019000008.jpg202154JPEG2025163019000009.jpg203154JPEG2025163019000010.jpg213154

[0057] In at least one embodiment, the recombinant human acid α-glucosidase has the wild-type GAA amino acid sequence set forth in SEQ ID NO: 1, as described in U.S. Patent No. 8,592,362, and has GenBank accession number AHE24104.1 (G1:568760974). In at least one embodiment, the recombinant human acid α-glucosidase has the wild-type GAA amino acid sequence encoded by SEQ ID NO: 2, the mRNA sequence of which has GenBank accession number Y00839.1. In at least one embodiment, the recombinant human acid α-glucosidase has the wild-type GAA amino acid sequence set forth in SEQ ID NO: 3. In at least one embodiment, the recombinant human acid α-glucosidase has the GAA amino acid sequence set forth in SEQ ID NO: 4, and has National Center for Biotechnology Information (NCBI) accession number NP_000143.2. In at least one embodiment, the recombinant human acid alpha-glucosidase is glucosidase alpha, the human acid alpha-glucosidase enzyme encoded by the most prevalent of nine observed haplotypes of the GAA gene.

[0058] In at least one embodiment, the recombinant human acid α-glucosidase is initially expressed as having the full-length 952 amino acid sequence of wild-type GAA set forth in SEQ ID NO: 1, and the recombinant human acid α-glucosidase undergoes intracellular processing to remove some amino acids, e.g., the first 56 amino acids. Thus, the recombinant human acid α-glucosidase secreted by the host cell can have a shorter amino acid sequence than the recombinant human acid α-glucosidase initially expressed within the cell. In at least one embodiment, the shorter protein can have the amino acid sequence set forth in SEQ ID NO: 5, which differs from SEQ ID NO: 1 only in that the first 56 amino acids, including the signal peptide and precursor peptide, have been removed, thus resulting in a protein having 896 amino acids. Other variations in the number of amino acids are also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more deletions, substitutions, and / or insertions relative to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5. In some embodiments, the rhGAA product comprises a mixture of recombinant human acid α-glucosidase molecules having different amino acid lengths.

[0059] In at least one embodiment, recombinant human acid α-glucosidase undergoes post-translational and / or chemical modifications at one or more amino acid residues in the protein. For example, methionine and tryptophan residues may undergo oxidation. As another example, N-terminal glutamine can form pyroglutamic acid. As another example, asparagine residues may undergo deamination to aspartic acid. As yet another example, aspartic acid residues may undergo isomerization to isoaspartic acid. As yet another example, unpaired cysteine ​​residues in the protein may form disulfide bonds with free glutathione and / or cysteine. Thus, in some embodiments, the enzyme is initially expressed as having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2 (or encoded by SEQ ID NO:2), SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and the enzyme undergoes one or more of these post-translational and / or chemical modifications. Such modified forms are also within the scope of the present disclosure.

[0060] Polynucleotide sequences encoding GAA and such mutant human GAA are also contemplated and may be used to recombinantly express rhGAA according to the present invention.

[0061] Preferably, no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10, or 5% of all recombinant human acid α-glucosidase molecules lack an N-glycan unit having one or more mannose-6-phosphate residues or lack the ability to bind to the cation-independent mannose-6-phosphate receptor (CIMPR). Alternatively, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, or <100% of the recombinant human acid α-glucosidase molecules contain at least one N-glycan unit having one or more mannose-6-phosphate residues or are capable of binding to CIMPR.

[0062] A recombinant human acid α-glucosidase molecule can have one, two, three, or four mannose-6-phosphate (M6P) groups on its glycan. For example, only one N-glycan in a recombinant human acid α-glucosidase molecule can have M6P (mono-phosphorylation), a single N-glycan can have two M6P groups (bis-phosphorylation), or two different N-glycans in the same recombinant human acid α-glucosidase molecule can each have a single M6P group. A recombinant human acid α-glucosidase molecule can also have an N-glycan that does not have an M6P group. In another embodiment, the N-glycans comprise, on average, greater than 2.5 moles / mole of M6P and greater than 4 moles / mole of sialic acid, such that the human acid α-glucosidase comprises, on average, at least 2.5 moles of mannose-6-phosphate residues per mole of recombinant human acid α-glucosidase and at least 4 moles of sialic acid per mole of recombinant human acid α-glucosidase. On average, at least about 3, 4, 5, 6, 7, 8, 9, or 10% of the total glycans in the recombinant human acid α-glucosidase may be in the form of mono-M6P glycans, for example, about 6.25% of the total glycans may have a single M6P group, on average, at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total glycans in the recombinant human acid α-glucosidase are in the form of bis-M6P glycans, and on average, less than 25% of the total recombinant human acid α-glucosidase does not contain phosphorylated glycans that are bound to CIMPR.

[0063] The recombinant human acid α-glucosidase can have an average content of M6P-bearing N-glycans ranging from 0.5 to 7.0 moles / mole recombinant human acid α-glucosidase, or any intermediate value within a subrange including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 moles / mole recombinant human acid α-glucosidase. The recombinant human acid α-glucosidase can be fractionated to provide recombinant human acid α-glucosidase preparations with different average numbers of M6P-containing or bis-M6P-containing glycans, allowing for further customization of recombinant human acid α-glucosidase targeting to lysosomes in target tissues by selecting specific fractions or selectively combining different fractions.

[0064] Up to 60% of the N-glycans in the recombinant human acid α-glucosidase can be fully sialylated, for example, up to 10%, 20%, 30%, 40%, 50%, or 60% of the N-glycans can be fully sialylated. In some embodiments, 4-20% of the total N-glycans are fully sialylated. In other embodiments, no more than 5%, 10%, 20%, or 30% of the N-glycans in the recombinant human acid α-glucosidase have sialic acid and terminal galactose residues (Gal). This range includes all intermediate values ​​and subranges, for example, 7-30% of the total N-glycans in the recombinant human acid α-glucosidase can carry sialic acid and terminal galactose. In still other embodiments, no more than 5, 10, 15, 16, 17, 18, 19, or 20% of the N-glycans in the recombinant human acid α-glucosidase have only terminal galactose and no sialic acid. This range includes all intermediate values ​​and subranges, for example, 8-19% of the total N-glycans in the recombinant human acid α-glucosidase in the composition may have only terminal galactose and no sialic acid.

[0065] In other embodiments of the present invention, 40, 45, 50, 55-60% of the total N-glycans in the recombinant human acid α-glucosidase are complex N-glycans; or 1, 2, 3, 4, 5, 6, 7% or less of the total N-glycans in the recombinant human acid α-glucosidase are hybrid N-glycans; 5, 10, or 15% or less of the high-mannose N-glycans in the recombinant human acid α-glucosidase are non-phosphorylated; at least 5% or 10% of the high-mannose N-glycans in the recombinant human acid α-glucosidase are phosphorylated mono-M6P; and / or at least 1 or 2% of the high-mannose N-glycans in the recombinant human acid α-glucosidase are phosphorylated bis-M6P. These values ​​include all intermediate values ​​and subranges. The recombinant human acid α-glucosidase may satisfy one or more of the above content ranges.

[0066] In some embodiments, the recombinant human acid α-glucosidase has an average of 2.0 to 8.0 moles of sialic acid residues per mole of recombinant human acid α-glucosidase. This range includes all intermediate values ​​and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 moles of residues / mole of recombinant human acid α-glucosidase. Without being bound by theory, it is believed that the presence of N-glycan units with sialic acid residues may prevent nonproductive clearance of the recombinant human acid α-glucosidase by the asialoglycoprotein receptor.

[0067] In one or more embodiments, rhGAA has M6P and / or sialic acid units at certain N-glycosylation sites of recombinant human lysosomal proteins. For example, rhGAA has seven potential N-linked glycosylation sites. These potential glycosylation sites are located at the following positions in SEQ ID NO:5: N84, N177, N334, N414, N596, N826, and N869. Similarly, with respect to the full-length amino acid sequence of SEQ ID NO:1, these potential glycosylation sites are located at the following positions: N140, N233, N390, N470, N652, N882, and N925. Other variants of rhGAA may have similar glycosylation sites depending on the position of the asparagine residue. Generally, the sequence ASN-X-SER or ASN-X-THR in the protein amino acid sequence indicates a potential glycosylation site, with the exception that X cannot be HIS or PRO.

[0068] In various embodiments, rhGAA has a specific N-glycosylation profile. In one or more embodiments, at least 20% of rhGAA is phosphorylated at the first N-glycosylation site (e.g., N84 of SEQ ID NO: 5 and N140 of SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA can be phosphorylated at the first N-glycosylation site. This phosphorylation can be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have a mono-M6P unit at the first N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have a bis-M6P unit at the first N-glycosylation site.

[0069] In one or more embodiments, at least 20% of the rhGAA is phosphorylated at the second N-glycosylation site (e.g., N177 of SEQ ID NO: 5 and N223 of SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA can be phosphorylated at the second N-glycosylation site. This phosphorylation can be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have mono-M6P units at the second N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have bis-M6P units at the second N-glycosylation site. In one or more embodiments, at least 5% of the rhGAA is phosphorylated at the third N-glycosylation site (e.g., N334 of SEQ ID NO:5 and N390 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the third N-glycosylation site. For example, the third N-glycosylation site may have a mixture of non-phosphorylated high-mannose glycans, di-, tri-, and tetra-antennary complex glycans, and hybrid glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the rhGAA is sialylated at the third N-glycosylation site.

[0070] In one or more embodiments, at least 20% of the rhGAA is phosphorylated at the fourth N-glycosylation site (e.g., N414 of SEQ ID NO: 5 and N470 of SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA can be phosphorylated at the fourth N-glycosylation site. This phosphorylation can be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have a mono-M6P unit at the fourth N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have a bis-M6P unit at the fourth N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, or 25% of the rhGAA is sialylated at the fourth N-glycosylation site.

[0071] In one or more embodiments, at least 5% of the rhGAA is phosphorylated at the fifth N-glycosylation site (e.g., N596 of SEQ ID NO:5 and N692 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the fifth N-glycosylation site. For example, the fifth N-glycosylation site may have a fucosylated di-antennary complex glycan as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA is sialylated at the fifth N-glycosylation site.

[0072] In one or more embodiments, at least 5% of the rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 of SEQ ID NO:5 and N882 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site may have a mixture of di-, tri-, and tetra-antennary complex glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA is sialylated at the sixth N-glycosylation site.

[0073] In one or more embodiments, at least 5% of rhGAA is phosphorylated at the seventh N-glycosylation site (e.g., N869 of SEQ ID NO:5 and N925 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of rhGAA is phosphorylated at the seventh N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60%, or 65% of rhGAA has any glycan at the seventh N-glycosylation site. In some embodiments, at least 30%, 35%, or 40% of rhGAA has a glycan at the seventh N-glycosylation site.

[0074] Recombinant human acid α-glucosidase is preferably produced by Chinese hamster ovary (CHO) cells, such as the CHO cell lines GA-ATB-200 or ATB-200-001-X5-14, or by subcultures or derivatives of such CHO cell cultures. DNA constructs expressing allelic variants of acid α-glucosidase or other variant acid α-glucosidase amino acid sequences, such as those at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 5, can be constructed and expressed in CHO cells. These variant acid α-glucosidase amino acid sequences may contain deletions, substitutions, and / or insertions relative to SEQ ID NO: 1 or SEQ ID NO: 5, e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more deletions, substitutions, and / or insertions compared to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5. Those skilled in the art will be able to select alternative vectors suitable for transformation of CHO cells for the production of such DNA constructs.

[0075] Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, available as part of the GCG Sequence Analysis Package (University of Wisconsin, Madison, Wisconsin), and can be used, for example, with default settings. For example, polypeptides having at least 90%, 95%, 98%, or 99% identity to a specific polypeptide described herein, and polynucleotides encoding such polypeptides, preferably exhibiting substantially the same function, are contemplated. Unless otherwise indicated, similarity scores are based on the use of BLOSUM62. When BLASTP is used, the percentage similarity is based on the BLASTP positive score, and the percentage sequence identity is based on the BLASTP identity score. BLASTP "identity" indicates the number and percentage of total residues in a pair of identical high-scoring sequences; and BLASTP "positive" indicates the number and percentage of residues that have a positive alignment score and are similar to each other. Amino acid sequences with these degrees of identity or similarity, or any intermediate degree of similarity to the amino acid sequences disclosed herein, are contemplated and encompassed by this disclosure. The polynucleotide sequence of a similar polypeptide can be deduced using the genetic code and obtained by conventional means, in particular by back-translating the amino acid sequence using the genetic code.

[0076] The present inventors have discovered that recombinant human acid α-glucosidase with superior targeting ability to the cation-independent mannose-6-phosphate receptor (CIMPR) and cellular lysosomes, as well as a glycosylation pattern that reduces non-productive clearance in vivo, can be produced using Chinese hamster ovary (CHO) cells. These cells can be induced to express recombinant human acid α-glucosidase with significantly higher levels of N-glycan units with one or more mannose-6-phosphate residues than conventional recombinant human acid α-glucosidase products, such as alglucosidase alfa. The recombinant human acid α-glucosidase produced by these cells, as exemplified by ATB200, has significantly more muscle cell-targeting mannose-6-phosphate (mono-M6P) and bis-mannose-6-phosphate (bis-M6P) N-glycan residues than conventional acid α-glucosidases, such as Lumizyme®. Without being bound by theory, it is believed that this extensive glycosylation allows the ATB200 enzyme to be more effectively taken up by target cells and therefore more efficiently removed from the circulation than other recombinant human acid α-glucosidases, such as alglucosidase alfa, which has a much lower M6P and bis-M6P content. ATB200 has been shown to efficiently bind to CIMPR, be efficiently taken up by skeletal and cardiac muscle, and have a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces non-productive clearance in vivo.

[0077] It is also contemplated that the unique glycosylation of ATB200 may contribute to the reduced immunogenicity of ATB200, for example, compared to alglucosidase alfa. As will be understood by those skilled in the art, glycosylation of proteins with conserved mammalian sugars generally increases the solubility of the product and reduces its aggregation and immunogenicity. Glycosylation indirectly alters protein immunogenicity by minimizing protein aggregation and shielding protein immunogenic epitopes from the immune system (Guidance for Industry-Immunogenicity Assessment for Therapeutic Protein Products, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, August 2014). Thus, in at least one embodiment, administration of recombinant human acid α-glucosidase does not induce anti-drug antibodies. In at least one embodiment, administration of recombinant human acid alpha-glucosidase reduces the incidence of anti-drug antibodies in a subject below the level of anti-drug antibodies induced by administration of alglucosidase alfa.

[0078] As described in co-pending International Patent Application PCT / US2015 / 053252, cells such as CHO cells can be used to produce the rhGAA described therein, and this rhGAA can be used in the present invention. Examples of such CHO cell lines are GA-ATB-200 or ATB-200-001-X5-14, or subcultures thereof that produce the rhGAA compositions described therein. Such CHO cell lines can contain multiple copies of a gene, such as 5, 10, 15, 20, or more copies per polynucleotide encoding GAA.

[0079] High M6P and bis-M6P rhGAA, such as ATB200 rhGAA, is produced by transforming CHO cells with a DNA construct encoding GAA. Although CHO cells have previously been used to produce rhGAA, it was not observed that transformed CHO cells could be cultured and selected to produce rhGAA with a high content of M6P and bis-M6P glycans that target CIMPR.

[0080] Surprisingly, it has been found that it is possible to transform a CHO cell line, select transformants that produce rhGAA containing a high content of glycans with M6P or bis-M6P targeting CIMPR, and stably express this high-M6P rhGAA. Accordingly, a method for producing these CHO cell lines is also described in co-pending International Patent Application PCT / US2015 / 053252. This method includes transforming CHO cells with DNA encoding GAA or a GAA variant, stably integrating the DNA encoding GAA into their chromosomes, selecting CHO cells that stably express GAA, and selecting CHO cells that express GAA with a high content of glycans with M6P or bis-M6P, and optionally selecting CHO cells with N-glycans that have a high sialic acid content and / or a low non-phosphorylated high mannose content. In at least one embodiment, the GAA has a low level of complex glycans with terminal galactose.

[0081] These CHO cell lines can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell lines and recovering the compositions from the culture of the CHO cells.

[0082] Recombinant human acid α-glucosidase or a pharmaceutically acceptable salt thereof can be formulated according to conventional procedures as a pharmaceutical composition suitable for administration to humans. For example, in a preferred embodiment, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active ingredient. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing pharmaceutical-grade sterile water, saline, or dextrose / water. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0083] The recombinant human acid α-glucosidase (or a composition or medicament containing the recombinant human acid α-glucosidase) is administered by an appropriate route. In one embodiment, the recombinant human acid α-glucosidase is administered intravenously. In another embodiment, the recombinant human acid α-glucosidase is administered by direct administration to a target tissue, such as the heart or skeletal muscle (e.g., intramuscularly) or the nervous system (e.g., direct injection into the brain; intracerebroventricularly; intrathecally). If necessary, multiple routes can be used simultaneously.

[0084] Recombinant human acid α-glucosidase (or a composition or medicament containing recombinant human acid α-glucosidase) is administered in a therapeutically effective amount (e.g., a dosage sufficient, when administered regularly, to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease). The therapeutically effective amount in treating a disease will depend on the nature and extent of the disease's impact and can be determined by standard clinical techniques. Additionally, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose employed will also depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. In at least one embodiment, recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of about 5 mg / kg to about 30 mg / kg, typically about 5 mg / kg to about 20 mg / kg. In at least one embodiment, the recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg. In at least one embodiment, the recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of about 20 mg / kg. The effective dose for a particular individual can be varied (e.g., increased or decreased) over time depending on the individual's needs. For example, the amount can be increased during times of physical illness or stress, or when anti-acid α-glucosidase antibodies are present or increase, or when disease symptoms worsen.

[0085] A therapeutically effective amount of recombinant human acid α-glucosidase (or a composition or medicament containing recombinant human acid α-glucosidase) is administered periodically and continuously, depending on the nature and extent of the disease's impact. As used herein, "periodic" administration refers to a therapeutically effective amount being administered periodically (as distinguished from a single administration). The interval can be determined by standard clinical techniques. In preferred embodiments, recombinant human acid α-glucosidase is administered monthly, every other month, weekly, twice weekly, or daily. The administration interval for a single individual need not be a fixed interval and can vary over time depending on the individual's needs. For example, the administration interval can be reduced during times of physical illness or stress, when anti-recombinant human acid α-glucosidase antibodies are present or increase, or when disease symptoms worsen. In some embodiments, a therapeutically effective amount of 5, 10, 20, 50, 100, or 200 mg enzyme / kg body weight is administered twice weekly, weekly, or every other week, with or without a chaperone.

[0086] The recombinant human acid α-glucosidase of the invention may be prepared for later use, such as in unit-dose vials or syringes, or in bottles or bags for intravenous administration. Kits containing the recombinant human acid α-glucosidase and optional excipients or other active ingredients, such as chaperones or other drugs, may be enclosed in packaging material and accompanied by instructions for reconstitution, dilution, or administration to treat a subject in need thereof, such as a patient suffering from Pompe disease.

[0087] In at least one embodiment, miglustat and recombinant human acid α-glucosidase are administered simultaneously. In at least one embodiment, miglustat and recombinant human acid α-glucosidase are administered sequentially. In at least one embodiment, miglustat is administered before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 3 hours before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 2 hours before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 2 hours before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1.5 hours before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1 hour before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 50 to about 70 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 55 to about 65 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 30 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 25 to about 35 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 27 to about 33 minutes prior to administration of recombinant human acid α-glucosidase.

[0088] In at least one embodiment, miglustat is administered simultaneously with the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 20 minutes before or after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 15 minutes before or after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 10 minutes before or after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 5 minutes before or after the administration of recombinant human acid α-glucosidase.

[0089] In at least one embodiment, miglustat is administered after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered up to 2 hours after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 30 minutes after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1 hour after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1.5 hours after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 2 hours after administration of recombinant human acid α-glucosidase.

[0090] Another aspect of the present invention provides a kit for combination treatment of Pompe disease in a patient in need thereof. The kit includes a pharmaceutically acceptable dosage form comprising miglustat, a pharmaceutically acceptable dosage form comprising recombinant human acid α-glucosidase as defined herein, and instructions for administering the pharmaceutically acceptable dosage form comprising miglustat and the pharmaceutically acceptable dosage form comprising recombinant acid α-glucosidase to a patient in need thereof. In at least one embodiment, the pharmaceutically acceptable dosage form comprising miglustat is an oral dosage form described herein, including, but not limited to, a tablet or capsule. In at least one embodiment, the pharmaceutically acceptable dosage form comprising recombinant human acid α-glucosidase is a sterile solution suitable for injection as described herein. In at least one embodiment, the instructions for administering the dosage form include instructions for administering the pharmaceutically acceptable dosage form comprising miglustat before administering the pharmaceutically acceptable dosage form comprising recombinant human acid α-glucosidase by intravenous infusion, as described herein.

[0091] Without being bound by theory, miglustat is believed to act as a pharmacological chaperone for recombinant human acid α-glucosidase ATB200, binding to its active site. Thus, as shown in Figure 1, miglustat was found to reduce the proportion of unfolded ATB200 protein, stabilize the active conformation of ATB200, prevent denaturation and irreversible inactivation at the neutral pH of plasma, and allow ATB200 to survive in the circulation long enough to reach and be taken up by tissues. However, binding of miglustat to the active site of ATB200 may also inhibit the enzymatic activity of ATB200 by preventing its natural substrate, glycogen, from accessing the active site. When miglustat and recombinant human acid α-glucosidase are administered to a patient under the conditions described herein, the concentrations of miglustat and ATB200 in the plasma and tissues are stabilized until ATB200 is taken up into the tissue and targets the lysosomes, but because the clearance of miglustat is rapid, hydrolysis of glycogen by ATB200 in the lysosomes is not excessively inhibited by the presence of miglustat, and the enzyme is believed to retain sufficient activity to be therapeutically useful.

[0092] All of the above embodiments may be combined, including specific embodiments relating to: the properties of pharmacological chaperones, e.g., miglustat; and the active site for which it is specific; the dosage, the route of administration of the pharmacological chaperone (miglustat), and the type of pharmaceutical composition, including the nature of the carrier and the use of commercially available compositions; A therapeutic protein drug product, which may be of medicinal nature, e.g., a counterpart of an endogenous protein whose expression is reduced or absent in a subject, preferably a recombinant human acid alpha-glucosidase, e.g., a recombinant human acid alpha-glucosidase expressed in Chinese hamster ovary (CHO) cells and comprising an increased content of N-glycan units having one or more alpha mannose-6-phosphate residues when compared to the content of N-glycan units having one or more alpha mannose-6-phosphate residues of an alglucosidase; and preferably having the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 (or encoded by SEQ ID NO: 2), SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; the number and type of N-glycan units in the recombinant human acid α-glucosidase, for example, complex N-glycans formed from N-acetylglucosamine, galactose, sialic acid, or combinations thereof, attached to the recombinant human acid α-glucosidase; the degree of phosphorylation of mannose units in recombinant human acid α-glucosidase to form mannose-6-phosphate and / or bis-mannose-6-phosphate; the dosage and route of administration of the replacement enzyme (recombinant human acid α-glucosidase) (e.g., intravenous administration, particularly intravenous infusion, or direct administration to the target tissue) and the type of formulation, including the carrier and therapeutically effective amount; dosing intervals for pharmacological chaperones (miglustat) and recombinant human acid α-glucosidase; the nature of the treatment response and the outcome of the combined treatment (e.g., enhanced outcome compared with the effect of each treatment given individually); The timing of administration of the combination treatment, e.g., simultaneous or sequential administration of miglustat and recombinant human acid alpha-glucosidase, e.g., when miglustat is administered before, after, or within a certain time period before or after the administration of recombinant human acid alpha-glucosidase; and The nature of the patient to be treated (e.g., a mammal such as a human) and the condition from which the individual suffers (e.g., an enzyme deficiency).

[0093] Any embodiment in the above list can be combined with one or more of the other embodiments in the list. [Example]

[0094] Further features of the present invention will become apparent from the following non-limiting examples which illustrate, by way of example, the principles of the invention.

[0095] Example 1: Limitations of existing Myozyme® and Lumizyme® rhGAA products To evaluate the ability of rhGAA in Myozyme® and Lumizyme®, the only currently approved drugs for the treatment of Pompe disease, these rhGAA preparations were injected onto a CIMPR column (which binds rhGAA bearing M6P groups) and subsequently eluted with a free M6 gradient. Fractions were collected in a 96-well plate, and GAA activity was assayed with 4MU-α-glucose substrate. The relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as a percentage of total enzyme.

[0096] Figures 2A-B illustrate the problems associated with conventional ERT (Myozyme® and Lumizyme®): 73% of Myozyme® rhGAA (Figure 2B) and 78% of Lumizyme® rhGAA (Figure 2A) did not bind to CIMPR (see the leftmost peak in each figure). Only 27% of Myozyme® rhGAA and 22% of Lumizyme® rhGAA contained M6P, which can target CIMPR on muscle cells.

[0097] The effective doses of Myozyme® and Lumizyme® correspond to the amount of rhGAA containing M6P, which targets CIMPR on muscle cells. However, most of the rhGAA in these two conventional products does not target the CIMPR receptor on target muscle cells. Administration of conventional rhGAA, most of which is not targeted to muscle cells, increases the risk of allergic reactions or immune induction to the non-targeted rhGAA.

[0098] Example 2: Preparation of CHO cells producing ATB200 rhGAA with a high content of mono- or bis-M6P-containing N-glycans CHO cells were transfected with DNA that expresses rhGAA, followed by selection of transformants that produced rhGAA. The DNA construct for transforming CHO cells with DNA encoding rhGAA is shown in Figure 3. CHO cells were transfected with DNA that expresses rhGAA, followed by selection of transformants that produced rhGAA.

[0099] After transfection, DG44 CHO (DHFR-) cells containing a stably integrated GAA gene were selected in hypoxanthine / thymidine-deficient (-HT) medium. Amplification of GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). Cell pools expressing large amounts of GAA were identified by GAA enzyme activity assay and used to establish individual clones producing rhGAA. Individual clones were generated on semi-solid medium plates, picked using the ClonePix system, and transferred to 24-deep-well plates. Individual clones were assayed for GAA enzyme activity to identify clones expressing high levels of GAA. Conditioned medium for determining GAA activity was prepared using 4-MU-α-glucopyranoside α-glucosidase substrate. Clones producing higher levels of GAA, as measured by the GAA enzyme assay, were further evaluated for viability, growth ability, GAA productivity, N-glycan structure, and stable protein expression. CHO cell lines, including the CHO cell line GA-ATB-200, that express rhGAA with enhanced mono-M6P or bis-M6P N-glycans were isolated using this procedure.

[0100] Example 3: Capture and purification of ATB200 rhGAA Multiple batches of rhGAA according to the present invention were produced in shake flasks and perfusion bioreactors using the CHO cell line GA-ATB-200, and CIMPR binding was measured. For purified ATB200 rhGAA from different production batches, CIMPR receptor binding (approximately 70%) similar to that shown in Figures 4B and 5A was observed, indicating that ATB200 rhGAA can be consistently produced. As shown in Figures 2A, 2B, 4A, and 4B, Myozyme® and Lumizyme® rhGAA showed significantly lower CIMPR binding than ATB200 rhGAA.

[0101] Example 4: Analytical Comparison of ATB200 to Lumizyme® ATB200 rhGAA was fractionated by terminal phosphate using weak anion exchange ("WAX") liquid chromatography. Elution profiles were generated by eluting ERT with increasing amounts of salt. The profiles were monitored by UV (A280 nm). ATB200 rhGAA was obtained and purified from CHO cells. Lumizyme® was obtained from a commercial source. Lumizyme® exhibited a high peak on the left side of its elution profile. ATB200 rhGAA exhibited four prominent peaks eluting to the right of Lumizyme® (Figure 6). This confirms that ATB200 rhGAA was more phosphorylated than Lumizyme®, as this assessment is based on terminal charge rather than CIMPR affinity.

[0102] Example 5: Oligosaccharide characteristics of ATB200 rhGAA Purified ATB200 rhGAA and Lumizyme® glycans were evaluated by MALDI-TOF to determine the individual glycan structures found on each ERT (Figure 7). The ATB200 sample was found to contain less non-phosphorylated high-mannose N-glycans than Lumizyme®. The higher M6P glycan content of ATB200 compared with Lumizyme® suggests that ATB200 rhGAA targets muscle cells more effectively. The high percentage of mono- and bis-phosphorylated structures determined by MALDI is consistent with the CIMPR profile, which indicated significantly greater binding of ATB200 to the CIMPR receptor. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule contained at least one natural bis-M6P N-glycan structure. The higher content of this bis-M6P N-glycan on ATB200 rhGAA directly correlated with high affinity binding to CIMPR in the M6P receptor plate binding assay (KD ∼2–4 nM), Figure 9A.

[0103] ATB200 rhGAA was also analyzed for site-specific N-glycan profiles using two different LC-MS / MS analytical techniques. In the first analysis, the protein was denatured, reduced, alkylated, and digested prior to LC-MS / MS analysis. During protein denaturation and reduction, 200 μg of protein sample, 5 μl of 1 mol / L Tris-HCl (final concentration 50 mM), 75 μL of 8 mol / L guanidine HCl (final concentration 6 M), 1 μL of 0.5 mol / L EDTA (final concentration 5 mM), 2 μL of 1 mol / L DTT (final concentration 20 mM), and Milli-Q® water were added to a 1.5 mL tube to provide a total volume of 100 μL. The sample was mixed and incubated at 56°C in a dry bath for 30 minutes. During alkylation, the denatured and reduced protein sample was mixed with 5 μL of 1 mol / L iodoacetamide (IAM, final concentration 50 mM) and then incubated in the dark for 30 minutes. After alkylation, 400 μL of pre-chilled acetone was added to the sample, and the mixture was frozen at -80°C for 4 hours. The sample was then centrifuged at 13,000 rpm for 5 minutes at 4°C, and the supernatant was removed. 400 μL of pre-chilled acetone was added to the pellet, which was then centrifuged at 13,000 rpm for 5 minutes at 4°C, and the supernatant was removed. The sample was air-dried on ice in the dark to remove acetone residue. 40 μL of 8 M urea and 160 μL of 100 μM NH4HCO3 were added to the sample to dissolve the protein. During trypsin digestion, 50 μg of protein was added to trypsin digestion buffer to a final volume of 100 μl, and 5 μL of 0.5 μg / mL trypsin (protein-to-enzyme ratio 20 / 1 w / w) was added. The solution was mixed well and incubated at 37°C overnight (16 ± 2 hours). The reaction was stopped by adding 2.5 μl of 20% TFA (final concentration 0.5%). Samples were then analyzed using a Thermo Scientific Orbitrap Velos Pro™ mass spectrometer.

[0104] In a second LC-MS / MS analysis, ATB200 samples were prepared following the same denaturation, reduction, alkylation, and digestion procedures, except that iodoacetic acid (IAA) was used as the alkylating reagent instead of IAM, and analyzed using a Thermo Scientific Orbitrap Fusion Lumos Tribid™ mass spectrometer.

[0105] For the third LC-MS / MS analysis, ATB200 samples were prepared following a similar denaturation, reduction, alkylation, and digestion procedure using iodoacetamide (IAM) as the alkylating reagent and analyzed using a Thermo Scientific Orbitrap Fusion mass spectrometer.

[0106] The results of the first and second analyses are shown in Figures 8B-8H, and the results of the third analysis are shown in Figure 8A. In Figures 8B-8H, the results of the first analysis are represented by the left bar (dark gray), and the results of the second analysis are represented by the right bar (light gray). In Figures 8B-8H, the glycan nomenclature follows Varki, A., Cummings, R.D., Esko, J.D., et al., Essentials of Glycobiology, 2nd edition (2009). In Figures 8A-8H, glycosylation sites are assigned for SEQ ID NO:5: N84, N177, N334, N414, N596, N826, and N869. With respect to the full-length amino acid sequence of SEQ ID NO:1, these potential glycosylation sites exist at the following positions: N140, N233, N390, N470, N652, N882, and N925.

[0107] As can be seen from Figures 8B–8H, the first two analyses yielded similar results, but there were some discrepancies between the results. This variation can be attributed to many factors, including the instrumentation used and the completeness of the N-glycan analysis. For example, if some species of phosphorylated glycans are not identified and / or quantified, the total number of phosphorylated glycans may be underestimated, and the percentage of rhGAA with phosphorylated glycans at that site may be underestimated. As another example, if some species of non-phosphorylated glycans are not identified and / or quantified, the total number of non-phosphorylated glycans may be overestimated, and the percentage of rhGAA with phosphorylated glycans at that site may be overestimated.

[0108] Figure 8A shows the N-glycosylation site occupancy of ATB200. As can be seen from Figure 8A, the first, second, third, fourth, fifth, and sixth N-glycosylation sites are mostly occupied by approximately 90% to a maximum of about 100% of the ATB200 enzymes, with glycans detected at each potential site. However, the seventh potential N-glycosylation site is glycosylated about half the time.

[0109] Figure 8B shows the N-glycosylation profile of the first site, N84. As can be seen from Figure 8B, the major glycan species is bis-M6P glycan. In both the first and second analyses, it was detected that 75% or more of ATB200 had bis-M6P glycan at the first site.

[0110] Figure 8C shows the N-glycosylation profile of the second site, N177. As can be seen from Figure 8C, the major glycan species are mono-M6P glycan and non-phosphorylated high-mannose glycan. In both the first and second analyses, it was detected that more than 40% of ATB200 had mono-M6P glycan at the second site.

[0111] Figure 8D shows the N-glycosylation profile of the third site, N334. As can be seen from Figure 8D, the major glycan species are non-phosphorylated high-mannose glycans, di-, tri-, and tetra-antennary complex glycans, and hybrid glycans. In both the first and second analyses, it was detected that more than 20% of ATB200 had a sialic acid residue at the third site.

[0112] Figure 8E shows the N-glycosylation profile of the fourth site, N414. As can be seen from Figure 8E, the major glycan species are bis-M6P and mono-M6P glycans. In both the first and second analyses, it was detected that 40% or more of ATB200 had bis-M6P glycan at the fourth site. In both the first and second analyses, it was also detected that 25% or more of ATB200 had mono-M6P glycan at the fourth site.

[0113] Figure 8F shows the N-glycosylation profile of the fifth site, N596. As can be seen from Figure 8F, the major glycan species is a fucosylated di-antennary complex glycan. In both the first and second analyses, it was detected that more than 70% of ATB200 had a sialic acid residue at the fifth site.

[0114] Figure 8G shows the N-glycosylation profile of the sixth site, N826. As can be seen from Figure 8F, the major glycan species are di-, tri-, and tetra-antennary complex glycans. In both the first and second analyses, it was detected that more than 80% of ATB200 had a sialic acid residue at the sixth site.

[0115] Figure 8H shows a summary of phosphorylation at each of the first six potential N-glycosylation sites. As can be seen from Figure 8H, both the first and second analyses detected high phosphorylation levels at the first, second, and fourth sites. Both analyses detected that 80% or more of ATB200 was mono- or di-phosphorylated at the first site, 40% or more of ATB200 was mono-phosphorylated at the second site, and 80% or more of ATB200 was mono- or di-phosphorylated at the fourth site.

[0116] Example 6: CIMPR affinity characteristics of ATB200 In addition to the higher percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of that interaction. Lumizyme® and ATB200 rhGAA receptor binding was measured using a CIMPR plate binding assay. Briefly, GAA was captured using a CIMPR-coated plate. Various concentrations of rhGAA were applied to the immobilized receptor, and unbound rhGAA was washed away. The amount of remaining rhGAA was determined by GAA activity. As shown in Figure 9A, ATB200 rhGAA bound to CIMPR significantly better than Lumizyme®.

[0117] 9B shows the relative content of bis-M6P glycans in Lumizyme®, conventional rhGAA, and ATB200 according to the present invention. In Lumizyme®, on average, only 10% of the molecules have bis-phosphorylated glycans. This is in contrast to ATB200, in which on average, all rhGAA molecules have at least one bis-phosphorylated glycan.

[0118] Example 7: ATB200 rhGAA was internalized more efficiently by fibroblasts than Lumizyme® The relative cellular uptake of ATB200 and Lumizyme® rhGAA was compared using normal and Pompe fibroblast cell lines. Comparisons included 5-100 nM ATB200 rhGAA according to the present invention compared with 10-500 nM conventional rhGAA Lumizyme®. After 16 hours of incubation, external rhGAA was inactivated with TRIS base, and cells were washed three times with PBS before harvesting. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and graphed against total cellular protein; the results are presented in Figure 10A-B.

[0119] ATB200 rhGAA was also shown to be efficiently internalized into cells (Figures 10A and 10B), demonstrating that ATB200 rhGAA was internalized into both normal and Pompe fibroblasts, demonstrating a higher degree of internalization than conventional Lumizyme® rhGAA. ATB200 rhGAA saturates cell receptors at approximately 20 nM, whereas Lumizyme® requires approximately 250 nM. The uptake efficiency constant (K) extrapolated from these results was 取り込み ) is 2-3 nM for ATB200 and 56 nM for Lumizyme®, as shown in Figure 10C. These results suggest that ATB200 rhGAA is a well-targeted treatment for Pompe disease.

[0120] Example 8: Population Pharmacokinetic (PK) Model of ATB200 and Miglustat Pharmacokinetic data for acid α-glucosidase (ATB200), including sampling times, dosing history, and plasma concentrations of acid α-glucosidase, are obtained from mice, rats, and monkeys administered ATB200 intravenously. Pharmacokinetic data for miglustat and duvoglustat in plasma and tissues are collected from humans or mice.

[0121] Modeling and simulations are performed using Phoenix® NLME™ v1.3. A compartmental PK model is constructed to evaluate the PK of ATB200 in plasma. The model includes: • Describe the relationship between plasma concentration and time; • Variance components characterizing the inter- and intra-animal variability in model parameters; and • Factors that account for uncertainty in the state of knowledge regarding key model elements.

[0122] The formula for the nonlinear mixed effects (NLME) model is: C pj =C(D i ,t j ,θ i )+ε ij θ i =(θ i1 ,…,θ im ) where C pij is the concentration at the jth collection in animal i, and D i represents the medication history for animal i, and θ i is the vector of PK parameters in animal i, and ε ij is the random error associated with the jth concentration in animal i.

[0123] The between-subject variability (BSV) in the parameters is modeled as a log-normal distribution: θ in =θ TVn exp(η in ) (η1,…,ηM)~MVN(0,Ω) where θ TVn is the population standard value for the nth PK parameter (e.g., clearance), and η in is the random between-animal effect in the nth parameter for animal i. The random effects (η1,…η m ) is the OMEGA(Ω) matrix with mean 0 and estimated variance ω 2 was normally distributed.

[0124] PK is assumed to be species independent and is scaled according to a generalized Dedrick approach that scales disposition according to the force of the animal's body weight: CL (p)i=a (p) BW i b V (p)i =c (p) BW i d where CL is total body clearance, V is volume of distribution, BW is body weight, p is peripheral, b and d are allometric indices, and a and c are typical values ​​for BW=1. In this scenario, the indices b and d can be compared to more generalized values ​​found in the literature (b=0.75 and d=1.0). Nominal BWs (0.025, 0.25, and 2.5 kg) are used in the analysis.

[0125] C ベースライン is species-specific and independent of ATB200 concentration, and is known in humans for Pompe disease, so baseline acid α-glucosidase concentrations are important for C ベースライン = rate of acid α-glucosidase synthesis / CL, which can be extrapolated to humans. To assess whether a one- or two-compartment model best fits the data, a base model is determined using Phoenix® FOCE-ELS. The sources of PK variability for acid α-glucosidase are also explored visually and by examining the influence of various wild-type / species / dose-related effects on PK.

[0126] For ATB200, a two-compartment model with linear elimination adequately characterized the concentration-time profile of acid α-glucosidase activity across all dose levels across animal species. The model includes theoretical allometric components to account for differences in body weight across animal species with respect to clearance (CL) and volume of distribution (Vc). The goodness-of-fit of the population PK model for ATB200 is shown in Figure 11. Population PK parameters for ATB200 in nonclinical studies are listed in Table 1.

[0127] JPEG2025163019000011.jpg82154

[0128] The concentration-time profiles of miglustat (200 mg) in patients with Pompe disease are compared with those obtained after administration of duvoglustat in normal, healthy volunteers (dose range: 50, 100, 250, 600, and 1000 mg). Dose-normalized plasma concentration-time profiles of miglustat and duvoglustat are shown in Figure 12. Because the concentration-time profiles of miglustat in patients with Pompe disease are similar to those observed over 24 hours after administration of duvoglustat in healthy subjects, PK data collected for duvoglustat in peripheral tissues were used as a surrogate for the exposure model to miglustat. A two-compartment model with linear elimination was used to characterize the concentration-time profiles of duvoglustat in tissues.

[0129] The goodness of fit of the PK model for duvoglustat is shown in Figures 13A and 13B. The final model PK parameters for duvoglustat in plasma and tissues are shown in Table 2.

[0130] JPEG2025163019000012.jpg96154

[0131] A population PK model of miglustat is constructed based on oral administration in Gaa knockout (KO) mice. The population PK parameters of miglustat in GaaKO mice are shown in Table 3. The goodness of fit is shown in Figure 14. The model has a residual additive error of 0.475 ng / mL.

[0132] JPEG2025163019000013.jpg59154

[0133] Example 9: Modeling of recombinant acid α-glucosidase (ATB200) pharmacokinetic (PK) parameters in humans Simulation was carried out using a pharmacokinetic model (Example 8) to predict the concentration-time profile of acid α-glucosidase in subjects with late-stage Pompe disease after administration of ATB200.The allometric function allows the correlation between body weight and clearance and volume of distribution, thus allowing the prediction of PK parameters in a typical human subject weighing 70 kg.This model is customized by including the endogenous synthesis rate of acid α-glucosidase in humans (Umapathysivam K, Hopwood JJ, Meikle PJ.Determination of acid α-glucosidase activity in blood spots as a diagnostic test for Pompe disease.Clin Chem.(2001)Aug;47(8):1378-83).

[0134] A single 20 mg / kg IV dose of ATB200 infused over 4 hours in humans is predicted to produce the concentration-time profile shown in Figure 15. The PK parameters in a typical 70 kg human and the resulting exposure parameters after IV infusion of 20 mg / kg of ATB200 over 4 hours are shown in Table 4.

[0135] JPEG2025163019000014.jpg63154

[0136] The predicted systemic clearance (CL) and volume of distribution (V) of ATB200 in a typical 70 kg patient are 0.768 L / h and 2.41 L, respectively.

[0137] According to the product label for Lumizyme® (alglucosidase alfa), the systemic clearance of acid α-glucosidase is 601 mL / h (0.601 L / h) at 52 weeks after repeated administration of Lumizyme® in patients with late-stage Pompe disease, and the half-life of Lumizyme® is 2.4 hours. Based on the above model, the systemic clearance of ATB200 in adult subjects with Pompe disease is predicted to be approximately 28% faster than that reported for Lumizyme®. Furthermore, the predicted AUC in humans after a 20 mg / kg dose of ATB200 is approximately 25% lower (AUC ) than the AUC reported after a 20 mg / kg dose of Lumizyme® (approximately 2700 μg h / mL). 0-inf :1822 mg·h / L).

[0138] Example 10: Exposure-Response Model of Glycogen Depletion Gaa knockout mice were administered acid α-glucosidase (ATB200) intravenously at doses of 5, 10, or 20 mg / kg and escalating oral doses of miglustat (1, 3, and 10 mg / kg) along with ATB200 at a dose of 5 or 10 mg / kg intravenous, or escalating oral doses of miglustat (1, 3, 5, 10, 20, and 30 mg / kg) along with ATB200 at a dose of 20 mg / kg intravenous. Glycogen levels were measured as previously described (Khanna, R, Flanagan, JJ, Feng, J, Soska, R, Frascella, M, Pellegrino, LJ et al. (2012). "The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease." PLoS One 7(7):e40776). The ratio of glycogen levels observed after each combination treatment to those observed after monotherapy (glycogen ratio) was calculated. Results are shown in Table 5.

[0139] JPEG2025163019000015.jpg187154

[0140] Additionally, Figures 15A-15C show the effects of administration of alglucosidase alfa (Lumizyme®) and ATB200 in Gaa knockout mice. Animals receive two IV bolus doses (biweekly). Two weeks after the final dose, tissues are collected and analyzed for acid α-glucosidase activity and glycogen content.

[0141] As can be seen from the results in Table 5, ATB200 was found to dose-dependently deplete tissue glycogen in acid α-glucosidase (Gaa) knockout mice. A 20 mg / kg dose of ATB200 consistently removed glycogen stores at a greater rate than the 5 mg / kg and 10 mg / kg dose levels in Gaa knockout mice. However, as seen in Figures 15A-15C, ATB200 administered at 5 mg / kg showed a similar reduction in glycogen in mouse heart and skeletal muscle (quadriceps and triceps) as Lumizyme® administered at 20 mg / kg, while ATB200 administered at 10 and 20 mg / kg showed a significantly better reduction in glycogen levels in skeletal muscle than Lumizyme®.

[0142] Furthermore, miglustat at 10 and 20 mg / kg doses coadministered with ATB200 at 20 mg / kg reduced glycogen levels in Gaa knockout mice to 118 and 122 μg / mg protein, respectively. Miglustat administered at 30 mg / kg resulted in a smaller decrease in glycogen. Without being bound by theory, it is believed that at higher concentrations of miglustat, inhibition of acid α-glucosidase in lysosomes may exceed the beneficial chaperone effect, thereby reducing glycogen degradation in lysosomes.

[0143] A pharmacokinetic model (Example 8) was used to predict exposure to acid α-glucosidase and miglustat timed to the tissue lysosomal glycogen level values ​​in Table 5. The steady-state exposure (AUC) ratio of miglustat / ATB200 (mean exposure 24 hours) was derived for each treatment combination tested, plotted against the corresponding glycogen ratio (Table 5), and fitted to a mathematical function. The exposure-response curves are shown in Figure 17.

[0144] As can be seen from the results in Figure 17, co-administration of 10 mg / kg and 20 mg / kg doses of miglustat with a 20 mg / kg dose of ATB200 maximizes glycogen depletion while providing good stability of plasma acid α-glucosidase activity. Lower doses of miglustat (1, 3, and 5 mg / kg) appear to result in suboptimal stabilization of acid α-glucosidase activity, while the highest dose of miglustat (30 mg / kg) appears to result in excessive inhibition of lysosomal α-glucosidase activity.

[0145] Based on the pharmacokinetic model (Example 8), the observed miglustat / ATB200 AUC ratio of 0.01159 (10 mg / kg miglustat co-administered with 20 mg / kg ATB200) is predicted to correspond to a miglustat dose of approximately 270 mg co-administered with 20 mg / kg ATB200 in a typical 70 kg human. AUC ratios of 0.01 and 0.02 would correspond to miglustat doses of 233 mg and 466 mg, respectively, co-administered with 20 mg / kg ATB200 in a typical 70 kg subject.

[0146] Example 11: Modeling miglustat / duvoglustat concentrations in humans Using a pharmacokinetic model (Example 8), plasma or tissue concentrations of duvoglustat (a surrogate for miglustat) were calculated based on the IC50 of miglustat in plasma and lysosomes. 50The IC value of miglustat at plasma pH (pH 7.0) was calculated based on the duration of time it remained above the threshold (the concentration that provided 50% of maximal inhibition of acid α-glucosidase activity). Inhibition of acid α-glucosidase activity was measured by a previously described method (Flanagan JJ, Rossi B, Tang K, Wu X, Mascioli K, et al. (2009) "The pharmacological chaperone 1-deoxynojirimycin increases the activity and lysosomal trafficking of multiple mutant forms of acid α-glucosidase." Hum Mutat 30:1683-1692). 50 The IC value was measured to be 170 μg / L, which is the IC at the pH of the lysosomal compartment (pH 5.2). 50 The value was measured as 377 μg / L.

[0147] The results of the model predictions are shown in Table 6. The predicted concentration-time profiles of miglustat in plasma and lysosomes after repeated dosing are shown in Figures 17 and 18, respectively.

[0148] JPEG2025163019000016.jpg75154

[0149] Based on the results shown in Table 6 and Figures 17 and 18, a 260 mg dose of miglustat is expected to bind and stabilize ATB200 in plasma for up to 18 hours, but inhibition of acid α-glucosidase activity in lysosomes is expected to last only 4 hours.

[0150] Example 12: Muscle physiology and morphology in Gaa knockout mice Gaa knockout mice are given two IV bolus doses of 20 mg / kg recombinant human acid α-glucosidase (alglucosidase alfa or ATB200) every other week. Miglustat is administered orally at a dose of 10 mg / kg to a subset of ATB200-treated animals 30 minutes prior to the ATB200 administration. Control mice are treated with vehicle alone. Soleus, quadriceps, and diaphragm tissues are collected 2 weeks after the last administration of recombinant human acid α-glucosidase. Soleus and diaphragm tissues are analyzed for glycogen levels by staining with periodic acid-Schiff reagent (PAS) and for lysosomal proliferation by measuring levels of the lysosome-associated membrane protein 1 (LAMP1) marker upregulated in Pompe disease. Semi-thin sections of quadriceps muscle embedded in epoxy resin (Epon) were stained with methylene blue and examined under an electron microscope (1000x) to determine the presence of vacuoles. Quadriceps muscle samples were analyzed immunohistochemically to determine the levels of autophagy markers microtubule-associated protein 1A / 1B type light chain 3 phosphatidylethanolamine conjugate (LC3A II) and p62, insulin-dependent glucose transporter GLUT4, and insulin-independent glucose transporter GLUT1.

[0151] In a similar experiment, Gaa knockout mice were given four IV bolus doses of 20 mg / kg recombinant human acid α-glucosidase (alglucosidase alfa or ATB200) every other week. Miglustat was administered orally at a dose of 10 mg / kg to a subset of ATB200-treated animals 30 minutes prior to the ATB200 dose. Control mice were treated with vehicle alone. Cardiac muscle tissue was collected 2 weeks after the last dose of recombinant human acid α-glucosidase and analyzed for glycogen levels by staining with periodic acid-Schiff reagent (PAS) and for lysosomal proliferation by measuring levels of LAMP1.

[0152] As can be seen in Figure 20, administration of ATB200 showed a decrease in lysosomal proliferation in cardiac and skeletal muscle (soleus) tissue compared to conventional treatment with alglucosidase alfa, and co-administration of miglustat with ATB200 showed a decrease in lysosomal proliferation approaching the levels seen in wild-type (WT) mice. Furthermore, as can be seen in Figure 21, administration of ATB200 showed a decrease in punctate glycogen levels in cardiac and skeletal muscle (soleus) tissue compared to conventional treatment with alglucosidase alfa, and co-administration of miglustat with ATB200 again showed a decrease approaching the levels seen in wild-type (WT) mice.

[0153] Similarly, as seen in Figure 22, coadministration of miglustat with ATB200 significantly reduced the number of vacuoles in muscle fibers in the quadriceps muscles of Gaa knockout mice compared with untreated mice and mice treated with alglucosidase alfa. As seen in Figure 23, the levels of both LC3II and p62 were increased in Gaa knockout mice compared with wild-type mice but were significantly reduced by treatment with ATB200 and miglustat, suggesting that the increased autophagy associated with acid α-glucosidase deficiency was reduced by coadministration of ATB200 and miglustat. Furthermore, the levels of the insulin-dependent glucose transporter GLUT4 and the insulin-independent glucose transporter GLUT1 were increased in Gaa knockout mice compared with wild-type mice but were again significantly reduced by treatment with ATB200 and miglustat. Elevated GLUT4 and GLUT1 levels associated with acid α-glucosidase deficiency may contribute to increased glucose uptake into muscle fibers, leading to increased glycogen synthesis both basally and after food ingestion. Accordingly, combined treatment with ATB200 and miglustat was found to improve skeletal muscle morphology and physiology in a mouse model of Pompe disease.

[0154] Example 13: Toxicity of ATB200 co-administered with miglustat in cynomolgus monkeys Naive cynomolgus monkeys from Cambodia were assigned to the dose groups shown in Table 7. The animals were acclimated to the laboratory for 18 days (females) to 19 days (males). On the last day of acclimation, the animals weighed between 2.243 kg and 5.413 kg and were 2 to 3 years old.

[0155] JPEG2025163019000017.jpg84154

[0156] The test dose levels were selected based on previous experiments in non-human primates to provide exposures (AUC) that are comparable to, or slightly higher (for the 25 mg / kg miglustat and 50 mg / kg ATB200 groups), or approximately 10-fold and 3-fold higher (for the 175 mg / kg miglustat and / or 100 mg / kg ATB200 groups) than the clinical AUCs expected in humans administered doses of 260 mg miglustat and 20 mg / kg ATB200 (approximately 20.9 hr μg / mL and approximately 1822 hr μg / mL, respectively), as predicted by the pharmacokinetic model of Example 8. In a previous study in non-human primates, an IV dose of 100 mg / kg ATB200 was found to produce an AUC of 5330 hr·μg / mL, and an oral dose of 175 mg / kg miglustat extrapolated to produce an AUC of 196 hr·μg / mL.

[0157] ATB200 was formulated in 25 mM sodium phosphate buffer, pH 6, containing 2.92 mg / mL sodium chloride, 20 mg / mL mannitol, and 0.5 mg / mL polysorbate 80 (formulation buffer). The test substance (ATB200 or miglustat) and control substance / vehicle (formulation buffer) were administered once every other week for 13 weeks, starting on Day 1 and ending on Day 85. ATB200 and control substance / vehicle were administered by 2-hour (±10-minute) intravenous (IV) infusion at 0 mg / kg (Group 1, control substance), 50 mg / kg (Group 2), or 100 mg / kg (Groups 3 and 5). When administered in combination, miglustat was administered intranasally in sterile water for injection (USP) at 25 mg / kg (Group 2) or 175 mg / kg (Groups 3 and 4) 30 minutes (±2 minutes) before the start of the ATB200 infusion. The dose in all groups was 10 mL / kg.

[0158] Parameters evaluated during the in-life phase of the experiment included body weight, food intake, clinical observations, detailed clinical observations, physical examination, electrocardiogram, ophthalmological evaluation, clinical pathology (hematology, coagulation, serum chemistry), anti-drug antibody (ADA) assessment, neutralizing ADA assessment, urinalysis, and plasma toxicokinetics (TK) of miglustat and ATB200 activity and total protein. Final necropsies of the animals were performed on day 99 (14 days after the last dose administration). At necropsy, gross observations and organ weights were recorded, and tissues were collected for microscopic examination.

[0159] All animals survived until scheduled euthanasia, and there were no changes attributable to ATB200, miglustat administration, or coadministration of ATB200 and miglustat during physical examination or during evaluation of food intake, clinical observations, detailed clinical observations, body weight, ophthalmology, or ECG parameters. Furthermore, there were no ATB200-, miglustat-, or ATB200 / miglustat-related changes in urinalysis, serum chemistry, hematology, or coagulation parameters, or during evaluation of gross observations, organ weights, or histopathology.

[0160] Total anti-drug antibodies (ADAs) and neutralizing antibodies (NAbs) Total anti-drug antibody (ADA) and neutralizing antibody (NAb) levels were measured in plasma. Blood samples (approximately 1.6 mL) were collected in K2EDTA tubes from all animals during acclimation, pre-dose (before miglustat administration), and on days 1, 85, and 99. Samples were kept on wet ice until processing. Plasma was obtained by centrifugation at 2°C to 8°C, and aliquots (approximately 0.2 mL) were transferred to polypropylene vials and stored frozen at -60°C to -86°C within 1 hour of blood collection. Samples were analyzed for ADA in samples collected from animals in groups 1, 2, 3, and 5 (miglustat-only samples were not analyzed). Neutralizing antibody analysis was performed using an enzymatic assay with the fluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4MU-Glc).

[0161] All animals in the ATB200-treated groups (Groups 2, 3, and 5) tested positive for anti-drug antibodies (ADA) on Days 85 and 99 (100% incidence). Titers ranged from 25,600 to 409,600 on Day 85 and from 51,200 to 819,200 on Day 99. There was no clear trend for titers to increase with increasing ATB200 dose level. In Group 2 (50 mg / kg ATB200 in combination with 25 mg / kg miglustat), five of eight animals tested positive for neutralizing antibodies (NAb) on Days 85 and 99. In Group 3 (100 mg / kg ATB200 in combination with 175 mg / kg miglustat), two of eight animals tested positive for NAb on Day 85 and four of eight animals tested positive on Day 99. In group 5 (100 mg / kg ATB200 monotherapy), 2 of 8 mice were NAb positive on day 85 and 3 of 8 were positive on day 99. There was no apparent effect of ADA on ATB200 exposure or other TK parameters.

[0162] ATB200 toxicokinetics The toxicokinetics of ATB200 was measured in blood samples collected in K2EDTA tubes from animals on days 1 and 85 at the following time points: For Groups 1, 2, 3, and 5: predose (before miglustat administration); 1 hour from start of infusion; 2 hours from start of infusion; 2.5 hours from start of infusion; 3 hours from start of infusion; 4 hours from start of infusion; 6 hours from start of infusion; 12 hours from start of infusion; 26 hours from start of infusion; 168 hours from start of infusion; 336 hours from start of infusion (collected before dosing on day 15); and For Group 4: Pre-dose (before administration of miglustat); 1.5 hours after administration of miglustat; 2.5 hours after administration of miglustat; 3.5 hours after administration of miglustat; 4.5 hours after administration of miglustat; 6.5 hours after administration of miglustat; 12.5 hours after administration of miglustat; 26.5 hours after administration of miglustat; 168.5 hours after administration of miglustat; 336.5 hours after administration of miglustat (collected before administration on day 15).

[0163] Plasma was obtained by centrifugation at 2°C to 8°C, and aliquots (approximately 0.1 mL) were transferred to polypropylene vials and stored frozen at -60°C to -86°C. Analysis of ATB200 acid α-glucosidase activity and total ATB200 protein was performed on 2-hour postdose samples from animals in Group 1 and on all samples collected from animals in Groups 2, 3, and 5. Total ATB200 protein was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS). Two characteristic peptides (TTPTFFPK and VTSEGAGLQLQK) were used as measures of ATB200. Results from these two peptides were consistent, indicating the presence of intact ATB200 in the analyzed plasma samples. Acid α-glucosidase activity was assayed using the fluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4MU-Glc).

[0164] Toxicokinetic (TK) data analysis was performed on the audit / validation datasets (concentration and time) from animals in groups 2, 3, and 5 using WinNonlin Phoenix, version 6.1 software (Pharsight Corporation). Individual subject plasma non-compartmental analysis concentration data were used to estimate TK parameters for acid α-glucosidase activity and ATB200 total protein (based on two signature peptides, TTPTFFPK and VTSEGAGLQLQK) after IV infusion. Dose levels were entered as actual ATB200 doses (mg) calculated based on the dose, body weight, and mean dose concentration for each individual animal. The start time of each dose (beginning of ATB200 infusion) was set to zero for all profiles in the dosing regimen. Nominal sample collection times were used for all analyses. Areas under the plasma concentration-time curve (AUC) were generated for ATB200 (total protein and activity assay datasets). 0-t ) was estimated by the log-linear trapezoidal rule. The regression used to estimate λz was based on uniformly weighted concentration data.

[0165] The following parameters were calculated for each ATB200 dataset (generated from the two signature peptides in the total ATB200 assay and the ATB200 activity assay): ●R 2 -λ zα The square of the correlation coefficient of the linear regression used to estimate , used when a set number of points is used to define the end stage (or specific time range) of the concentration versus time profile; ●R 2 adj-λ zβ λ, adjusted for the number of points used to estimate z The square of the correlation coefficient of the linear regression used to estimate . Used when the number of points used to define the terminal phase of the concentration versus time profile can be variable; λ z Number of points - λ z The number of linear regression analysis points used to estimate ; λ zα -t 最大値 the elimination rate constants at the first three subsequent time points; λ zβ -terminal discharge rate constant; ●t 1 / 2α -t 最大値 Half-lives based on the first three time points thereafter; ●t 1 / 2β -λ z Terminal elimination half-life (0.693 / λ z ); ●t 最大値 - time of maximum concentration of the analyte in plasma; ●C 最大値 - Maximum observed concentration of the analyte in plasma; AUC 0-t - Area under the plasma concentration-time curve (AUC) measured from time 0 (pre-dose) to the time point with the last measurable concentration; AUC 0-∞ -AUC extrapolated to infinite time; AUC ext - The part of the AUC extrapolated to time infinity is the total AUC 0-∞ Expressed as % of; ●CL T -Total clearance (λ zβ Based on total dose (mg) from actual body weight; ●CL T / F-total clearance (λ zβ Based on total dose (mg) from actual body weight divided by bioavailable fraction; ●V ss - apparent volume of distribution at equilibrium; ●V z -Based on the final stage (λ zβ Based on the volume of distribution; based on the total dose (mg) from actual body weight; ●V z / F-based on the final stage (λ zβ volume of distribution (based on ); based on total dose (mg) from actual body weight divided by bioavailable fraction; and ●Accumulation rate-AR C最大値 = C on day 1 of 85 最大値Ratio of ;AR AUC = C on day 1 of 85 0-t Ratio of.

[0166] ATB200 concentrations and TK parameters were similar in males and females. Plasma concentrations following a 2-hour IV ATB200 infusion of 50 mg / kg in combination with 25 mg / kg miglustat were measurable from 12 to 26 hours post-dose. At the 100 mg / kg dose level (with or without 175 mg / kg miglustat), ATB200 concentrations were measurable from 26 to 168 hours post-dose. Single-dose toxicokinetic parameters (Day 1) are shown in Table 8.

[0167] JPEG2025163019000018.jpg182154

[0168] Repeated dose toxicokinetic parameters (day 85) are shown in Table 9.

[0169] JPEG2025163019000019.jpg183154

[0170] Maximum ATB200 plasma concentration (t 最大値 The time to ATP production was approximately 2 hours after administration in all three dose groups. ATB200 plasma concentrations and TK parameters on days 1 and 85 were consistent between the two evaluated signature peptides, TTPTFFPK and VTSEGAGLQLQK, as measured by total ATB200 protein assay. C was significantly higher than C when measured by acid α-glucosidase activity assay. 最大値 and AUC 0-t Exposure measured by the assay was relatively low. This is expected because the total protein assay measures both active and inactive enzyme concentrations, whereas the acid α-glucosidase activity assay measures only the active enzyme concentration. ATB200 exposure increased with administration of dose levels between 50 and 100 mg / kg. 最大値 Initial t on day 1 based on the first three time points past 1 / 2αThe mean terminal half-life (t) on day 1 ranged from 1.28 to 3.07 hours (males and females combined). 1 / 2β ) ranged from 1.70 to 11.1 hours (longer t 1 / 2β (Values ​​were affected in animals with measurable concentrations up to 168 hours after dosing). A similar range of values ​​was observed after dosing on day 85. Little or no accumulation was observed with repeated dosing once every other week. The addition of 175 mg / kg miglustat to a 100 mg / kg ATB200 dose appeared to decrease ATB200 clearance and increase plasma exposure approximately two-fold compared to 100 mg / kg ATB200 monotherapy.

[0171] Because no adverse test substance-related changes were identified, the no-observed-adverse-effect level (NOAEL) for ATB200 in cynomolgus monkeys was 100 mg / kg / infusion, the highest dose tested, when administered by 2-hour infusion every other week for 13 weeks with or without miglustat administration. At this dose level, the mean sex-averaged AUC 0-t and C 最大値 (total protein) were 7830 (TTPTFFPK) and 7790 (VTSEGAGLQLQK) hr·μg / mL and 2020 (TTPTFFPK) or 2010 (VTSEGAGLQLQK) μg / mL, respectively, for ATB200 alone, and 13900 (TTPTFFPK) or 13800 (VTSEGAGLQLQK) hr·μg / mL and 2270 (both peptides) μg / mL, respectively, when combined with 175 mg / kg miglustat.

[0172] Miglustat toxicokinetics The toxicokinetics of miglustat were measured in blood samples taken from animals in K2EDTA tubes on days 1 and 85 at the following time points: For Groups 1, 2, 3, and 5: pre-dose (before administration of miglustat); 15 minutes after administration of miglustat; 0 hours (before the start of the infusion); 0.5 hours from the start of the infusion; 1 hour from the start of the infusion; 2 hours from the start of the infusion; 4 hours from the start of the infusion; 6 hours from the start of the infusion; 12 hours from the start of the infusion; 26 hours from the start of the infusion; 50 hours from the start of the infusion; 74 hours from the start of the infusion; and For group 4: pre-administration (before administration of miglustat); 15 minutes after administration of miglustat; 30 minutes after administration of miglustat; 1 hour after administration of miglustat; 1.5 hours after administration of miglustat; 2.5 hours after administration of miglustat; 4.5 hours after administration of miglustat; 6.5 hours after administration of miglustat; 12.5 hours after administration of miglustat; 26.5 hours after administration of miglustat; 50.5 hours after administration of miglustat; 74.5 hours after administration of miglustat.

[0173] Plasma was obtained by centrifugation at 2°C to 8°C, and aliquots (approximately 0.2 mL) were transferred to polypropylene vials and stored frozen at -60°C to -86°C. Analysis of miglustat concentrations was performed using an LC-MS / MS method similar to that described for duvoglustat concentrations by Richie Khanna, Allan C. Powe Jr., Yi Lun, Rebecca Soska, Jessie Feng, Rohini Dhulipala, Michelle Frascella, Anadina Garcia, Lee J. Pellegrino, Su Xu, Nastry Brignol, Matthew J. Toth, Hung V. Do, David J. Lockhart, Brandon A. Wustman, and Kenneth J. Valenzano. “The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid Alpha-Glucosidase,and Promotes Glycogen Reduction in Transgenic Mouse Model of Pompe Disease.”PLOS ONE(1 July 2014)9(7):e102092.

[0174] Analysis of miglustat toxicokinetic (TK) data was performed on the audit / validation datasets (concentration and time) from animals in groups 2, 3, and 4 using WinNonlin Phoenix®, version 6.1 software (Pharsight Corporation). TK parameters were estimated using non-compartmental analysis of individual plasma concentration data. Miglustat TK parameters were estimated by the log-linear trapezoidal rule. The regression used to estimate λz was based on uniformly weighted concentration data. The following parameters were calculated: ●R 2 adj-λ z λ, adjusted for the number of points used to estimate zThe square of the correlation coefficient of the linear regression used to estimate . Used when the number of points used to define the terminal phase of the concentration versus time profile can be variable; λ z Number of points - λ z The number of linear regression analysis points used to estimate ; λ z -terminal discharge rate constant; ●t 1 / 2 -λ z Terminal elimination half-life (0.693 / λ z ); ●t 最大値 - time of maximum concentration of the analyte in plasma; ●C 最大値 - Maximum observed concentration of the analyte in plasma; AUC 0-t - Area under the plasma concentration-time curve (AUC) measured from time 0 (pre-dose) to the time point with the last measurable concentration; AUC 0-∞ -AUC extrapolated to infinite time; AUC ext - The part of the AUC extrapolated to time infinity is the total AUC 0-∞ Expressed as % of; ●CL T / F - total clearance divided by bioavailable fraction based on total dose (mg) from actual body weight; ●V z / F - Volume of distribution based on end-stage divided by bioavailable fraction based on total dose (mg) from actual body weight; ●Accumulation rate-AR C最大値 = C on day 1 of 85 最大値 ratio; and AR AUC = C on day 1 of 85 0-t Ratio of.

[0175] There was no consistent effect of gender on miglustat TK parameters. Plasma concentrations of miglustat after nasogastric (NG) administration of 25 mg / kg in combination with 50 mg / kg ATB200 or 175 mg / kg NG administration (with or without 100 mg / kg ATB200) were measurable up to 74.5 hours (last measured time point). Single-dose (Day 1) and repeat-dose (Day 85) toxicokinetic parameters are shown in Table 10.

[0176] JPEG2025163019000020.jpg141154

[0177] t 最大値 ranged from approximately 2 to 4 hours after administration. Miglustat exposure increased at dose levels of 25 to 175 mg / kg. Mean t 1 / 2 (males and females combined) were consistent on days 1 and 85, ranging from 6.66 to 8.23 ​​hours. Little to no accumulation was observed with repeated NG administration once every other week. Overall miglustat exposure (i.e., AUC 0-t There was no observable effect of ATB200 co-administration on EGFR deficiency, EGFR deficiency, or TK parameters.

[0178] Because no adverse test substance-related changes were identified, the no-observed-adverse-effect level (NOAEL) for miglustat in cynomolgus monkeys was 175 mg / kg / dose, the highest dose tested, when administered nasogastrically every other week for 13 weeks with or without ATB200. At this dose level, the mean sex-averaged AUC 0-t and C 最大値 were 204,000 hr·ng / mL and 14,700 ng / mL, respectively, for miglustat alone, and 216,000 hr·ng / mL and 22,000 ng / mL, respectively, for miglustat in combination with 100 mg / kg ATB200.

[0179] Example 14: Protocol for clinical trials of recombinant acid α-glucosidase (ATB200) administered alone and co-administered with miglustat Experimental design: This is an open-label, fixed-sequence, dose-escalation, first-in-human study to evaluate the safety, tolerability, and pharmacokinetics (PK) of intravenous (IV) recombinant acid α-glucosidase (ATB200, lyophilized powder reconstituted with sterile water for injection and diluted for injection with 0.9% sodium chloride) alone and co-administered with oral miglustat (hard gelatin capsules, 65 mg). The study will be conducted in two stages. Stage 1 will evaluate safety, tolerability, and PK after sequential single ascending doses of ATB200 administered every 2 weeks as an approximately 4-hour intravenous infusion at three dosing periods: 5, 10, and 20 mg / kg. In Stage 2, safety, tolerability, and PK will be evaluated after the following single and multiple escalating dose combinations: 20 mg / kg ATB200 co-administered with 130 mg miglustat (two 65 mg capsules) every 2 weeks, with miglustat taken orally 1 hour before an approximately 4-hour intravenous infusion of ATB200 for three doses; then 20 mg / kg ATB200 co-administered with 260 mg miglustat (four 65 mg capsules), with miglustat taken orally 1 hour before an approximately 4-hour intravenous infusion of ATB200 for three doses.

[0180] Twelve enzyme replacement therapy (ERT)-experienced subjects with Pompe disease (approximately six ambulatory and six nonambulatory) will be enrolled in Stage 1. These same subjects will continue the study in Stage 2. At least four ambulatory subjects will be enrolled and dosed before any nonambulatory subjects are enrolled. ERT-experienced (ambulatory) subjects have received ERT for 2-6 years prior to enrollment, can walk at least 200 meters in the 6-minute walk test (6MWT), and have an FVC of 30-80% of predicted normal. ERT-experienced (nonambulatory) subjects are defined as those who are completely wheelchair-bound, unable to walk unassisted, and have been receiving ERT for 2 or more years prior to enrollment. Treatment assignments are shown in Table 11.

[0181] JPEG2025163019000021.jpg55154

[0182] Subjects should fast for at least 2 hours before and 2 hours after oral administration of miglustat. IV infusion of ATB200 should begin 1 hour after oral administration of miglustat.

[0183] Experimental procedure The study consisted of screening, baseline, stage 1 (3 periods, fixed sequence, single ascending doses of ATB200 only), and stage 2 (2 periods, fixed sequence, multiple doses of 20 mg / kg ATB200 co-administered with multiple ascending doses of miglustat).

[0184] screening: All subjects will provide informed consent and undergo a review of eligibility criteria. Evaluations of all subjects will include a medical history, including prior infusion-related reactions (lARs) and fall history; a review of prior and concomitant medications and non-pharmacological therapies; vital signs (heart rate [HR], respiratory rate [RR], blood pressure [BP], and temperature); height; weight; a comprehensive physical examination (PE); a 12-lead electrocardiogram (ECG); clinical safety assessments (serum chemistry, hematology, and urinalysis); a urine pregnancy test; a urine sample for hexose tetrasaccharide (Hex4); and GAA genotyping (for subjects unable to provide a GAA genotyping report at screening). Blood samples will also be collected for exploratory immunogenicity assessments (total and neutralizing antibodies, exploratory cytokines / other biomarkers of immune system activation, cross-reactivity with alglucosidase alfa, and immunoglobulin E [IgE]). Subjects who meet all inclusion criteria and none of the exclusion criteria will be assigned to Stage 1, as described in Table 11.

[0185] Baseline: Safety assessments for all subjects included a review of eligibility criteria; history of infusion-related reactions (lARs) and falls, adverse events (AEs) and serious AEs (SAEs), review of prior and concomitant medications and non-pharmacological therapies; vital signs (HR, RR, BP, and temperature); weight; summary PE; ECG; Rasch-built Pompe-specific activity (R-PAct) scale; Rotterdam Handicap Scale; and Fatigue Severity Scale; clinical safety assessments (serum chemistry, hematology, and urinalysis); urine pregnancy test; pharmacodynamic (PD) assessments (Hex4 and creatinine phosphokinase [CPK]); immunogenicity assessments (total and neutralizing antibodies, antibody cross-reactivity with alglucosidase alfa, test cytokines and other biomarkers of immune system activation, cross-reactivity to alglucosidase alfa, and IgE as appropriate); pulmonary function tests (PFTs); exercise function testing; and muscle strength testing for all subjects.

[0186] Stage 1, Periods 1, 2 and 3: This stage includes: Safety: Review of AEs, including serious adverse events (SAEs) and IARs; review of concomitant medications and non-pharmacological therapies; vital signs (HR, RR, BP, and temperature); weight; summary PE; ECG; clinical safety study evaluations (serum chemistry, hematology, and urinalysis); and urine pregnancy test ●PD: urinary Hex4 and serum CPK Immunology: Blood samples for anti-recombinant acid alpha-glucosidase antibody titers (anti-recombinant acid alpha-glucosidase total and neutralizing antibody titers and antibody cross-reactivity with alglucosidase alpha) and for measurement of pro-inflammatory cytokines and other immune system activation biomarkers. IgE measurements will also be performed if necessary. ● Continuous 24-hour pharmacokinetics (PK): During Period 1 (Visit 3, Day 1), Period 2 (Visit 4, Day 15), and Period 3 (Visit 5, Day 29), blood samples for plasma acid alpha-glucosidase activity levels and total acid alpha-glucosidase protein concentrations will be taken for all subjects.

[0187] Stage 2, Periods 4 and 5: Safety: Review of AEs, including SAEs and IARs; review of concomitant medications and non-pharmacological therapies; vital signs (HR, RR, BP, and temperature); weight; PE; ECG; clinical safety study evaluations (serum chemistry, hematology, and urinalysis); and urine pregnancy test. ●PD: urinary Hex4 and serum CPK Immunology: Blood samples for anti-recombinant acid alpha-glucosidase antibody titers (anti-recombinant acid alpha-glucosidase total and neutralizing antibody titers and antibody cross-reactivity with alglucosidase alpha) and for measurement of pro-inflammatory cytokines and other immune system activation biomarkers. IgE measurements will also be performed if necessary. Continuous 24-hour PK: During Period 4 (Visit 6, Day 43 and Visit 8, Day 71) and Period 5 (Visit 9, Day 85 and Visit 11, Day 113), blood samples for plasma acid alpha-glucosidase activity levels, total acid alpha-glucosidase protein concentration, and miglustat concentration will be taken for all subjects.

[0188] End of pharmacokinetic phase: Safety: Review of AEs, including SAEs and IARs; review of concomitant medications and non-pharmacological therapies; vital signs (HR, RR, BP, and temperature); weight; PE; ECG; clinical safety study evaluations (serum chemistry, hematology, and urinalysis); and urine pregnancy test. ●PD: urinary Hex4 and serum CPK Immunology: Blood samples for anti-recombinant acid alpha-glucosidase antibody titers (anti-recombinant acid alpha-glucosidase total and neutralizing antibody titers and antibody cross-reactivity with alglucosidase alpha) and for measurement of pro-inflammatory cytokines and other immune system activation biomarkers. IgE measurements will also be performed if necessary.

[0189] Subjects who withdraw early from the study will come in for an early termination visit and undergo all of the assessments that should be performed at the end of the PK visit. They will not receive study drug. If any of the sentinel subjects withdraw early from the study, that subject will be replaced by the next ambulatory subject enrolled in the study (e.g., if subject 1 withdraws, subject 3 [ambulatory] will replace that subject as the sentinel subject).

[0190] Subjects who complete this study and / or other eligible subjects will be offered the opportunity to participate in a long-term extension study where they will continue to be evaluated for safety and tolerability of ATB200 co-administered with miglustat. Additionally, functional assessments relevant to Pompe disease will be performed periodically in the extension study.

[0191] Safety Monitoring Safety will be monitored continuously by medical monitors and investigators, and periodically by the Safety Steering Committee (SSC).

[0192] Sentinel administration The first two ambulatory subjects in this study will serve as sentinel subjects and will be the first two subjects to be dosed in each period of the study (Periods 1-5). If a sentinel subject withdraws early from the study, that subject will be replaced by another ambulatory subject. Note: At least four ambulatory subjects will be dosed with 5 mg / kg ATB200 before dosing any non-ambulatory subjects.

[0193] In Stage 1 (Periods 1, 2, and 3), subjects receive single ascending doses of ATB200 (5 mg / kg [Period 1], 10 mg / kg [Period 2], and 20 mg / kg [Period 3]).

[0194] After dosing of the two sentinel subjects for each experimental period in Stage 1, evaluation of available safety data (PE, vital signs, AEs, infusion reactions, ECG, and available locally performed laboratory tests) will be conducted by the medical monitor and investigator within 24–48 hours. An SSC will be convened for formal safety review when central safety study data are available for both sentinel subjects at each dose level. If the SSC determines there are no safety concerns that would prevent dosing at the dose assigned to that period, 10 additional subjects will be enrolled and dosed. An SSC will also be convened for safety review when safety data (including central study safety data) are available for all subjects at all three Stage 1 dose levels.

[0195] In Stage 2 (Periods 4 and 5), two sentinel subjects will be dosed, and safety will be assessed after the first dose, as for each period in Stage 1. If the SSC determines that there are no safety concerns preventing the additional dose of ATB200 at 20 mg / kg coadministered with 130 mg miglustat (Period 4) or ATB200 at 20 mg / kg coadministered with 260 mg miglustat (Period 5), 10 additional subjects will receive three biweekly doses at the dose assigned to that period. The SSC will be held again at the end of Stage 2 when all safety data (including central safety study data) are available for all subjects. The SSC will also be held on an ad hoc basis if an SAE or safety concern is identified.

[0196] The SSC may recommend a review of any of the following: Continue the experiment without any changes ● Make changes (corrections) and continue the experiment Temporary suspension of administration • Permanent discontinuation of medication.

[0197] If, in the opinion of the SSC, there are no AEs or safety concerns in the sentinel subject that would preclude continued study dosing, then all remaining subjects will continue to be dosed at that dose level. Subject safety will continue to be closely monitored on an ongoing basis by the medical monitor and study investigator, and periodically by the SSC.

[0198] Target number (planned): Twelve adult ERT-experienced subjects with Pompe disease (approximately 6 ambulatory and 6 non-ambulatory) will be enrolled in Stage 1. These same subjects will continue in the study in Stage 2.

[0199] Diagnosis and eligibility criteria: At the screening visit, adult ERT-experienced subjects with Pompe disease will be evaluated using the eligibility criteria outlined below. Each subject must meet all of the inclusion criteria and none of the exclusion criteria. Waivers of the inclusion / exclusion criteria will not be granted.

[0200] Participation criteria ERT experience required (ambulatory) 1. Male and female subjects aged 18 to 65 years; 2. Subjects must sign informed consent prior to any experiment-related procedures; 3. Subjects of childbearing potential must agree to use medically accepted methods of contraception during the study and for 30 days after the last ATB200 + miglustat co-administration; 4. The subject has a diagnosis of Pompe disease based on documented deficiency of acid α-glucosidase enzyme activity or by GAA genotyping; 5. The subject has received ERT with alglucosidase alfa in the past 2–6 years; 6. The subject is currently receiving alglucosidase alfa once every two weeks; 7. Subject received and completed the last two infusions without any drug-related adverse events resulting in dose interruption; 8. Subjects must be able to walk 200-500 meters on the 6MWT; and 9. Upright forced vital capacity (FVC) should be 30% to 80% of predicted normal. ERT experienced subjects (non-ambulatory) 10. Male and female subjects aged 18 to 65 years; 11. Subject must sign informed consent prior to any experiment-related procedures; 12. Subjects of childbearing potential must agree to use medically accepted methods of contraception during the study and for 30 days after the last ATB200 + miglustat co-administration; 13. The subject has a diagnosis of Pompe disease based on documented deficiency of acid α-glucosidase enzyme activity or by GAA genotyping; 14. The subject has received ERT with alglucosidase alfa for ≥2 years; 15. The subject is currently receiving alglucosidase alfa once every two weeks; 16. The subject has received and completed the last two infusions without any drug-related adverse events resulting in dose interruption; and 17. The subject is completely wheelchair-bound and unable to walk unassisted.

[0201] Exclusion criteria ERT experience required (ambulatory) 1. The subject has received, or is expected to receive during the study, a Pompe disease therapeutic trial other than alglucosidase alfa within 30 days prior to the baseline visit; 2. The subject has received treatment with a prohibited substance (miglitol (e.g., Glyset®)); miglustat (e.g., Zavesca®); acarbose (e.g., Precose®, Glucobay®); voglibose (e.g., Volix®, Vocarb®, and Volibo®); albuterol and clenbuterol; or any investigational / experimental drug) within 30 days prior to the baseline visit; 3. The subject, if female, is pregnant or lactating at the time of screening; 4. The subject, regardless of gender, plans to conceive a child during the experiment. 5. Subject requires invasive ventilatory support. 6. The subject uses non-invasive ventilatory support for 6 hours or more while awake; 7. The subject has a medical or other excusable condition or circumstance that, in the investigator's opinion, poses an undue safety risk to the subject or may impair their ability to comply with protocol requirements; 8. Subject has a history of anaphylaxis to alglucosidase alfa; 9. The subject has a history of high persistent anti-recombinant acid alpha-glucosidase antibody titers; 10. The subject has a history of allergy or hypersensitivity to miglustat or other iminosugars; 11. The subject has a history of lupus, autoimmune thyroiditis, scleroderma, or rheumatoid arthritis; and 12. The subject has a history of bronchial asthma. ERT experienced subjects (non-ambulatory) 13. The subject has received, or is expected to receive during the study, a Pompe disease therapeutic trial other than alglucosidase alfa within 30 days prior to the baseline visit; 14. The subject has received treatment with a prohibited substance (miglitol (e.g., Glyset®)); miglustat (e.g., Zavesca®); acarbose (e.g., Precose®, Glucobay®); voglibose (e.g., Volix®, Vocarb®, and Volibo®); albuterol and clenbuterol; or any investigational / experimental drug) within 30 days prior to the baseline visit; 15. The subject, if female, is pregnant or lactating at the time of screening; 16. The subject, regardless of gender, plans to conceive a child during the experiment; 17. The subject has a medical or other excusable condition or circumstance that, in the investigator's opinion, poses an undue safety risk to the subject or may impair their ability to comply with protocol requirements; 18. The subject has a history of anaphylaxis to alglucosidase alfa; 19. The subject has a history of high and persistent anti-recombinant acid alpha-glucosidase antibody titers; 20. The subject has a history of allergy or hypersensitivity to miglustat or other iminosugars; 21. The subject has a history of lupus, autoimmune thyroiditis, scleroderma, or rheumatoid arthritis; and 22. The subject has a history of bronchial asthma.

[0202] Investigational drug, dose, and mode of administration: Stage 1 (consisting of three doses administered at two-week intervals) ● Period 1: Single IV infusion of 5 mg / kg ATB200; Period 2: A single IV infusion of 10 mg / kg ATB200 in all subjects who completed Period 1; and ● Period 3: A single IV infusion of 20 mg / kg ATB200 in all subjects who completed Period 2. Stage 2 (consisting of two dosing periods, each containing 3 weeks of study drug dose, 2 weeks apart) Period 4: All subjects who completed Period 3 (repeated every 2 weeks for a total of 3 doses) will receive 130 mg of miglustat orally 1 hour prior to a single IV infusion of 20 mg / kg ATB200; and Period 5: All ERT-experienced subjects who completed Period 4 (repeated every 2 weeks for a total of 3 doses) will receive 260 mg of miglustat orally 1 hour prior to a single IV infusion of 20 mg / kg ATB200. NOTE: Subjects should fast for 2 hours before and 2 hours after administration of oral miglustat. Total duration of the study: up to 22 weeks (up to 4 weeks of screening period, followed by approximately 18 weeks of study treatment [Stages 1 and 2]) Single-dose PK observation period (Stage 1, Periods 1, 2 and 3): 6 weeks Multiple-dose PK observation period (Stage 2, Periods 4 and 5): 12 weeks Safety, tolerability, and immunogenicity observation period (Periods 1, 2, 3, 4, and 5): 18 weeks.

[0203] Evaluation criteria: Primary: Safety rating: ●PE Vital signs including temperature, RR, HR, and BP AEs including IARs ●12-lead ECG • Clinical safety testing evaluation: serum chemistry, hematology and urinalysis.

[0204] PK of Plasma ATB200 and Miglustat: Plasma acid α-glucosidase activity levels and total acid α-glucosidase protein concentration PK parameters: Maximum observed plasma concentration (C 最大値 ), the time to reach the maximum observed plasma concentration (t 最大値 ), the area under the plasma-drug concentration-time curve from time 0 to the last measurable concentration (AUC 0-t ), the area under the plasma drug concentration-time curve extrapolated from time 0 to infinity (AUC 0-∞ ), half-life (t 1 / 2 ), and total clearance after IV administration (CL T ) Plasma acid α-glucosidase activity and total acid α-glucosidase protein C for all dosing regimens 最大値 and AUC 0-∞ Percentage of Plasma miglustat PK parameters: C 最大値 , t 最大値 , AUC 0-t , AUC 0-∞ and t 1 / 2 , the apparent total clearance of the drug after oral administration (CL T / F) and final volume of distribution after oral administration (Vz / F) for each dose level Plasma miglustat C at each dose level最大値 and AUC 0-∞ Ratio of.

[0205] Functional assessment (performed at baseline) For ambulatory subjects ●Motor function test ● 6-minute walk test (6MWT) ●10-meter walking test Walking, Stair, Gower, and Chair Scores ●Time Up and Go (TUG) Upper and lower limb muscle strength testing (Medical Laboratory Criteria [MRC] and handheld dynamometer) PFTs (FVC, MIP, MEP, and SNIP) For those who cannot walk Muscle strength test - upper limbs only MRC performed using only the upper limbs and a handheld dynamometer • Pulmonary function tests (PFTs) (forced vital capacity [FVC], maximum inspiratory pressure [MIP], maximum expiratory pressure [MEP], nasal inspiratory pressure [SNIP]).

[0206] Patient-reported outcomes (conducted at baseline) Fatigue Severity Scale ●Rotterdam Handicap Scale ●Rasch-built Pompe specific activity (R-PAct).

[0207] Examination Anti-ATB200 antibody titer (total antibody and neutralizing antibody) Cross-reactivity of anti-recombinant acid α-glucosidase antibodies to alglucosidase alpha Pro-inflammatory cytokines and other biomarkers of immune system activation • PD markers (Hex4 and CPK).

[0208] Analysis method: Statistical methods: Descriptive statistics for PK parameters are provided. Summary statistics are provided for all variables that are not PK parameters. Acid α-glucosidase activity and total acid α-glucosidase protein exposure (C 最大値 , AUC 0-t , and AUC 0-∞ ) ratio. ANOVA of acid α-glucosidase activity and total acid α-glucosidase protein exposure (C) for 20 mg / kg ATB200 alone vs. 20 mg / kg ATB200 + 130 mg miglustat vs. 20 mg / kg ATB200 + 260 mg miglustat. 最大値 , AUC 0-t and AUC 0-∞ ) ratio. ANOVA of acid α-glucosidase activity and total acid α-glucosidase protein exposure (C) between ambulatory and non-ambulatory subjects for 20 mg / kg ATB200 + 130 mg miglustat and 20 mg / kg ATB200 + 260 mg miglustat. 最大値 , AUC 0-t and AUC 0-∞ ) ratio. The proportion of miglustat exposure between 130 mg and 260 mg within each study population and overall (C 最大値 , AUC 0-t and AUC 0-∞ ) Dose proportionality assessment. The impact of immunogenicity results on PK, PD, and safety will be evaluated.

[0209] Interim analysis: An interim analysis will be conducted when at least 50% (n=6) of subjects have completed Stage 2 of the study. Up to two additional interim analyses may be conducted in this study.

[0210] First penalty kick result: The PK summaries of the GAA activity and GAA total protein of interest are shown in Tables 12 and 13, respectively.

[0211] In Tables 12-15 and Figures 24-26, single dose (SD) measurements were made after a single dose of miglustat and ATB200, and multiple dose (MD) measurements were made after the third biweekly dose of miglustat and ATB200.

[0212] JPEG2025163019000022.jpg103154

[0213] JPEG2025163019000023.jpg116154

[0214] Figure 24A shows the concentration-time profiles of mean plasma GAA activity after administration of 5 mg / kg, 10 mg / kg, and 20 mg / kg of ATB200. Figure 24B also provides the concentration-time profiles of mean plasma GAA activity after administration of 5 mg / kg, 10 mg / kg, and 20 mg / kg of ATB200, but with plasma GAA activity displayed on a logarithmic scale. As can be seen from Figures 24A-24B and Table 12, ATB200 demonstrated slightly greater than dose-proportional exposure to plasma GAA activity.

[0215] Figure 24C shows the concentration-time profiles of mean plasma GAA activity after administration of 20 mg / kg ATB200 alone, and 20 mg / kg ATB200 with 130 mg or 260 mg miglustat. Figure 24D also provides the mean plasma GAA activity after administration of 20 mg / kg ATB200 alone, with 130 mg miglustat, or with 260 mg miglustat, with plasma GAA activity displayed on a logarithmic scale.

[0216] Figure 25A shows the concentration-time profiles of mean plasma GAA total protein after administration of 5 mg / kg, 10 mg / kg, and 20 mg / kg ATB200. Figure 25B also provides the concentration-time profiles of mean plasma GAA total protein after administration of 5 mg / kg, 10 mg / kg, and 20 mg / kg ATB200, but with plasma GAA total protein displayed on a logarithmic scale. As can be seen from Figures 25A-25B and Table 13, ATB200 demonstrated slightly greater than dose-proportional exposure to plasma GAA total protein.

[0217] Figure 25C shows the concentration-time profiles of mean plasma GAA total protein after administration of 20 mg / kg ATB200 alone, 20 mg / kg ATB200 and 130 mg miglustat, and 20 mg / kg ATB200 and 260 mg miglustat. Figure 25D also provides the mean plasma GAA total protein after administration of 20 mg / kg ATB200 alone, with 130 mg miglustat, or with 260 mg miglustat, but with plasma GAA total protein displayed on a logarithmic scale.

[0218] As shown in Table 13, co-administration of miglustat increased the total GAA protein plasma half-life by approximately 30% compared to ATB200 administered alone. The volume of distribution ranged from 3.5 to 5.7 L for all treatments, suggesting that glycosylation of ATB200 enables efficient distribution of ATB200 to tissues.

[0219] The PK summary of miglustat is shown in Table 14.

[0220] JPEG2025163019000024.jpg84154

[0221] FIG. 26 shows the concentration-time profile of miglustat in plasma in human subjects after administration of 130 mg or 260 mg of miglustat.

[0222] As can be seen from Table 14 and Figure 26, plasma miglustat administered orally 1 hour before ATB200 infusion reached peak concentrations 2 hours after infusion, demonstrating dose-proportional kinetics.

[0223] To determine the partial AUC, analyses were performed on various parts of the plasma concentration curves of GAA activity and total protein. Table 15 shows the partial AUCs for GAA activity and total protein from 0 to t 最大値 , t 最大値 ~6h,t 最大値 ~10h,t 最大値 ~12h, and t 最大値 Provides an overview of the partial AUC from ~24hr.

[0224] JPEG2025163019000025.jpg157154

[0225] As can be seen from Table 15, the pAUCt of 20 mg / kg ATB200 + miglustat compared to 20 mg / kg ATB200 alone 最大値-24h The mean increases in GAA activity were 21.4%, 17.8%, and 40.2% for 130 mg SD, 130 mg MD, and 260 mg SD, respectively.

[0226] Similarly, the pAUCt of 20 mg / kg ATB200 + miglustat compared with 20 mg / kg ATB200 alone 最大値-24h The mean increases in GAA total protein were 12.5%, 16.4%, and 37.5% for 130 mg SD, 130 mg MD, and 260 mg SD, respectively.

[0227] Therefore, partial AUC analysis showed that co-administration of miglustat significantly increased the end-phase partial AUC(t 最大値-24h ) by approximately 15% for the 130 mg dose of miglustat and approximately 40% for the 260 mg dose of miglustat.

[0228] Early biomarker results Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine phosphokinase (CPK) levels were monitored in a human patient who was switched from Lumizyme® to ATB200. The patient received increasing doses of ATB200 (5, 10, and 20 mg / kg) followed by co-administration of ATB200 (20 mg / kg) and miglustat (130 and 260 mg). High levels of CPK enzymes may indicate damage or stress to muscle tissue, heart, or brain. Elevated ALT and AST are markers of liver and muscle damage, respectively, due to Pompe disease. Initial analysis of ALT, AST, and CPK levels is shown in Figures 38-41.

[0229] As can be seen in Figures 38-41, two patients showed an early trend toward improvement in all three biomarkers, and two patients remained stable. One patient showed a 44%, 28%, and 34% decrease in CPK, AST, and ALT, respectively. Another patient showed a 31%, 22%, and 11% decrease in CPK, AST, and ALT, respectively.

[0230] To date, there have been no serious adverse events (SAEs). AEs were generally mild and transient. To date, after more than 100 infusions in all enrolled patients, there have been no infusion-related reactions. All patients had anti-rhGAA antibodies at baseline, which were generally stable. Cytokines remained low and stable throughout the infusion.

[0231] Example 15: GAA and LAMP1 levels in wild-type and Pompe fibroblasts Immunofluorescence microscopy was used to detect GAA and LAMP1 levels in wild-type and Pompe fibroblasts harboring a common splicing mutation. As shown in Figure 27, GAA is located in distinct lysosomal compartments in wild-type fibroblasts. Figure 27 also shows abundant GAA signal in Pompe fibroblasts, and both GAA and LAMP1 signals in Pompe fibroblasts appear to be localized to the ER and Golgi rather than distal lysosomes. This is evidence of altered GAA protein trafficking in Pompe fibroblasts.

[0232] Example 16: Improved cellular dysfunction and muscle function in Gaa knockout mice Impairment of lysosomal glycogen catabolism due to GAA deficiency has been shown to cause substantial cellular dysfunction, as evidenced by prominent and persistent autophagy and the proliferation and accumulation of membrane-bound intracellular compartments filled with stored glycogen (N. Raben et al.). Our immunohistological data indicate that protein trafficking is significantly altered for many proteins, including several key proteins essential for muscle membrane stability, such as dystrophin, α- and β-dystroglycan, various sarcoglycans, and others, including the dystrophin glycoprotein complex, as well as proteins involved in muscle repair, such as dysferlin. These critical muscle proteins require proper protein trafficking to the muscle cell membrane where they function. As shown in Figure 28, our immunohistological data reveal that a significant portion of these important muscle proteins have intracellular localization in the muscles of a Gaa knockout (KO) mouse model of Pompe disease. These data suggest that incorrect trafficking of these important muscle proteins induces pseudomuscular dystrophy, ultimately leading to muscle weakness and loss.

[0233] Alglucosidase alfa (Myozyme®) and ATB200 with and without 10 mg / kg miglustat were evaluated in GaaKO mice at an equivalent ERT dose (20 mg / kg) on ​​a biweekly dosing schedule. After two doses, alglucosidase alfa modestly reduced accumulated lysosomal glycogen in skeletal muscle compared with vehicle-treated mice (Figures 32A-32C) and had negligible effects on autophagy (Figures 30A-30B) or lysosomal proliferation (Figures 29A-29B). In contrast, substantially better lysosomal glycogen clearance was observed with ATB200 / miglustat under the same conditions (Figures 32A-32D). ATB200 / miglustat appeared to improve overall muscle physiology, as evidenced by reduced LC3 II levels (Figures 30A-30B), a well-established autophagy biomarker, and clearance of accumulated intracellular vesicles stained for LAMP1 (Figures 29A-29B), a known resident lysosomal membrane protein, and dysferlin (Figures 31A-31B), a known cell surface protein involved in muscle repair. Furthermore, ATB200 / miglustat significantly improved muscle architecture, resembling myofibers from wild-type mice.

[0234] Figures 29-32 also show that two different batches of ATB200 (early and later generation manufacturing processes) produced comparable results. In Figures 32A-32D, * indicates statistical significance compared to Myozym® alone.

[0235] Example 17: Muscle function in Gaa knockout mice In a long-term study with 12 biweekly doses, 20 mg / kg ATB200 plus 10 mg / kg miglustat progressively increased functional muscle strength in GaaKO mice from baseline, as measured by both grip strength and the wire hang test (Figures 33A-33B). Alglucosidase alfa (Lumizyme®)-treated mice receiving the same ERT dose (20 mg / kg) were observed to decline under the same conditions throughout the majority of the study (Figures 33A-33B). Similar to the short-term study, ATB200 / miglustat had substantially better glycogen clearance than alglucosidase alfa after 3 months (Figures 34A-34C) and 6 months (Figures 34D-G) of treatment. ATB200 / miglustat also reduced autophagy and the intracellular accumulation of LAMP1 and dysferlin after 3 months of treatment compared to alglucosidase alfa (Figure 35). In Figure 33A, * indicates statistical significance compared to Lumizyme alone (p<0.05, two-tailed t-test). In Figures 34A-34G, * indicates statistical significance compared to Lumizyme® alone (p<0.05, one-way ANOVA analysis using Dunnett's multiple comparisons method).

[0236] Collectively, these data demonstrate that ATB200 / miglustat efficiently targeted muscle, reversed cellular dysfunction, and improved muscle function. Importantly, the clear improvement in muscle structure and the reduction in autophagy and intracellular accumulation of LAMP1 and dysferlin may be a good proxy for improved muscle physiology, which correlates with improved functional muscle strength. These results suggest that monitoring autophagy and these important muscle proteins in GaaKO mice is a rational and practical method for assessing the efficacy of therapeutic treatments for Pompe disease, which may prove useful biomarkers from muscle biopsies in clinical trials.

[0237] Figure 40 shows that 6 months of ATB200 administration with or without miglustat reduces the intracellular accumulation of dystrophin in GaaKO mice. ATB200 ± miglustat reduced dystrophin accumulation to a greater extent than Lumizyme®.

[0238] Example 18: Effect of sialic acid content on ATB200 in Gaa knockout mice Two batches of ATB200 with different sialic acid contents were evaluated for pharmacokinetics and efficacy in GaaKO mice. Table 16 summarizes the characteristics of the two batches.

[0239] JPEG2025163019000026.jpg42154

[0240] As can be seen from Table 16, Batch B had a higher sialic acid content than Batch A, but a slightly lower M6P content than Batch A.

[0241] 36 shows the concentration-time profile of GAA activity in plasma in GaaKO mice after a single IV bolus administration of ATB200. The half-lives of batches A and B are shown in Table 17 below.

[0242] JPEG2025163019000027.jpg27154

[0243] As can be seen from Table 17, Batch B has a shorter half-life than Batch A. Although the decrease in half-life was small, this decrease in half-life was statistically significant (p<0.05 in a two-sided t-test).

[0244] In a related experiment, GaaKO mice were given IV bolus tail vein injections of ATB200 (Batches A and B) and Lumizyme® every other week for a total of two injections. Fourteen days after the last dose, tissue glycogen levels were measured. As shown in Figures 37A-37D, Batch B was generally more effective than Batch A in reducing glycogen at similar doses. Both Batch A and B were superior to Lumizyme® in reducing glycogen. In Figures 37A-37D, * indicates statistical significance compared to Lumizyme® (p<0.05, t-test), and ^ indicates statistical significance between Batch A and B at the same dose (p<0.05, t-test).

[0245] The embodiments described herein are intended to illustrate the compositions and methods of the present invention and are not intended to limit the scope of the present invention. It is intended to include various modifications and variations that are consistent with the present specification as a whole and are readily apparent to those skilled in the art. The scope of the appended claims should not be limited by the specific embodiments described in the examples, but should be given the broadest interpretation consistent with the overall description.

[0246] Patents, patent applications, publications, product descriptions, GenBank accession numbers, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. 1. A recombinant acid α-glucosidase for use in the treatment of Pompe disease in combination with miglustat, the recombinant acid α-glucosidase being expressed in Chinese hamster ovary (CHO) cells and comprising an increased content of N-glycan units having one or two mannose-6-phosphate residues compared to the content of N-glycan units having one or two mannose-6-phosphate residues of alglucosidase alfa.

2. 2. The recombinant human acid alpha-glucosidase for use according to claim 1, wherein the recombinant human acid alpha-glucosidase comprises a sequence that is at least 95% identical to SEQ ID NO:1 or SEQ ID NO:

5.

3. 3. The recombinant human acid alpha-glucosidase for use according to claim 1 or 2, wherein at least 30% of the recombinant human acid alpha-glucosidase comprises one or more N-glycan units having one or two mannose-6-phosphate residues.

4. 4. The recombinant human acid α-glucosidase for use according to any one of claims 1 to 3, wherein the recombinant human acid α-glucosidase comprises an average of 0.5 to 7.0 moles of N-glycan units having one or two mannose-6-phosphate residues per mole of recombinant human acid α-glucosidase.

5. 5. The recombinant human acid α-glucosidase for use according to any one of claims 1 to 4, wherein the recombinant human acid α-glucosidase comprises, on average, at least 2.5 moles of mannose-6-phosphate residues per mole of recombinant human acid α-glucosidase and at least 4 moles of sialic acid residues per mole of recombinant human acid α-glucosidase.

6. 6. The recombinant human acid α-glucosidase for use according to any one of claims 1 to 5, wherein the recombinant human acid α-glucosidase comprises seven potential N-glycosylation sites, at least 50% of the molecules of the recombinant human acid α-glucosidase comprise N-glycan units having two mannose-6-phosphate residues at a first site, at least 30% of the molecules of the recombinant human acid α-glucosidase comprise N-glycan units having one mannose-6-phosphate residue at a second site, at least 30% of the molecules of the recombinant human acid α-glucosidase comprise N-glycan units having two mannose-6-phosphate residues at a fourth site, and at least 20% of the molecules of the recombinant human acid α-glucosidase comprise N-glycan units having one mannose-6-phosphate residue at a fourth site.

7. The recombinant human acid α-glucosidase for use according to any one of claims 1 to 6, wherein the recombinant human acid α-glucosidase is produced by Chinese hamster ovary cell line GA-ATB-200 or ATB-200-001-X5-14 or a subculture thereof.

8. 8. The recombinant human acid alpha-glucosidase for use according to any one of claims 1 to 7, wherein the recombinant human acid alpha-glucosidase is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and the miglustat is administered orally at a dose of about 200 mg to about 600 mg.

9. The recombinant human acid alpha-glucosidase for use according to any one of claims 1 to 8, wherein said miglustat is administered before administration of said recombinant human acid alpha-glucosidase.

10. The recombinant human acid alpha-glucosidase for use according to any one of claims 1 to 9, wherein the miglustat is administered about 1 hour before the administration of the recombinant human acid alpha-glucosidase.

11. 11. The recombinant human acid alpha-glucosidase for use according to any one of claims 1 to 10, wherein the recombinant human acid alpha-glucosidase is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and the miglustat is administered orally at a dose of about 233 mg to about 500 mg.

12. 12. The recombinant human acid alpha-glucosidase for use according to any one of claims 1 to 11, wherein the recombinant human acid alpha-glucosidase is administered intravenously at a dose of about 5 mg / kg to about 20 mg / kg, and the miglustat is administered orally at a dose of about 50 mg to about 200 mg.

13. 13. The recombinant human acid alpha-glucosidase for use according to any one of claims 1 to 12, wherein the recombinant human acid alpha-glucosidase is administered intravenously at a dose of about 20 mg / kg, and the miglustat is administered orally at a dose of about 260 mg.

14. The recombinant human acid alpha-glucosidase for use according to any one of claims 1 to 13, wherein said miglustat is administered prior to administration of said recombinant human acid alpha-glucosidase.

15. The recombinant human acid alpha-glucosidase for use according to any one of claims 1 to 14, wherein the miglustat is administered about 1 hour before the administration of the recombinant human acid alpha-glucosidase.

16. 1. A kit for combination treatment of Pompe disease in a patient in need thereof, comprising: a pharmaceutically acceptable dosage form comprising miglustat; a pharmaceutically acceptable dosage form comprising recombinant human acid alpha-glucosidase; and instructions for administering said pharmaceutically acceptable dosage form comprising miglustat and said pharmaceutically acceptable dosage form comprising recombinant acid alpha-glucosidase to a patient in need thereof; The kit comprises a recombinant acid α-glucosidase expressed in Chinese hamster ovary (CHO) cells and comprising an increased content of N-glycan units having one or two mannose-6-phosphate residues compared to the content of N-glycan units having one or two mannose-6-phosphate residues of alglucosidase alfa.

17. 17. The kit of claim 16, wherein the pharmaceutically acceptable dosage form comprising the recombinant human acid α-glucosidase is configured for intravenous administration at a dose of about 5 mg / kg to about 20 mg / kg, and the pharmaceutically acceptable dosage form comprising miglustat comprises a dose of about 200 mg to about 600 mg and is configured for oral administration.

18. 18. The kit of claim 16 or 17, wherein the instructions include instructions for administering the miglustat about 1 hour before administering the recombinant human acid alpha-glucosidase.

19. 19. The kit of any one of claims 16 to 18, wherein the pharmaceutically acceptable dosage form comprising the recombinant human acid α-glucosidase is configured for intravenous administration at a dose of about 5 mg / kg to about 20 mg / kg, and the pharmaceutically acceptable dosage form comprising miglustat comprises a dose of about 50 mg to about 200 mg and is configured for oral administration.

20. 20. The kit of any one of claims 16 to 19, wherein the pharmaceutically acceptable dosage form comprising the recombinant human acid α-glucosidase is configured for intravenous administration at a dose of about 20 mg / kg, and the pharmaceutically acceptable dosage form comprising miglustat comprises a dose of about 260 mg and is configured for oral administration.

21. 21. The kit of any one of claims 16 to 20, wherein the instructions comprise instructions for administering the pharmaceutically acceptable dosage form comprising miglustat about 1 hour before administration of the pharmaceutically acceptable dosage form comprising the recombinant human acid alpha-glucosidase.

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