High-concentration α-glucosidase composition for the treatment of Pompe disease

Abstract

To provide novel compositions for treating Pompe disease, and methods for administering such compositions to a subject.SOLUTION: The present application provides compositions comprising high concentrations of acid α-glucosidase in combination with an active site-specific chaperone for the acid α-glucosidase, and methods for treating Pompe disease in a subject in need thereof, including a method of administering such compositions to the subject. The present application also provides methods for increasing the in vitro and in vivo stability of an acid α-glucosidase enzyme formulation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 61 / 607,920 filed 7 March 2012 and U.S. Provisional Patent Application No. 61 / 750,718 filed 9 January 2013, each claiming priority and each incorporated herein by whole by reference.

[0002] 1. Preface The present invention relates to methods for treating, preventing, and / or curing Pompe disease. The present invention also relates to compositions and drugs that can be labeled for use in the treatment of Pompe disease. [Background technology]

[0003] 2.Background technology Pompe disease (acid maltase deficiency) is caused by a deficiency of the enzyme acid alpha-glucosidase (GAA). GAA metabolizes glycogen, the stored form of sugar used for energy, into glucose. The accumulation of glycogen leads to a progressive muscle disease throughout the body, affecting various body tissues, particularly the heart, skeletal muscle, liver, and nervous system. According to the National Institute of Neurological Disorders and Stroke, Pompe disease is estimated to occur in approximately 1 in 40,000 births.

[0004] There are three recognized types of Pompe disease: infantile, adolescent, and adult-onset (see, for example, Hirschhorn and Reuser, In:Scriver CR, Beaudet AL, Sly W, Valle D, editors; Metabolic and Molecular Bases of Inherited Disease, Vol. III, New York: McGraw-Hill; 2001. pp. 3389-420, 2001: 3389-3420). Infantile Pompe disease is the most severe, presenting with symptoms including severe muscle tone deficiency, weakness, enlarged liver and heart, and cardiomyopathy. Swallowing may be difficult, and the tongue may protrude and enlarge. Most children die before the age of two from respiratory or cardiac complications, but a subset of infantile-onset patients survive longer (non-classical infant patients). Juvenile-onset Pompe disease is initially found in early to late childhood and involves progressive weakness of the respiratory muscles in the trunk, diaphragm, and lower extremities, as well as exercise intolerance. Most juvenile-onset Pompe patients do not survive beyond 20 or 30 years of life. Adult-onset symptoms include generalized muscle weakness, as well as weakness of the respiratory muscles in the trunk, lower extremities, and diaphragm. Some adult patients do not have major symptoms or exercise limitations.

[0005] Diagnosing Pompe disease is difficult unless identified during prenatal screening. Diagnosing adult-onset Pompe disease is even more challenging due to the wide variation in the number, severity, and type of symptoms experienced by patients, which may suggest more common conditions such as muscular dystrophy. Diagnosis is supported by measuring alpha-glucosidase activity and / or detecting pathological levels of glycogen in biological samples. The only currently approved treatment is enzyme replacement therapy with recombinant alpha-glucosidase.

[0006] Pompe disease is one of several symptoms of glycogen-related disorders. Other examples include debranching enzyme deficiency (Cori's-Forbes disease; glycogen storage disease type III); branching enzyme deficiency (glycogen storage disease type IV; Anderson's disease); muscle phosphorylase deficiency (McCardle's disease, glycogen storage disease type V); phosphofructokinase deficiency-M isoform (Tauri's disease; glycogen storage disease type VII); phosphorylase b kinase deficiency (glycogen storage disease type VIII); phosphoglycerate kinase A isoform deficiency (glycogen storage disease type IX); and phosphoglycerate M-mutase deficiency (glycogen storage disease type X). [Overview of the project] [Means for solving the problem]

[0007] 3. Means for solving the problem The present invention relates to a method for treating Pompe disease (e.g., infant-onset Pompe disease), which involves administering an acid α-glucosidase (GAA) enzyme (e.g., recombinant human GAA (rhGAA)) to an individual in need of such treatment in combination with an active site-specific chaperone (ASSC) for the GAA enzyme (e.g., 1-deoxynojirimycin (DNJ, 1-DNJ)).

[0008] The present invention further provides a method for increasing the in vivo and in vitro stability of GAA enzymes in a suitable three-dimensional structure. In one embodiment, a combination of an acid α-glucosidase (GAA) enzyme (e.g., recombinant human GAA (rhGAA)) and an ASSC for a GAA enzyme (e.g., 1-deoxynojirimycin or 1-deoxynojirimycin-HCl) is administered to an individual requiring such treatment. When combined with the ASSC, the GAA enzyme is stereostemically stabilized and is highly suitable for withstanding, for example, heat and pH challenges.

[0009] In a given embodiment, the GAA enzyme is combined with ASSC at a high concentration, for example, at a concentration of approximately 5 to 250 mg / mL.

[0010] In a given embodiment, the GAA enzyme is combined with ASSC at a high concentration, for example, at a concentration selected from the group consisting of approximately 25 mg / mL, approximately 80 mg / mL, approximately 115 mg / mL, approximately 160 mg / mL, approximately 200 mg / mL, and approximately 240 mg / mL.

[0011] In a given embodiment, the GAA enzyme is combined with ASSC, and the ASSC is present at a concentration of approximately 5 mg / mL to approximately 200 mg / mL.

[0012] In a given embodiment, the GAA enzyme is combined with ASSC, and the ASSC is present at a concentration selected from the group consisting of approximately 32 mg / mL and approximately 160 mg / mL.

[0013] In a given embodiment, the GAA enzyme is combined with ASSC, and the ASSC is present at a concentration of approximately 0.5 mM to approximately 20 mM.

[0014] In a given embodiment, the GAA enzyme is combined with ASSC as a co-formulation.

[0015] In a given embodiment, the GAA enzyme is combined with ASSC in a co-formulation, and the co-formulation further comprises an excipient. In a given embodiment, the excipient is selected from the group consisting of polyethyl glycol (PEG), PEG-400, arginine, arginine and glutamic acid, proline, γ-cyclodextrin, and combinations thereof.

[0016] In certain embodiments, the formulations of the present invention maintain physical and chemical stability over long periods despite high protein concentrations and possess a viscosity suitable for subcutaneous administration. The formulations of the present invention are established, at least in part, based on the remarkable discovery that GAA enzyme combined with ASSC can maintain solubility at high concentrations (e.g., 25 mg / mL), maintain a viscosity suitable for injection (e.g., subcutaneous administration), and remain non-aggregated.

[0017] In a given embodiment, the composition of the present invention contains more than approximately 5 mg / mL of GAA enzyme.

[0018] In certain embodiments, the composition of the present invention comprises about 25 mg / mL of GAA enzyme and about 10 mM of DNJ.

[0019] In certain embodiments, the composition of the present invention comprises about 25 mg / mL of GAA enzyme and about 1 mM of DNJ.

[0020] An advantage of the formulations of the present invention is to provide a high concentration of protein without increasing protein aggregation, which typically occurs as the protein concentration increases. In one embodiment, the formulation of the present invention has less than about 1% aggregate protein.

[0021] According to one aspect of the present invention, there is provided a method of improving the delivery of GAA to the tissues of an individual having Pompe disease, such as muscle tissue. The method comprises administering GAA in combination with ASSC subcutaneously to the individual. In some embodiments, the combination of GAA and ASSC is administered at a dose sufficient to result in a peak concentration of GAA in the tissue of the subject within 24 hours from the administration of the dose. In certain embodiments, the combination of GAA and ASSC is administered at a dose sufficient to result in a peak concentration of GAA in the tissue of the subject within about 10 to about 50 hours, or within about 45, 40, 35, 30, 25, or less than that time from the administration of the dose. In some embodiments, the dose does not result in toxic levels of GAA in the liver of the individual.

[0022] In various non-limiting embodiments, the ASSC for the GAA enzyme is a small molecule inhibitor of the GAA enzyme that comprises a reversible and competitive inhibitor of the GAA enzyme.

[0023] In one embodiment, ASSC has the formula: JPEG0007905382000001.jpg48135(wherein, R1 is H or a linear or branched alkyl, cycloalkyl, alkoxyalkyl or aminoalkyl containing 1 to 12 carbon atoms, which are optionally substituted with -OH, -COOH, -Cl, -F, -CF3, -OCF3, -O-C(=O)N-(alkyl)2; R2 is H or a linear or branched alkyl, cycloalkyl or alkoxylalkyl containing 1 to 9 carbon atoms), and includes its pharmaceutically acceptable salts, esters and prodrugs. In one embodiment, ASSC is as defined above and R1 is H. In another embodiment, ASSC is as defined above and R2 is H.

[0024] In a specific non-limiting embodiment, ASSC is of the following formula: JPEG0007905382000002.jpg41135 represents 1-deoxynojirimycin (1-DNJ), or a pharmaceutically acceptable salt, ester or prodrug of 1-deoxynojirimycin. In one embodiment, the salt is hydrochloride (i.e., 1-deoxynojirimycin-HCl).

[0025] In a specific non-limiting embodiment, ASSC is of the following formula: JPEG0007905382000003.jpg63135 represents N-butyl-deoxynojirimycin (NB-DNJ; Zavesca (registered trademark), Actelion Pharmaceuticals Ltd, Switzerland), or a pharmaceutically acceptable salt, ester or prodrug of NB-DNJ.

[0026] In a specific non-limiting embodiment, ASSC is of the following formula: JPEG0007905382000004.jpg41135 represents C 10 H 19 NO4, or C 10 H 19A pharmaceutically acceptable salt, ester, or prodrug of NO4. In one embodiment, the salt is a hydrochloride salt.

[0027] In a non-limiting specific embodiment, the ASSC is given by the following formula: C represented by JPEG0007905382000005.jpg63135 12 H 23 NO4, or C 12 H 23 A pharmaceutically acceptable salt, ester, or prodrug of NO4. In one embodiment, the salt is a hydrochloride salt. [Brief explanation of the drawing]

[0028] [Figure 1] This document describes the stability of recombinant human GAA (Myozyme®, Genzyme Corp.) at ER pH (7.4) or lysosomal pH (5.2) in the presence or absence of 100 μM 1-deoxynojirimycin hydrochloride (1-DNJ-HCl), as determined by a thermal stability assay. The thermal stability assay utilizes heat to induce protein denaturation, which is monitored using a SYPRO Orange dye that fluoresces after binding to hydrophobic amino acids (not exposed within the folded protein). Protein structures requiring more heat for denaturation are, by definition, more stable. As shown above, Myozyme® is generally far more stable at lysosomal pH (5.2) compared to ER pH (7.4). However, enzyme stability at pH 7.4 is significantly increased after the addition of 100 μM 1-deoxynojirimycin compared to Myozyme® alone. [Figure 2A]This study demonstrates the effect of 1-DNJ-HCl on recombinant human GAA (Myozyme®, Genzyme Corp.) enzyme activity at plasma pH (7.4) or lysosomal pH (5.2) at 37°C. GAA activity was evaluated to assess the ability of GAA ASSC to extend rhGAA activity over a certain period of time. Myozyme® (45 nM) was incubated at 37°C for 24 hours in pH 7.4 or pH 5.2 buffer with or without 50 μM 1-DNJ. Samples were assayed for GAA enzyme activity using 4-MU-α-glucose at 0, 3, 6, and 24 hours, and residual GAA activity was expressed as a percentage of the initial activity. These results indicate that 1-DNJ restores the loss of GAA enzyme activity at plasma pH (7.4). [Figure 2B] Figure 2A shows parallel SYPRO Orange thermal stability experiments to determine whether the loss of enzyme activity, particularly Myozyme® activity, at ERpH (7.4) correlates with protein unfolding and denaturation. Myozyme® (0.9 μM) was incubated at 37°C in pH 7.4 or pH 5.2 buffer with or without 100 μM 1-DNJ-HCl, and protein folding was monitored hourly for 24 hours. Figures 2A and 2B show that GAA denaturation correlates with the loss of enzyme activity (compare the curves in the two figures with the diamond-shaped curves). More importantly, these results indicate that 1-DNJ can prevent GAA denaturation and thus prevent the loss of enzyme activity at plasma pH. [Figure 3]The results of GAA activity tests in ERT-receiving GAA KO mice with and without co-administration of 1-DNJ-HCl are shown. Myozyme® was administered via IV infusion at a dose of 10 mg / kg once weekly for up to 3 weeks, either alone or in combination with 10, 100, or 1000 mg / kg of 1-DNJ-HCl 30 minutes before Myozyme® administration and 8, 16, and 24 hours later. These results indicate that Myozyme® tissue uptake (as a measure of GAA activity) decreased 7 days after injection. Co-administration of 1-DNJ-HCl and Myozyme® promotes a dose-dependent increase in Myozyme® uptake up to 7 days after injection. The effect of 1-DNJ-HCl was more evident and significant at 4 and 7 days after injection, regardless of whether Myozyme® was administered once, twice, or three times per week (p<0.05 t-test, compared to Myozyme® alone). [Figure 4] This study shows that 1-DNJ-HCl inhibits GAA with an IC50 of approximately 1 μM. [Figure 5] The results of a thermal stability assay that utilizes heat to induce protein denaturation are shown, and such denaturation is monitored using the SYPRO Orange dye, which fluoresces after binding to hydrophobic amino acids (not exposed within the folded protein). 1-DNJ-HCl increases the thermal stability of GAA, as evidenced by the dose-dependent increase in the melting temperature of GAA. [Figure 6]The results for GAA activity in rats over 24 hours after IV administration of 10 mg / kg rhGAA or saline with and without 3 mg / kg or 30 mg / kg 1-DNJ-HCl are shown. rhGAA or saline was administered 30 minutes after 1-DNJ-HCl administration. In this example, 1-DNJ-HCl suppressed the loss of enzyme activity after administration, thereby extending the in vivo half-life of rhGAA. The in vivo half-life of rhGAA was extended from 1.4 ± 0.2 hours (0 mg / kg 1-DNJ-HCl) to 2.1 ± 0.2 hours (3 mg / kg 1-DNJ-HCl) and 3.0 ± 0.4 hours (30 mg / kg 1-DNJ-HCl). [Figure 7] This study shows the GAA activity in cardiac and diaphragmatic tissue in GAA KO mice treated with ERT monotherapy and ERT / ASSC combination therapy (rhGAA + 1-DNJ-HCl). rhGAA uptake in the heart and diaphragm is increased when co-administered with 1-DNJ-HCl. [Figure 8] This study demonstrates that 1-DNJ-HCl prevents rhGAA enzyme inactivation in blood. Myozyme® (0.5 μM) was incubated at 37°C in citrate-anticoagulated whole blood in or without 50 μM 1-DNJ-HCl. Aliquots were collected at 0, 2, 4, 8, and 24 hours and centrifuged to obtain plasma. These plasma samples were then diluted in potassium acetate buffer (pH 4.0) and assayed for GAA activity using 4-methylumbeliferyl-α-glucose (4-MUG) fluorescence-generating substrate. GAA activity measured for individual samples at each time point was normalized to 0 hours and expressed as a percentage of initial activity. Data from four independent experiments were analyzed to obtain the mean (and standard deviation), which was then plotted against time to assess the loss of enzyme activity over this time course. [Figure 9]This study demonstrates that low 1-DNJ-HCl concentrations prevent rhGAA enzyme inactivation in the blood. Myozyme® (0.5 μM) was incubated at 37°C in citrate-treated whole blood at various 1-DNJ-HCl concentrations (0–100 μM). Aliquots were collected at 0, 3, and 6 hours and centrifuged to obtain plasma. These plasma samples were then diluted in potassium acetate buffer (pH 4.0) and assayed for GAA activity using 4-methylumbeliferyl-α-glucose (4-MUG) fluorescence-generating substrate. GAA activity measured for individual samples at each time point was normalized to 0 hours and expressed as a percentage of initial activity. Residual GAA enzyme activity was plotted against time to evaluate the loss of enzyme activity associated with 1-DNJ-HCl concentration over this time course. [Figure 10] The experimental design for Example 9 is shown below. [Figure 11] This study demonstrates that Myozyme® co-administration with 1-DNJ-HCl resulted in significantly higher tissue glycogen reduction in GAA KO mice compared to Myozyme® alone. Glycogen reduction with Myozyme® alone was 93±1%, 41±4%, 69±3%, and 18±4% in the heart, diaphragm, soleus muscle, and quadriceps muscle, respectively, compared to untreated mice. Glycogen reduction with Myozyme® co-administration with 1-DNJ-HCl was 96±0.6%, 66±5%, 82±3%, and 23±3%, respectively. [Figure 12] This study demonstrates that combining 1 mM DNJ with 25 mg / mL Myozyme® reduces aggregation of Myozyme® at a neutral pH of 7.4 and 37°C. Aggregation was evaluated after a 4-week incubation period. [Figure 13] This study demonstrates that co-administration of 30 mg / kg DNJ, equivalent in dose to 10 mg / kg Myozyme®, via tail vein injection increased the circulating plasma half-life and tissue uptake of Myozyme® within the quadriceps muscle. [Figure 14]This study demonstrates that subcutaneous administration of DNJ co-prescription with 20 mg / kg Myozyme® increased circulating levels of rhGAA compared to administration of 20 mg / kg Myozyme® without DNJ. [Figure 15] This shows the rhGAA activity in the skin at the injection site three days after the final subcutaneous administration of rhGAA or a co-formulation of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 16A] This shows the rhGAA activity in the ventral skin three days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 16B] This shows the glycogen levels in the ventral skin 14 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 17A] This shows the rhGAA activity in the heart of mice 3 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ as described in Example 13. [Figure 17B] This shows the intracardiac glycogen levels 14 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 18A] This shows the rhGAA activity in the tongue three days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 18B] This shows the intralingual glycogen levels in mice 14 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ, as described in Example 13. [Figure 19A] This shows the rhGAA activity in the diaphragm three days after the final subcutaneous administration of rhGAA or a co-formulation of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 19B] This shows the glycogen levels in the diaphragm 14 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 20A] This shows the rhGAA activity in the biceps muscle three days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 20B] This shows the glycogen levels in the biceps muscle 14 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 21A] This shows the rhGAA activity in the triceps muscle three days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 21B] This shows the glycogen levels in the triceps muscle 14 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 22A] This shows the rhGAA activity in the gastrocnemius muscle three days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 22B] This shows the glycogen levels in the gastrocnemius muscle 14 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 23A] This shows the rhGAA activity in the quadriceps muscle three days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 23B] This shows the glycogen levels in the quadriceps muscle 14 days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 24] This shows the rhGAA activity in the soleus muscle three days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 25] This shows the rhGAA activity in the liver three days after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 26]This shows a comparison of rhGAA activity in various tissues, tested 3 days after the final subcutaneous administration of rhGAA or a co-formulation of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 27A] This shows the plasma rhGAA activity 2 hours (A) after the final subcutaneous administration (rhGAA or rhGAA and 1-DNJ) of the preparations described in Example 13 to mice. [Figure 27B] This shows the plasma rhGAA activity 4 hours (B) after the final subcutaneous administration (B) of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13. [Figure 28] The plasma rhGAA activity and plasma GAA protein levels (measured by Western blotting) two hours after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13, are shown. [Figure 29] The plasma rhGAA activity and plasma GAA protein levels (measured by Western blotting) four hours after the final subcutaneous administration of rhGAA or a combination of rhGAA and 1-DNJ to mice, as described in Example 13, are shown. [Modes for carrying out the invention]

[0029] 5. Modes for Carrying Out the Invention This invention is at least in part based on the discovery that combining an acid α-glucosidase (GAA) enzyme (e.g., recombinant human GAA (rhGAA)) with an ASSC for a GAA enzyme (e.g., 1-deoxynojirimycin) results in a remarkable increase in GAA activity in vivo compared to either treatment alone. This invention is also at least in part based on the discovery that a GAA enzyme (e.g., rhGAA) stabilizes to a proper conformation after the addition of an ASSC for the GAA enzyme, both in vitro and in vivo. This invention is also at least in part based on the discovery that combining an ASSC with high concentrations of GAA reduces GAA aggregation, which normally occurs at high GAA concentrations.

[0030] For clarity, and not for the purpose of limitation, this detailed description is divided into the following subsections. (i) Definition; (ii) Pompe disease; (iii) Obtaining GAA and ASSC; (iv) Treatment of Pompe disease using ERT and ASSC; (v) Pharmaceutical compositions; (vi) Stability in vitro; and (vii) Stability in vivo.

[0031] 5.1 Definition Terms used herein have the common meaning in the art in the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide practitioners with further guidance in describing the compositions and methods of the present invention, as well as their preparation and use.

[0032] According to the present invention, the "subject" or "patient" is a human or a non-human animal. While the animal subject is preferably a human, the compounds and compositions of the present invention also have applications in veterinary medicine for the treatment of domesticated species such as dogs, cats, and various other pets; domesticated species such as cattle, horses, sheep, goats, and pigs; wild animals such as wild animals or animals in zoos; and birds such as chickens, turkeys, quail, and songbirds.

[0033] The term "enzyme replacement therapy" or "ERT" refers to the introduction of non-natural, purified enzymes into individuals who are deficient in those enzymes. The administered enzymes can be obtained from natural sources or through recombinant expression. The term also refers to the introduction of purified enzymes into individuals who otherwise require or would benefit from the administration of purified enzymes, for example, those suffering from protein deficiencies. The introduced enzymes may be purified recombinant enzymes produced in vitro, or they may be purified enzymes from isolated tissues or fluids, such as placenta or milk, or plants.

[0034] The term "stabilizing the appropriate three-dimensional structure" refers to the ability of a compound, peptide, or other molecule to stabilize a wild-type protein, or a mutant protein that can perform the wild-type function of such a wild-type protein in vitro and in vivo, so that the structure of the wild-type or mutant protein can be maintained in its native or appropriate form. This effect may manifest in practice through one or more of the following: (i) extension of the protein's shelf life; (ii) increased activity per unit / quantity of the protein; or (iii) increased potency in vivo. This can be observed experimentally and by similar means during expression, through increased yield from the ER; or increased resistance to unfolding due to increased temperature (e.g., determined by a thermal stability assay); or through the presence of a chaotropic agent.

[0035] As used herein, the term “active site” refers to a region of a protein that has a specific biological activity. For example, an active site may be a site that contributes to an amino acid residue that binds to a substrate or other binding partner and directly participates in the formation and cleavage of chemical bonds. In the present invention, active sites may include catalytic sites of enzymes, antigen-binding sites of antibodies, ligand-binding domains of receptors, binding domains of regulatory factors, or receptor-binding domains of secreted proteins. Active sites may also include transactivation, protein-protein interaction, or DNA-binding domains of transcription factors and regulatory factors.

[0036] As used herein, the term "active site-specific chaperone" refers to any molecule, including proteins, peptides, nucleic acids, and carbohydrates, that specifically and reversibly interacts with the active site of a protein to improve the formation of a stable molecular structure. As used herein, "active site-specific chaperone" does not include endogenous common chaperones present in the cellular ER, such as Bip, calnexin, or calreticulin, nor common nonspecific chemical chaperones, such as heavy water, DMSO, or TMAO.

[0037] As used herein, the term “purified” refers to unrelated materials, including natural materials from which materials were obtained, i.e., materials isolated under conditions that reduce or eliminate the presence of contaminants. For example, purified proteins are preferably substantially free of other proteins or nucleic acids associated within the cell; purified nucleic acid molecules are preferably substantially free of proteins or other unrelated nucleic acid molecules that may be found together with such nucleic acid molecules within the cell. As used herein, the term “substantially free” is used operationally in the context of analytical testing of materials. Purified materials substantially free of contaminants are preferably at least 95% pure; more preferably at least 97% pure, and even more preferably at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, compositional analysis, biological assay, and other methods known in the art. In certain embodiments, “purified” means that the level of contaminants is below a level acceptable by regulatory authorities for safe administration to humans or non-human animals.

[0038] As used herein, the terms “mutant” and “mutation” mean any detectable change in genetic material, e.g., DNA, or any process, mechanism, or result of such change. Mutants and mutations include gene mutations in which the structure of a gene (e.g., DNA sequence) is altered, any gene or DNA resulting from any mutation process, and any expression products (e.g., RNA, protein, or enzyme) expressed by a modified gene or DNA sequence.

[0039] As used herein, the term “mutant protein” refers to a protein translated from a gene containing a gene mutation that results in a change in the protein sequence. In certain embodiments, such mutations make it impossible for the protein to achieve its native three-dimensional structure under conditions normally present in the ER. This failure to achieve the three-dimensional structure leads to degradation of these proteins rather than transport to their proper locations within the cell via normal pathways in the protein transport system. Other mutations may result in decreased activity or a more rapid metabolic turnover.

[0040] As used herein, the term “wild-type gene” refers to a nucleic acid sequence that encodes a protein capable of having normal biological functional activity in vivo. Wild-type nucleic acid sequences may include nucleotide changes that differ from published known sequences, insofar as they result in amino acid substitutions that have little or no effect on biological activity. The term “wild-type” may also include nucleic acid sequences that have been engineered to encode a protein capable of increased or enhanced activity compared to endogenous or native proteins.

[0041] As used herein, the term "wild-type protein" refers to any protein encoded by a wild-type gene that is capable of having functional biological activity when expressed or introduced in vivo. The term "normal wild-type activity" refers to the normal physiological function of a protein within a cell. Such functionality can be tested by any known means for establishing the functionality of a protein.

[0042] The term "genetically modified" refers to cells that express a specific gene product after the introduction of a nucleic acid containing a coding sequence that encodes a gene product, along with regulatory elements that control the expression of that coding sequence. The introduction of nucleic acid can be achieved by any method known in the art, including gene targeting and homologous recombination. As used herein, this term also includes cells that have been engineered, for example, by gene activation techniques, to express or overexpress endogenous genes or gene products that are not normally expressed by those cells.

[0043] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically acceptable and typically do not produce undesirable reactions when administered to humans, whether or not they are used in connection with the pharmaceutical compositions of the present invention. Preferably, as used herein, the term “pharmaceutically acceptable” means that it is authorized by federal or state regulatory authorities or listed in the United States Pharmacopeia or other commonly recognized pharmacopoeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle administered with the compound. Such pharmaceutical carriers may be sterile liquids such as water and oil. Water or aqueous saline solutions, as well as aqueous solutions of dextrose and glycerol, are preferred as carriers, particularly for use as injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin, 18th edition.

[0044] The terms “therapeutically effective dose” and “effective amount” refer to the amount of compound sufficient to produce a therapeutic response. In embodiments in which ASSC and GAA are administered as a complex, the terms “therapeutically effective dose” and “effective amount” may refer to the amount of the complex sufficient to produce a therapeutic response. A therapeutic response may be any response that the user (e.g., a clinician) would recognize as an effective response to the treatment. Therefore, a therapeutic response would generally be the recovery of one or more symptoms or signs of a disease or illness.

[0045] It should be noted that the concentration of ASSCs, which are inhibitory during the in vitro production, transport, or storage of purified therapeutic proteins, may still constitute an "effective amount" for the purposes of the present invention after in vivo administration, due to the dilution of ASSCs (and consequently, the shift in binding due to changes in equilibrium), bioavailability, and metabolism.

[0046] The term "alkyl" refers to a straight-chain or branched-chain hydrocarbon consisting only of carbon and hydrogen atoms that contains no unsaturation and is bonded to the remainder of the molecule by a single bond, such as methyl, ethyl, N-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl).

[0047] The term "alkenyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and may be straight-chain or branched-chain, for example, ethenyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl. 20 20

[0048] The term "cycloalkyl" refers to an unsaturated non-aromatic monocyclic- or polycyclic hydrocarbon ring system such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. Examples of polycyclic cycloalkyl groups include bridged cyclic groups such as perhydronaphthyl, adamantyl and norbornyl groups, or spiro bicyclic groups such as spiro(4,4)nona-2-yl.

[0049] The term "aryl" refers to an aromatic radical having from about 6 to about 14 carbon atoms such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, etc.

[0050] The term "heterocyclic" refers to a stable 3- to 15-membered ring radical consisting of carbon atoms and 1 to 5 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For the purposes of the present invention, the heterocyclic radical may be a monocyclic or bicyclic ring system, which may include fused or bridged ring systems, and the nitrogen, carbon, oxygen or sulfur atoms within the heterocyclic radical may optionally be oxidized to various oxidation states. In addition, when present, the nitrogen atoms may optionally be quaternized; and the ring radical may be partially or fully saturated (i.e., heterocyclic aromatic or heteroaryl aromatic).

[0051] Heterocyclic radicals can be bonded to the main structure at either a heteroatom or carbon atom, resulting in the formation of a stable structure.

[0052] The term "heteroaryl" refers to a heterocyclic ring in which the ring is aromatic.

[0053] The substituents in "substituted alkyl," "substituted alkenyl," "substituted cycloalkyl," "substituted aryl," and "substituted heteroaryl" may be the same or different, and one or more of them may be hydrogen, halogen, acetyl, nitro, carboxyl, oxo(=O), CF3, -OCF3, NH2, -C(=O)-alkyl2, OCH3, or optionally selected from the group of substituted groups selected from alkyl, alkoxy, and aryl.

[0054] The term "halogen" refers to the radicals of fluorine, chlorine, bromine, and iodine.

[0055] 5.2 Pompe disease Pompe disease is an autosomal recessive LSD characterized by deficient acid α-glucosidase (GAA) activity, which impairs lysosomal glycogen metabolism. This enzyme deficiency leads to lysosomal glycogen accumulation, progressive skeletal muscle weakness, impaired cardiac function, respiratory failure, and / or CNS damage in the later stages of the disease. Gene mutations in the GAA gene result in decreased enzyme expression or the generation of mutant forms of the enzyme with altered stability and / or biological activity, ultimately leading to the disease (see Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid α-Glucosidase (Acid Maltase) Deficiency, The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th edition, pp. 2443-2464). The three recognized clinical forms of Pompe disease (infant, adolescent, and adult) correlate with levels of residual α-glucosidase activity (Reuser AJ et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61-S69). ASSCs (also referred to elsewhere as "pharmacological chaperones") represent a promising new therapeutic approach for treating genetic disorders such as lysosomal storage disorders (e.g., Pompe disease).

[0056] Infant Pompe disease (type I or A) is the most common and severe form, characterized by growth retardation, generalized hypotonia, cardiac hypertrophy, and cardiorespiratory failure within the first two years of life. Juvenile Pompe disease (type II or B) is of intermediate severity and is characterized by a predominance of muscular symptoms without cardiomegaly. Individuals with juvenile Pompe disease usually die before reaching the age of 20 due to respiratory failure. Adult Pompe disease (type III or C) often presents as a slowly progressive myopathy in the teens or late sixties (Felice KJ et al., 1995, Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature, Medicine 74, 131-135).

[0057] Pompe demonstrated that α-glucosidase undergoes extensive post-translational modification through glycosylation, phosphorylation, and proteolysis. Optimal glycogen catalysis requires the conversion of a 110-kilodalton (kDa) precursor to mature forms of 76 and 70 kDa via lysosome proteolysis.

[0058] As used herein, the term "Pompe disease" refers to all types of Pompe disease. The formulations and administration regimens disclosed herein can be used, for example, to treat type I, type II, or type III Pompe disease.

[0059] 5.3 Acquisition of GAA and ASSC GAA can be obtained from cells that endogenously express GAA, or GAA may be recombinant human GAA (rhGAA) as described herein. In one non-limiting embodiment, rhGAA is full-length wild-type GAA. In another non-limiting embodiment, rhGAA comprises a subset of amino acid residues present in wild-type GAA, such subset comprising amino acid residues of wild-type GAA that form an active site for substrate binding and / or substrate reduction. Accordingly, the present invention envisions rhGAA as a fusion protein comprising a wild-type GAA active site for substrate binding and / or substrate reduction and other amino acid residues that may or may not be present in wild-type GAA.

[0060] GAA can be obtained from commercial sources or by synthesis techniques known to those skilled in the art. Wild-type enzymes can be expressed in recombinant cell expression systems (e.g., mammalian or insect cells - see generally U.S. Patent No. 5,580,757 granted to Desnick et al.; U.S. Patents No. 6,395,884 and 6,458,574 granted to Selden et al.; U.S. Patent No. 6,461,609 granted to Calhoun et al.; U.S. Patent No. 6,210,666 granted to Miyamura et al.; U.S. Patent No. 6,083,725 granted to Selden et al.; U.S. Patent No. 6,451,600 granted to Rasmussen et al.; U.S. Patent No. 5,236,838 granted to Rasmussen et al.; and U.S. Patent No. 5,879,680 granted to Ginns et al.), human placenta, or livestock milk (Reuser et al. It can be purified from (see U.S. Patent No. 6,188,045, granted to al.). After injection, the exogenous enzyme is expected to be taken up by tissues via nonspecific or receptor-specific mechanisms. Generally, the uptake efficiency (without using ASSC) is not high, and the circulation time of the exogenous protein is short (Ioannu et al., Am.J.Hum.Genet. 2001;68:14-25). In addition, the exogenous protein is unstable and subject to rapid intracellular degradation in vitro.

[0061] Other synthetic techniques for obtaining GAA suitable for pharmaceutical use include, for example, U.S. Patent Nos. 7,560,424 and 7,396,811 granted to Lebowitz et al. These references can be found in U.S. Patent Publication No. 2009 / 0203575, U.S. Patent Publication No. 2009 / 0029467, U.S. Patent Publication No. 2008 / 0299640, U.S. Patent Publication No. 2008 / 0241118, U.S. Patent Publication No. 2006 / 0121018 and U.S. Patent Publication No. 2005 / 0244400, U.S. Patent No. 7,423,135, U.S. Patent No. 6,534,300 and U.S. Patent No. 6,537,785; in International Publication No. 2005 / 077093 and U.S. Patent Publication No. 2007 / 0280925, and U.S. Patent Publication No. 2004 / 0029779. These references are incorporated herein by reference in their entirety.

[0062] In one embodiment, GAA is alglucosidase α, consisting of human enzymatic acid α-glucosidase (GAA), which is encoded by the most dominant of nine observed haplotypes of this gene and is produced by recombinant DNA technology within a Chinese hamster ovary cell line. Alglucosidase α is available from Genzyme Corporation (Cambridge, MA) as Myozyme® and Lumizyme®.

[0063] ASSCs can be obtained using synthesis techniques known to those skilled in the art. For example, ASSCs such as 1-DNJ, which may be used in this application, can be prepared as described in U.S. Patent No. 6,274,597 and U.S. Patent No. 6,583,158, and U.S. Patent Application Publication No. 2006 / 0264467, respectively, which are incorporated herein by whole reference.

[0064] In one embodiment of the present application, ASSC is α-homonojirimycin and GAA is hrGAA (e.g., Myozyme® or Lumizyme®). In an alternative embodiment, ASSC is castanospermine and GAA is hrGAA (e.g., Myozyme® or Lumizyme®). ASSCs (e.g., α-homonojirimycin and castanospermine) can be obtained from synthetic libraries that provide potential ASSC sources according to the present invention (see, for example, Needels et al., Proc.Natl.Acad.Sci.USA 1993;90:10700-4; Ohlmeyer et al., Proc.Natl.Acad.Sci.USA 1993;90:10922-10926; Lam et al., PCT International Publication No. 92 / 00252; Kocis et al., PCT International Publication No. 94 / 28028). Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH) and Microsource (New Milford, Conn.). A library of rare chemicals is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available from, for example, Pan Laboratories (Bothell, Wash.) or Myco Search (NC), or can be readily produced. In addition, naturally and synthetically produced libraries and compounds are readily modified according to Res. 1986;155:119-29.

[0065] In one embodiment, ASSCs useful to the present invention are lysosomal enzyme inhibitors and include glucose and galactose imino sugar derivatives as described in Asano et al., J. Med. Chem. 1994; 37: 3701-06; Dale et al., Biochemistry 1985; 24: 3530-39; Goldman et al., J. Nat. Prod. 1996; 59: 1137-42; Legler et al., Carbohydrate Res. 1986; 155: 119-29. These derivatives include those available from commercial sources such as Toronto Research Chemicals, Inc. (North York, On. Canada) and Sigma.

[0066] 5.4 Treatment of Pompe disease using ERT and ASSC The present invention provides a method for using GAA (e.g., rhGAA) in combination with ASSC for GAA. One embodiment of the present invention provides a combination therapy of GAA (e.g., hrGAA ERT) and ASSC. For example, the ASSC chaperone 1-deoxynojirimycin-HCl binds to mutant GAA, increasing the ability of GAA to be stabilized into a proper three-dimensional structure.

[0067] One embodiment of the present invention provides a method for treating Pompe subset patients with IVS 1(-13T>G) splicing deficiency using ASSC and hrGAA enzyme replacement therapy. In cell lines derived from late-onset Pompe patients with this common splicing mutation, 1-deoxynojirimycin-HCl increased GAA levels both alone and in combination with hrGAA.

[0068] In one non-limiting embodiment of the present invention, 1-deoxynojirimycin-HCl, or a pharmaceutically acceptable salt thereof, may be administered to subjects at doses of approximately 10 mg / kg to 1000 mg / kg, preferably orally, before, simultaneously with, or after administration of GAA. In one non-limiting embodiment, 1-deoxynojirimycin-HCl and recombinant human GAA exhibit remarkable efficacy in treating cellular enzyme activity, glycogen depletion, and Pompe disease. In rats, the plasma half-life of recombinant human GAA (rhGAA) was doubled when 1-deoxynojirimycin-HCl (30 mg / kg po) was administered in a dosing regimen that included administration 30 minutes prior to rhGAA injection. In GAA KO mice, rhGAA uptake was approximately doubled in the heart and diaphragm when 1-deoxynojirimycin-HCl (100 mg / kg po) was administered in a dosing regimen that included administration prior to rhGAA injection. These results demonstrate that co-administration of ASSC and rhGAA dramatically increases enzyme exposure and tissue uptake in vivo.

[0069] For example, one embodiment of the present invention provides a method for treating Pompe disease, which includes administering GAA (e.g., rhGAA) in combination with ASSC (e.g., 1-DNJ-HCl) at a dose of about 1 to about 5000 mg / kg twice a week, once a week, or once every two weeks for up to about 10 weeks, at regular intervals before and after GAA infusion. For example, ASSC may be administered within 2 hours of infusion, and then once, twice, three, four, five, or six times within 24 hours after infusion, at regular intervals.

[0070] In one particular embodiment, GAA is Myozyme®, administered once weekly via infusion, and ASSC (e.g., 1-DNJ-HCl) is administered at 10 mg / kg, 100 mg / kg, or 1000 mg / kg 30 minutes prior to the infusion, and then 8, 16, and 24 hours after each Myozyme® infusion.

[0071] In another specific embodiment, GAA is Lumizyme®, administered once weekly via infusion, and ASSC (e.g., 1-DNJ-HCl) is administered at 10 mg / kg, 100 mg / kg, or 1000 mg / kg 30 minutes prior to the infusion, and then 8, 16, and 24 hours after each Lumizyme® infusion.

[0072] While not bound by any particular theory, acid α-glucosidase (GAA) is thought to function by removing terminal glucose residues from lysosomal glycogen. Several gene mutations reduce GAA transport and maturation. Pharmacological chaperone 1-DNJ selectively binds to the enzyme to achieve the correct conformation and stabilizes such enzymes, thereby increasing GAA levels and restoring proper protein transport to lysosomes.

[0073] In an alternative embodiment, the ASSC is administered as described in International Publication No. 2008 / 134628, which is incorporated herein by reference in its entirety.

[0074] In some embodiments, the route of administration is subcutaneous. Other routes of administration may be oral or parenteral, including via intravenous, intra-arterial, intraperitoneal, intraocular, intramuscular, buccal, intrarectal, vaginal, orbital, intracerebral, intradermal, intracranial, intraspinal, intraventricular, subarachnoid, intracisional, intrasacral, intrapulmonary, intranasal, transmucosal, transdermal, or inhalation. Methods, apparatus, and drug preparations for intrapulmonary delivery are described, for example, in U.S. Patent No. 5,785,049, U.S. Patent No. 5,780,019, and U.S. Patent No. 5,775,320, which are incorporated herein by reference. In some embodiments, the method of intradermal delivery is by ionophoretic delivery via a patch; an example of such delivery is taught in U.S. Patent No. 5,843,015, which is incorporated herein by reference.

[0075] Administration may be by periodic injection of bolus of the preparation, or as a sustained-release dosage form over a long period, or by intravenous or intraperitoneal administration from a reservoir that is external (e.g., an IV bag) or internal (e.g., a bioerosive implant, a bioprosthetic organ, or a population of transplanted GAA-producing cells). See, for example, U.S. Patent No. 4,407,957 and U.S. Patent No. 5,798,113, which are incorporated herein by reference. Methods and devices for intrapulmonary delivery are described, for example, U.S. Patent No. 5,654,007, U.S. Patent No. 5,780,014 and U.S. Patent No. 5,814,607, which are incorporated herein by reference. Other useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposome delivery, needle delivery injection, needle-free injection, nebulizers, aerosolizers, electroporation, and transdermal patches. Needle-free injector devices are described in U.S. Patent Nos. 5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications of which are incorporated herein by reference. Any GAA preparation described herein can be administered by these methods.

[0076] The delivery of the formulation may be continuous over a predetermined administration period ranging from several hours to several weeks, from one to several months, or up to one year or more. In certain embodiments, the dosage form is adapted for the delivery of GAA over a long period. Such delivery devices may be adapted for the administration of GAA over several hours (e.g., 2 hours, 12 hours, or 24 to 48 hours or more), several days (e.g., 2 to 5 days or more, about 100 days or more), several months, or several years. In some of these embodiments, the device is adapted for delivery over a period ranging from about one month to about 12 months or more. The GAA delivery device may be adapted to administer GAA to an individual over time ranges, for example, approximately 2 hours to 72 hours, 4 hours to 36 hours, 12 hours to 24 hours; approximately 2 days to 30 days, 5 days to 20 days, 7 days to 100 days or more, 10 days to 50 days; approximately 1 week to 4 weeks; approximately 1 month to 24 months or more, 2 months to 12 months, 3 months to 9 months; or other time ranges including incremental ranges within these ranges as needed.

[0077] In a given embodiment, the method of the present invention involves administering a dose of approximately 0.1 to approximately 50 mg / kg of GAA to an individual, for example, subcutaneously, with the dose being administered once daily, once every two days, once every three days, once every four days, once every five days, or once every six days. In a given embodiment, the formulation of the present application is administered once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, or seven times weekly.

[0078] In a given embodiment, the method of the present application comprises administering a co-formulation containing GAA and ASSC to an individual, the co-formulation being administered subcutaneously. In a given embodiment, the dose of GAA in the co-formulation is approximately 0.1 to approximately 5000 mg / kg, or approximately 10 to approximately 4000 mg / kg, or approximately 25 to approximately 3000 mg / kg, or approximately 50 to approximately 2000 mg / kg, or approximately 100 to approximately 1000 mg / kg, or approximately 200 to approximately 500 mg / kg.

[0079] In a given embodiment, the dose of GAA in the co-formulation is approximately 0.1 to approximately 100 mg / kg, or approximately 1 to approximately 80 mg / kg, or approximately 5 to approximately 50 mg / kg, or approximately 10 to approximately 40 mg / kg, or approximately 15 to approximately 25 mg / kg.

[0080] In a given embodiment, the dose of rhGAA in the co-formulation is approximately 20 mg / kg.

[0081] In a given embodiment, the dose of ASSC in the co-formulation is approximately 0.1 to approximately 5000 mg / kg, or approximately 10 to approximately 4000 mg / kg, or approximately 25 to approximately 3000 mg / kg, or approximately 50 to approximately 2000 mg / kg, or approximately 100 to approximately 1000 mg / kg, or approximately 200 to approximately 500 mg / kg.

[0082] In a given embodiment, the dose of ASSC in the co-formulation is approximately 0.1 to approximately 100 mg / kg, or approximately 1 to approximately 80 mg / kg, or approximately 5 to approximately 50 mg / kg, or approximately 10 to approximately 40 mg / kg, or approximately 15 to approximately 25 mg / kg.

[0083] In a given embodiment, the dose of ASSC in the co-formulation is approximately 30 mg / kg.

[0084] In some embodiments, the dose does not result in toxic levels of GAA in the individual's liver. In some embodiments, GAA is administered in a dose sufficient to produce a peak concentration of GAA in the target tissue, e.g., muscle tissue, within about 24 hours of dose administration. In some embodiments, GAA is administered in a dose sufficient to produce a peak concentration of GAA in the target tissue within about 10 to about 50 hours, or within about 45, 40, 35, 30, 25, or shorter hours, of dose administration. In some embodiments, the GAA formulation is a single-dose formulation. In some embodiments, the GAA formulation is a multi-dose formulation.

[0085] The GAA preparation of the present invention may be formulated so that the required total dose is 1 milliliter or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 milliliters or more, administered by a single subcutaneous injection. The preparation may also be formulated for subcutaneous administration at several different injection sites. To enable injection volumes of 1 or 2 milliliters, the GAA preparation of the present invention may be formulated at a concentration such that the preferred dose is delivered in a volume of 1 to 2 milliliters. Subcutaneous injection of the GAA preparation has the advantage of being convenient for the patient, particularly by enabling self-administration, while also providing a longer plasma half-life compared to, for example, intravenous administration. The extended plasma half-life results in the maintenance of effective plasma GAA levels for a longer period, and the benefit is that it can increase the uptake of GAA into clinically affected tissues as a result of increased exposure of those tissues to the injected GAA. This allows for a more beneficial effect on the patient and / or a reduction in the frequency of administration. Furthermore, as described herein, a variety of devices designed for patient convenience, such as refillable injection pens and needleless injectors, can be used with the GAA preparations of the present invention. Since GAA co-formulated with ASSC is stable for extended periods at room temperature, the preparations can be kept in a cartridge alongside the patient's body, enabling repeated or continuous low-dose administrations to provide a steady state of enzyme administration. Such administrations can avoid the potential side effects of high-dose enzyme replacement therapy (ERT) administered during intravenous infusion.

[0086] 5.5 Pharmaceutical Compositions The compounds and compositions of the present invention can be formulated as pharmaceutical compositions by mixing them with pharmaceutically acceptable carriers or excipients.

[0087] In one embodiment, ASSC and GAA are formulated as a single composition (i.e., a co-formulation). Such a composition enhances the stability of GAA both during storage (i.e., in vitro) and after administration to the subject in vivo, thereby increasing its circulating half-life, tissue uptake, and thus increasing the therapeutic efficacy of GAA. The formulation is preferably suitable for parenteral administration, including intravenous, subcutaneous, and intraperitoneal administration, but formulations suitable for other routes of administration, such as oral, intranasal, or transdermal, are also conceivable.

[0088] This invention relates to a liquid pharmaceutical formulation (e.g., a formulation containing GAA and ASSC) having improved properties compared to formulations approved in the art. The invention is based on the remarkable discovery that combining ASSC with GAA can increase the concentration of GAA in the formulation to an amount that would normally result in the formation of GAA aggregates in the absence of ASSC. Despite the high concentration of GAA, the formulation of the present invention can maintain the solubility and stability of GAA during, for example, manufacturing, storage, and / or repeated freeze / thawing steps, or prolonged exposure to an increased air-liquid interface. In addition, despite having a high concentration of GAA, the formulation of the present invention maintains low levels of protein aggregation (e.g., less than about 5%, 4%, 3%, 2%, or less than about 1%). Furthermore, the formulation of the present invention also remarkably maintains a low viscosity within a range suitable for subcutaneous injection, despite having a high concentration of GAA.

[0089] The present invention also provides a highly potent and concentrated GAA formulation that achieves solubilization of GAA in a small volume by combining GAA with ASSC. The formulation of the present invention is particularly useful when the delivery device is relatively small (e.g., an implantable system), delivery for a relatively long duration is required, or when a high effective dose of GAA is required to achieve the desired therapeutic effect. Therefore, it is possible to deliver a consistent amount of GAA over a long period (e.g., days, weeks, months, etc.) without the need to refill or replace the delivery device, thereby reducing the risk of infection and tissue damage, improving patient compliance, and achieving consistent and accurate administration.

[0090] In certain embodiments of the present invention, therapeutic doses of GAA (even high doses) can be administered to a subject by using only very small volumes of GAA (e.g., on the order of microliters or nanoliters per day). In a given body tissue, such as the subcutaneous space, delivery of small volumes promotes better absorption of GAA by local tissue and minimizes local tissue damage, trauma, or edema.

[0091] In a given embodiment, the formulation of the present invention contains a high concentration of GAA such that the liquid formulation does not exhibit significant emulsification, aggregation, or precipitation.

[0092] In another embodiment, the formulation of the present invention contains a high concentration of GAA so as to be suitable for subcutaneous administration, for example, without significant pain sensation (felt pain) (determined, for example, by a visual rating scale (VAS) score).

[0093] In certain embodiments, the formulations of the present invention contain high GAA concentrations, such as approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 115 mg / mL, approximately 150 mg / mL, approximately 160 mg / mL, approximately 200 mg / mL, approximately 240 mg / mL, or approximately 250 mg / mL. For example, as described in Example 10 below, in one aspect of the present invention, the liquid pharmaceutical formulation contains a human recombinant wild-type GAA concentration of approximately 25 mg / mL. The formulations of the present invention may also contain GAA concentrations of approximately 1 mg / mL to approximately 500 mg / mL, approximately 5 mg / mL to approximately 500 mg / mL, approximately 5 mg / mL to approximately 250 mg / mL, approximately 10 mg / mL to approximately 200 mg / mL, approximately 20 mg / mL to approximately 100 mg / mL, or approximately 1 mg / mL to approximately 60 mg / mL.Intermediate concentrations and ranges of the concentrations cited above are also intended to be part of the present invention (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 15 6, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 mg / mL).

[0094] In a given embodiment, the formulation of the present invention contains GAA at a concentration of 5 mg / mL or more.

[0095] In a given embodiment, the formulation of the present invention contains ASSC in an amount effective in reducing or inhibiting the aggregation of GAA in the formulation. Such amounts of ASSC include, for example, about 0.005 mM to 100 mM, or about 0.05 mM to about 90 mM, or about 0.1 mM to about 80 mM, or about 0.5 mM to about 70 mM, or about 1 mM to about 60 mM, or about 2 mM to about 50 mM, or about 3 mM to about 40 mM, or about 4 mM to about 30 mM, or about 5 mM to about 20 mM. In a given embodiment, ASSC is present in the formulation of the present invention at a concentration of about 0.5 to about 20 mM. In a given embodiment, ASSC is present in the formulation of the present invention at a concentration of about 1 mM. In a given embodiment, ASSC is present in the formulation of the present invention at a concentration of about 10 mM.

[0096] In a given embodiment, the formulation of the present invention contains ASSC in an amount effective in reducing or inhibiting the aggregation of GAA in the formulation. Such amounts of ASSC include, for example, about 5 to about 500 mg / mL, or about 10 to about 250 mg / mL, or about 20 to about 200 mg / mL, or about 30 to about 150 mg / mL, or about 40 to about 100 mg / mL, or about 50 to about 75 mg / mL. In a given embodiment, ASSC is present in an amount of about 5 to about 200 mg / mL.

[0097] In a given embodiment, ASSC is present in the formulation at a concentration of approximately 32 mg / mL or approximately 160 mg / mL.

[0098] In a predetermined embodiment of the present invention, a liquid formulation containing DNJ and GAA is prepared by dissolving DNJ in water to achieve a concentration of 10 mM DNJ. GAA may be reconstituted in 1.8 ml of water and dialyzed overnight in phosphate-buffered saline (pH 7.4). Then, 4.4 microliters of DNJ (10 mM) may be added to 400 microliters of GAA to achieve a GAA concentration of 25 mg / mL.

[0099] In another embodiment, GAA and ASSC are formulated in separate compositions. In this embodiment, the chaperone and supplement protein may be administered via the same route, for example, by intravenous infusion, or via different routes, for example, the supplement protein by intravenous infusion and the ASSC by oral administration.

[0100] Suitable pharmaceutical formulations for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to allow for easy injection. The formulations must be stable under manufacturing and storage conditions and must be stored in a manner that resists contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and polyethyl glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained by coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prevention of microbial activity can be achieved by various antimicrobial and antifungal agents such as parabens, chlorobutanol, calcareous salts, benzyl alcohol, and sorbic acid.

[0101] In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Extended absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition. Sterile injectable solutions may be prepared by incorporating the required amounts of GAA and ASSC in a suitable solvent, along with various other components listed above as needed, and then filtering or final sterilization. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary components listed above. In the case of sterile powders for preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which provide a powder of the active ingredient and any additional desired components from its previously sterile filtered solution.

[0102] The formulation may preferably contain one or more excipients. Pharmaceutically acceptable excipients that may be included in the formulation include buffers such as citrate buffer, phosphate buffer (e.g., monosodium phosphate, sodium hydrogen phosphate, and combinations thereof), acetate buffer, and bicarbonate buffer; amino acids, urea, alcohol, ascorbic acid, phospholipids; proteins such as serum albumin, collagen, and gelatin; salts such as EDTA or EGTA and sodium chloride; liposomes; polyvinylpyrrolidone; sugars such as dextran, mannitol, sorbitol, and glycerol; propylene glycol and polyethyl glycol (e.g., PEG-4000, PEG-6000); glycerol; glycine or other amino acids; and lipids. Buffer systems used in the formulation include citrate, acetate, bicarbonate, and phosphate buffer.

[0103] In a given embodiment, the formulation of the present invention further comprises an excipient selected from the group consisting of polyethyl glycol (PEG), PEG-400, arginine, arginine and glutamic acid, proline, γ-cyclodextrin, and combinations thereof.

[0104] In a given embodiment, the buffer and / or excipient is present in the formulation at a concentration of about 1 to about 50% by weight / volume (w / v), or about 2 to about 40% w / v, or about 3 to about 30% w / v, or about 4 to about 20% w / v, or about 5 to about 10% w / v.

[0105] In a given embodiment, the buffer and / or excipient is present in the formulation at a concentration of about 1 to about 500 mM, or about 10 to about 400 mM, or about 20 to about 300 mM, or about 30 to about 250 mM, or about 40 to about 200 mM, or about 50 to about 150 mM, or about 60 to about 100 mM.

[0106] In a given embodiment, the formulation includes a phosphate buffer solution present at a concentration of approximately 26 mM.

[0107] In a given embodiment, the formulation contains a citrate buffer solution present at a concentration of approximately 150 mM.

[0108] In a given embodiment, the formulation contains PEG-400 present at a concentration of approximately 5% w / v.

[0109] In a given embodiment, the formulation contains arginine present at a concentration of approximately 100 mM.

[0110] In a given embodiment, the formulation contains arginine at a concentration of about 50 mM and glutamic acid at a concentration of about 50 mM.

[0111] In a given embodiment, the formulation contains proline present at a concentration of approximately 250 mM.

[0112] In a given embodiment, the formulation contains γ-cyclodextrin present at a concentration of approximately 10% w / v.

[0113] The formulation may also include nonionic detergents. Preferred nonionic detergents include polysorbate 20, polysorbate 80, Triton X-100, Triton X-114, Nonidet P-40, octyl α-glycoside, octyl β-glycoside, Brij 35, Pluronic, and Tween 20.

[0114] For lyophilization of protein and chaperone preparations, the protein concentration may be 0.1 to 10 mg / mL. Fillers such as glycine, mannitol, albumin, and dextran may be added to the lyophilized mixture. In addition, possible cryoprotective substances such as disaccharides, amino acids, and PEG may be added to the lyophilized mixture. Any buffer, excipient, and detergent listed above may be added.

[0115] The route of administration may be oral or parenteral, including intravenous, subcutaneous, intraarterial, intraperitoneal, intraocular, intramuscular, buccal, intrarectal, vaginal, orbital, intracerebral, intradermal, intracranial, intraspinal, intraventricular, subarachnoid, cisterna magna, intrasacral, intrapulmonary, intranasal, transmucosal, transdermal, or by inhalation.

[0116] The parenteral formulations described above may be administered by periodic injection of bolus of the preparation, or by intravenous or intraperitoneal administration from an external (e.g., IV bag) or internal (e.g., bioerosional implant, bioprosthetic organ, or population of transplanted cells that produce replacement proteins) reservoir. See, for example, U.S. Patent No. 4,407,957 and U.S. Patent No. 5,798,113, which are incorporated herein by reference. Methods and devices for intrapulmonary delivery are described, for example, U.S. Patent No. 5,654,007, U.S. Patent No. 5,780,014 and U.S. Patent No. 5,814,607, which are incorporated herein by reference. Other useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposome delivery, needle delivery injection, needle-free injection, nebulizers, aerozoizers, electroporation, and percutaneous patches. Needle-free injector devices are described in U.S. Patent Nos. 5,879,327; 5,520,639; 5,846,233; and 5,704,911, the specifications of which are incorporated herein by reference. Any of the formulations described above can be administered by these methods.

[0117] 5.6 In vitro stability Ensuring the stability of GAA preparations during their shelf life is a major challenge. For example, the patient instructions for Myozyme® and Lumizyme® note that the vials are for single use only and that unused products should be discarded. The instructions further indicate that Myozyme® and Lumizyme® must be reconstituted, diluted, and administered by a healthcare professional, and that administration should be carried out without delay. Myozyme® and Lumizyme® must be stored at 2-8°C, and the products are stable at these temperatures for only 24 hours.

[0118] When ASSC and GAA are present in the same composition, the formulated composition of the present invention provides a more stable composition. In addition to stabilizing the administered protein in vivo, ASSC reversibly binds to GAA in vitro, stabilizing the three-dimensional structure of GAA, thereby preventing aggregation and degradation and extending the shelf life of the formulation. Analysis of ASSC / supplementary protein interactions can be evaluated using techniques known in the art, such as differential scanning calorimeter or circular dichroism.

[0119] For example, when an aqueous injection formulation of the composition is supplied in a stoppered vial suitable for drawing out the contents using a needle and syringe, the presence of ASSC inhibits the aggregation of GAA. The vial may be for single use or multiple use. The formulation may also be supplied as a pre-filled syringe, a pen-type syringe, or a needleless administration device. In another embodiment, the formulation may be in a dry or lyophilized state, which would need to be reconstituted into a liquid state using a standard or supplied physiological diluent. In this case, the presence of ASSC stabilizes GAA during and after GAA reconstitution, preventing aggregation. In embodiments where the formulation is in a liquid for intravenous administration, such as in a sterile bag connected to an intravenous administration line or catheter, the presence of ASSC provides the same benefit.

[0120] In addition to stabilizing the administered supplement protein, the presence of ASSC allows GAA preparations to be stored at a neutral pH of approximately 7.0–7.5. This benefits proteins that normally need to be stored at lower pH levels to maintain stability. For example, lysosomal enzymes such as GAA typically maintain a stable three-dimensional structure at low pH (e.g., below 5.0). However, long-term storage of supplement enzymes at low pH levels can accelerate the degradation of the enzyme and / or preparation.

[0121] As described above, the liquid formulations of the present invention have advantageous stability and storage properties. The stability of the liquid formulations is independent of the storage method and includes, but is not limited to, formulations that have been frozen, lyophilized, spray-dried, or in which the active ingredient is suspended. Stability may be measured for a selected period and at a selected temperature. In one aspect of the present invention, the protein in the liquid formulation is stable in liquid form for at least about 1 week; at least about 2 weeks; at least about 3 weeks; at least about 1 month; at least about 2 months; at least about 3 months; at least about 4 months, at least about 5 months; at least about 6 months; at least about 12 months; and at least about 18 months. Intermediate values ​​and ranges of the periods cited above, for example, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 months are also intended to be part of the present invention. In addition, ranges of values ​​using any combination of the values ​​cited above are intended to be included as upper and / or lower limits. In a given embodiment, the formulation is stable at room temperature (approximately 30°C), or approximately 37°C, or approximately 40°C, for at least approximately one month, and / or stable at approximately 2-8°C for at least approximately one year, or more preferably stable at approximately 2-8°C for at least approximately two years. Furthermore, the formulation is preferably stable after freezing (e.g., to -80°C) and thawing, which will hereafter be referred to as the "freeze / thaw cycle".

[0122] The stability of proteins in a liquid formulation (e.g., protein stability and / or reduction of contamination) may also be defined as the percentage of protein monomers, aggregates, or fragments, or combinations thereof, in the formulation. A protein "maintains its physical stability" in a formulation if such a protein does not substantially show signs of aggregation, precipitation, and / or denaturation, as measured by visual inspection of color and / or clarity, by UV light scattering, or by size exclusion chromatography, non-denaturing PAGE, or other methods for determining size. In one aspect of the present invention, a stable liquid formulation is one in which less than about 10%, less than about 5%, or less than 1% of protein exists as aggregates.

[0123] In one embodiment, the physical stability of a liquid formulation is determined by measuring the turbidity of the formulation after a stirring stress assay, for example, a 24-hour or 48-hour stirring stress assay. For example, the stirring stress assay may be performed by placing a suitable volume of the liquid formulation in a beaker equipped with a magnetic stirrer, for example (multipoint HP, 550 rpm), removing aliquots at any suitable time, for example T0 to T48 (hours), and optionally performing a suitable assay on the aliquots. Under the same conditions, a sample of the formulation without stirring serves as a control.

[0124] Turbidity measurements can be performed using a laboratory turbidity measurement system manufactured by Hach (Germany), and the results are reported in nephelometric units (NTU).

[0125] The stability of the composition (e.g., protein stability and / or reduction of contaminants) may also be measured, for example, by measuring proteolysis or the growth or presence of contaminants. Proteolysis can be determined, for example, by reverse-phase HPLC, non-denaturing PAGE, ion-exchange chromatography, peptide mapping, or similar methods.

[0126] The stability of GAA in the presence of ASSC at the concentrations described herein may be measured, for example, as a percentage of aggregation or decomposition over a given time and compared to one or more criteria. For example, a preferred criterion is a composition that is identical to the test conditions except that the GAA is not in contact with ASSC. The stability of GAA at one concentration is compared. Suitability can be demonstrated by a combination of GAA and ASSC at a particular concentration that has stability equivalent to or higher than that in the absence of ASSC.

[0127] 5.7 In vivo stability As mentioned above regarding in vitro formulations, the presence of ASSC for GAA has the advantage of maintaining effective supplemental protein levels for a longer period by extending the plasma half-life of exogenous GAA, resulting in increased exposure of clinically affected tissues to GAA and, consequently, increased protein uptake into the tissues. This provides patients with beneficial effects such as increased reassurance, reduced administration frequency, and / or reduced dosage. This will also reduce treatment costs.

[0128] ASSC will also stabilize and enhance the expression of endogenous mutant GAA, which is deficient as a result of mutations that prevent proper folding and processing within the ER, such as in conformational disorders like Pompe disease, in addition to stabilizing wild-type supplemental GAA.

[0129] The present invention should not be limited to the specific embodiments described herein and the following examples. In fact, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description and the accompanying examples and drawings, in addition to those described herein. These modifications are included within the scope of the accompanying claims. [Examples]

[0130] Example 1: In vitro thermal stability of rhGAA and 100 μM 1-DNJ-HCl The stability of recombinant human GAA (Myozyme®, Genzyme Corp.) with and without 100 μM ASSC 1-deoxynojirimycin hydrochloride (1-DNJ-HCl) was determined by a thermal stability assay using heat to induce protein denaturation. Denaturation was monitored using a SYPRO Orange dye that fluoresces after binding to hydrophobic amino acids (which are not exposed within the folded protein).

[0131] Thermal stability was tested for both formulations at pH 7.4, matching the pH of the ER. As shown in Figure 1, the formulation containing 100 μM 1-DNJ-HCl at pH 7.4 required significantly more heat to denature and was therefore more stable compared to the formulation without ASSC at pH 7.4.

[0132] Example 2: Residual activity and thermal stability of rhGAA and 50 μM 1-DNJ-HCl Residual GAA activity was determined for four formulations: (1) Myozyme® alone at pH 7.4; (2) Myozyme® + 50 μM 1-DNJ-HCl at pH 7.4; (3) Myozyme® alone at pH 5.2; (4) Myozyme® + 50 μM 1-DNJ-HCl at pH 5.2.

[0133] Activity was measured over 24 hours based on the percentage of initial activity (t=0). GAA enzyme activity of samples was assayed at 0, 3, 6, and 24 hours based on the hydrolysis of the fluorescence-generating substrate 4-MU-α-glucose. GAA activity was expressed as the percentage of initial activity, i.e., residual activity.

[0134] As shown in Figure 2A, formulation (1) (without ASSC) lost activity over time, retaining only about 20% of its initial activity 24 hours after administration. In contrast, formulation (2) maintained almost all, though not all, of its initial activity over 24 hours. Both formulations at pH 5.2 (formulations (3) and (4) above) maintained almost all of their initial activity over 24 hours.

[0135] To determine whether the loss of initial enzyme activity correlates with insufficient maintenance of proper three-dimensional structure, SYPRO Orange thermal stability experiments were performed on the above samples as outlined in Example 1. However, in this thermal stability experiment, the concentration of 1-DNJ-HCl was increased to 100 μM in formulations (2) and (4). Based on this experiment, the percentage of folded GAA was estimated and plotted in Figure 2B. The decrease in the amount of folded GAA over 24 hours for formulation (1) in Figure 2B correlates with the loss of activity shown in Figure 2A for this general formulation.

[0136] Example 3: In vivo Myozyme® uptake in GAA KO mice with and without oral administration of 1-DNJ-HCl Five groups of GAA KO mice were administered one of the following formulations: (1) Untreated control; (2) 10 mg / kg of Myozyme® IV once a week for up to 3 weeks (3) Injection of Myozyme® as in (2), + 10 mg / kg of 1-DNJ-HCl; (4) Injection of Myozyme® as in (2), + 100 mg / kg of 1-DNJ-HCl; (5) Injection of Myozyme® as in (2), + 1000 mg / kg of 1-DNJ-HCl; Tissue homogenates were prepared for analysis. Enzyme activity was determined by a 4-MUG fluorescence-generating substrate assay. The results are shown in Figure 3.

[0137] These results indicate that Myozyme® tissue uptake (a measure of GAA activity) decreased 7 days after injection across all groups. Co-administration of 1-DNJ-HCl and Myozyme® promoted a dose-dependent increase in Myozyme® uptake up to 7 days after injection. The effect of 1-DNJ-HCl was more evident and significant at days 4 and 7 after injection of any of 1, 2, or 3 doses (p<0.05 t-test, vs. Myozyme® alone).

[0138] Example 4: In vivo Myozyme® uptake in GAA KO mice with and without oral administration of 1-DNJ-HCl Generally, the thermal stability experiments described in Example 1 were performed for four compositions: (1) Composition consisting solely of Myozyme®; (2) Myozyme® + 1 μM 1-DNJ-HCl; (3) Myozyme® + 10 μM 1-DNJ-HCl; (4) Myozyme (registered trademark) + 100 μM 1-DNJ-HCl; As is evident from the dose-dependent increase in the melting temperature of GAA shown in Figure 5, DNJ-HCl increases the thermal stability of GAA.

[0139] Example 5: In vivo half-life of rhGAA in rats when administered as monotherapy or in combination with 1-DNJ-HCl Four groups of rats were administered one of the following dosing regimens. (1) Physiological saline + water; (2) 10 mg / kg of rhGAA + water; (3) 10 mg / kg of rhGAA + 3 mg / kg of 1DNJ-HCl; (4) 10 mg / kg rhGAA + 30 mg / kg 1DNJ-HCl; rhGAA or physiological saline was administered 30 minutes after 1-DNJ-HCl administration. GAA activity was generally determined as described in Example 3. The results over 24 hours are shown in Figure 6. 1-DNJ-HCl suppressed the loss of enzyme activity after administration, thereby extending the in vivo half-life of rhGAA. The in vivo half-life of rhGAA was extended from 1.4 ± 0.2 hours (0 mg / kg 1-DNJ-HCl) to 2.1 ± 0.2 hours (3 mg / kg 1-DNJ-HCl) and 3.0 ± 0.4 hours (30 mg / kg 1-DNJ-HCl).

[0140] Example 6: GAA enzyme activity in GAA KO mice Three groups of GAA KO mice were administered one of the following formulations: (1) Control (no treatment); (2) 10 mg / kg of rhGAA; (3) 10 mg / kg of rhGAA and 100 mg / kg of 1-DNJ-HCl for 48 hours, 30 minutes before rhGAA infusion and every 8 hours after infusion.

[0141] Cardiac and diaphragmatic tissue homogenates were collected, and rhGAA activity was measured using a fluorescence-generating substrate (4-MUG). The results are shown in Figure 7.

[0142] Example 7: 1-DNJ-HCl stabilizes rhGAA and prevents enzyme inactivation in the blood. The ability of 1-DNJ-HCl to stabilize rhGAA (e.g., Myozyme®) in whole (sodium citrate anticoagulant) blood at 37°C was evaluated to mimic the environment in which ERT was exposed during several hours of infusion. The results, as shown in Figure 8, indicate that rhGAA is unstable under these conditions, with approximately 40% of the enzyme inactivated by 4 hours, about 70% by 8 hours, and almost 100% by 24 hours (plotted by red diamond lines). These results suggest that a significant proportion of rhGAA doses may become inactive, as these infusions typically exceed 6 hours and sometimes exceed 12 hours. Furthermore, because Myozyme® has a long plasma half-life (reported to be over 3 hours), there is a high possibility that a considerable amount of the enzyme will remain in circulation for several hours after infusion, and such enzymes are also susceptible to inactivation. On the other hand, when rhGAA was incubated with 50 μM 1-DNJ-HCl under identical experimental conditions, the enzyme remained fully active throughout the entire experiment (plot of the blue square line). These results indicate that 1-DNJ-HCl stabilized rhGAA and prevented enzyme inactivation in whole blood. Importantly, these data also show that while plasma proteins present in the blood are not sufficient to prevent loss of rhGAA enzyme activity, pharmacological chaperones like 1-DNJ-HCl can prevent enzyme inactivation.

[0143] Example 8: 1-DNJ-HCl stabilizes rhGAA and prevents enzyme inactivation in the blood. rhGAA was measured in whole blood using various concentrations of 1-DNJ-HCl (0-100 μM) to determine the minimum concentration of 1-DNJ-HCl required to prevent rhGAA enzyme inactivation (Figure 9). As expected, high 1-DNJ-HCl concentrations (50 and 100 μM) were best for stabilizing rhGAA and preventing enzyme inactivation. However, it is interesting that even low 1-DNJ-HCl concentrations (as low as 2.5 μM) maintained rhGAA activity with only about a 20% loss over a 6-hour period. These results suggest that moderate 1-DNJ-HCl concentrations (e.g., 10-25 μM) may be sufficient to stabilize rhGAA in the blood during infusion. Based on human plasma PK data, these concentrations can be readily obtained in a clinical setting.

[0144] Example 9: Myozyme® co-administered with 1-DNJ-HCl resulted in a significantly higher reduction in tissue glycogen in GAA KO mice compared to Myozyme® alone. Male GAA KO mice aged 12 weeks were administered a single dose of Myozyme® (40 mg / kg) every week for 8 weeks via bolus tail vein injection. To prevent anaphylaxis, diphenhydramine (10 mg / kg intraperitoneally) was administered 10 minutes prior to the third and fourth Myozyme® injections. In addition, 30 minutes prior to Myozyme® administration, the mice received either water or 30 mg / kg of 1-DNJ-HCl via oral gavage. The mice were euthanized 14 days after the final Myozyme® administration. The experimental design is shown in Figure 10.

[0145] Next, glycogen levels were measured in the heart, diaphragm, soleus muscle, and quadriceps muscle. Myozyme® co-administered with 1-DNJ-HCl resulted in a significantly higher reduction in tissue glycogen in GAA KO mice compared to Myozyme® alone (Figure 11). Briefly, homogenates were prepared by homogenizing approximately 50 mg of tissue in 200 μL of deionized water on ice for 3-5 seconds using a microhomogenizer. The supernatant was thermally denatured (99°C for 10 minutes) to remove endogenous amyloglucosidase activity. Then, denatured lysates (4 μL) were analyzed in duplicate by adding 36 μL of water to 10 μL of 800 U / mL amyloglucosidase (Sigma Aldrich, St. Louis, MO) and 36 μL of water, and incubating at 50°C for 1 hour. The reaction was stopped by inactivation at 100°C for 10 minutes. Finally, 200 μL of glucose reagent (Sigma) was added, and the absorbance was read at 340 nM on Spectramax. Standard curves for type III rabbit liver glycogen (Sigma) in the range of 5 μg / mL to 400 μg / mL were created daily, and the absorbance was converted to absolute glycogen units. Simultaneously, the amount of protein in the tissue homogenate was determined using a Micro BCA protein assay (Pierce, Rockford, IL) according to the manufacturer's instructions. The glycogen content of each sample was normalized to protein, and the data was finally expressed as milligrams of glycogen per milligram of protein (μg / mg protein).

[0146] Example 10: DNJ reduces aggregation of Myozyme® in a composition containing a high concentration of Myozyme®. Liquid formulations containing DNJ and GAA were prepared by dissolving DNJ in water to achieve a concentration of 10 mM DNJ. GAA was reconstituted in 1.8 ml of water and dialyzed overnight in phosphate-buffered saline (pH 7.4). Then, 4.4 microliters of DNJ (10 mM) were added to 400 microliters of GAA to achieve a GAA concentration of 25 mg / mL.

[0147] 25 mg / mL Myozyme® was incubated in pH 7.4 phosphate-buffered saline with or without 1 mM DNJ. After incubation at 37°C for 4 weeks, the aggregation of Myozyme® was evaluated. As shown in Figure 12, the aggregation of Myozyme® was reduced when 1 mM DNJ was combined with 25 mg / mL Myozyme®.

[0148] Example 11: DNJ increases the circulating half-life and tissue uptake of Myozyme®. Sprague-Dawley rats were administered either 10 mg / kg Myozyme® or 30 mg / kg DNJ mixed with 10 mg / kg Myozyme® via the tail vein. GAA activity was measured in plasma and quadriceps muscle tissue. Baseline GAA activity in the quadriceps muscle was subtracted from the GAA measured after administration of GAA or DNJ and GAA (approximately 16 nMol / mg protein / hour).

[0149] As shown in Figure 13, administration of 30 mg / kg DNJ together with 10 mg / kg Myozyme® via the tail vein increased the circulating plasma half-life and tissue uptake of Myozyme® in the quadriceps muscle.

[0150] Example 12: Solubility of Lumizyme® in the presence of 1-DNJ The solubility of Lumizyme® (alglucosidase α) was tested in the presence of 1-DNJ and different excipients.

[0151] method A vial of Lumizyme® containing 52.5 mg protein, 210 mg mannitol, 0.5 mg polysorbate 80, 9.9 mg Na2HPO4x7H2O, and 31.2 mg NaH2PO4xH2O was dissolved in the minimum volume of water (1 mL added) to prepare a 1.2 mL protein solution. Five 240 μL aliquots (each containing 10.5 mg of protein) were transferred to an Amicon Ultra 0.5 mL 30 kDa cutoff centrifuge filter and centrifuged in an Eppendorf centrifuge tube at 14,000 x g for 10 minutes to obtain approximately 50 μL of residues (retentate).

[0152] The original excipients present in the composition, namely mannitol and polysorbate 80, were replaced with new excipients, and the protein solubility was tested (while maintaining the same buffering conditions). Using the original phosphate buffer, the following excipient solutions for replacement were prepared: 1. PEG400 (5% w / v) 2. Arginine (100mM) 3. Arginine (50mM) + Glutamic acid (50mM) 4. Proline (250mM) 5. γ-cyclodextrin (10%) All samples except for sample 2 (100 mM arginine) contained the small molecule ligand 1-deoxynojirimycin hydrochloride (1-DNJ-HCl, AT2220 HCl, 2220 HCl) in a ligand-to-protein ratio of 1:1 (w / w), while sample 2 contained the ligand in a lower ligand-to-protein ratio of 1:5 (w / w).

[0153] To replace the excipients, 5.2 mg Na2HPO4 and 27.1 mg NaH2PO4 were dissolved in 10.3 mL of water to prepare a washing / excipient exchange phosphate buffer of approximately 26 mM. 8 mL of the buffer for excipient exchange was supplemented with 160 mg / mL of small molecule ligand (to prepare solutions for excipients 1, 3, 4, and 5), while 2 mL was supplemented with 32 mg / mL of ligand for excipient 2 (100 mM arginine). Each excipient was dissolved in 2 mL of this buffer at the concentrations listed above.

[0154] The excipient solution was added to each separate filter device to achieve a total volume of 0.5 mL. Each filter device was centrifuged in a benchtop Eppendorf centrifuge tube at 14,000 xg for 15 minutes to maintain a volume of approximately 25 μL. The unit was refilled to 0.5 mL with the same excipient solution and centrifuged again (this process was repeated twice). This procedure efficiently replaced mannitol and polysorbate 80 with the aforementioned excipients, diluting the original components at approximately 1:1000. The total filtrate for each excipient was collected and examined for any protein leakage through the filter.

[0155] The concentrated protein solution was collected from each centrifuge filter by inverting the filter into a clean tube and centrifugating at 1000xg for 2 minutes. The volume of each sample was determined (66 μL, corresponding to a target protein concentration of 160 mg / mL), and it was confirmed that no visible precipitate was observed in each sample. The samples were transferred back to their respective filter devices and centrifuged for a further 5 minutes. The samples were then collected by inverting the filters into a clean tube and centrifuged at 1000xg for 2 minutes to provide approximately 37 μL of residues (corresponding to >280 mg / mL protein, based on the total initial concentration of 10.5 mg of protein).

[0156] The samples were vortex-stirred and incubated at room temperature for 1 hour to equilibrate the liquid and solid phases, then centrifuged at 14,000xg for 10 minutes. Again, no visible precipitate was observed. Two to three 10 μL aliquots were taken from the supernatant of each sample (depending on the available volume of the liquid phase) and diluted to 1:1000 in two steps. Protein concentration was measured by UV absorbance at 280 nM, and maximum protein solubility was calculated according to a calibration curve with high reproducibility. A calibration curve was prepared by dissolving 6.8 mg of Myozyme® (alglucosidase α) lyophilized powder (containing 1.16 mg of protein) in 1.16 mL of water, followed by serial dilution in deionized water. Absorbance was measured at 280 nM using 0.5 mL aliquots in a 1 cm light-pass quartz semi-micro cuvette.

[0157] result: As shown in Table 1, the excipients tested in this study increased the solubility of proteins in the presence of the small molecule ligand, 1-DNJ-HCl. The maximum value observed was for a mixture of arginine and glutamic acid (242 mg / mL), and the minimum value was for γ-cyclodextrin (114 mg / mL). The solubility of the protein without excipients was determined to be approximately 80 mg / mL. While not bound by any particular theory, the increase in solubility may be due to the efficient interaction of the amino acid excipients with the hydrophobic and hydrophilic sites on the protein surface, where such interactions competitively interfere with protein-protein association.

[0158] JPEG0007905382000006.jpg68161

[0159] Example 13: Effects of repeated subcutaneous administration of co-formulated rhGAA and 1-DNJ on tissue uptake and glycogen reduction in GAA KO mice This study investigated whether GAA knockout mice (GAA KOs) could tolerate repeated subcutaneous (SQ) injections of rhGAA or a combination of rhGAA and 1-DNJ, whether repeated SQ injections of rhGAA increased tissue uptake of rhGAA and decreased glycogen levels in GAA KO mice, and whether repeated SQ injections of a combination of rhGAA and 1-DNJ increased tissue uptake of rhGAA and decreased glycogen levels compared to SQ injections of rhGAA alone.

[0160] method This study used seven groups of 12-week-old male GAA knockout mice. Each group of mice was administered one of the following treatments: (1) Physiological saline only (no drug control); (2) Subcutaneous delivery (SQ) of Lumizyme® alone (20 mg / kg); or (3) Co-prescribed Lumizyme® (20 mg / kg) and 1-DNJ (30 mg / kg) SQ.

[0161] Each treatment was administered to its corresponding treatment group for two weeks. The treatments were administered on Mondays and Thursdays of each week. An SQ injection was administered between the scapulae of each treated mouse. A total of four doses were administered to each research animal. Diphenhydramine was administered intraperitoneally (IP) prior to the third and fourth doses.

[0162] rhGAA and glycogen levels were determined according to the following sampling protocol. Blood was collected from each study animal after the fourth dose for plasma pharmacokinetic analysis. GAA activity, Western blot, and 1-DNJ levels were determined for plasma samples collected 2 and 4 hours after the final SQ dose.

[0163] Three days after the final dose of this study (i.e., three days after dose 4), tissue samples were collected to determine rhGAA uptake. Tissue samples included the heart, diaphragm, tongue, brain, spleen, liver, biceps, triceps, quadriceps, soleus, gastrocnemius, ventral skin, and dorsal skin from the SQ injection site. Tissue samples were also collected 14 days after the final dose of the study (i.e., 14 days after dose 4) to determine glycogen concentration. Table 2 summarizes the treatments administered to the study subjects and the timing of sample collection.

[0164] JPEG0007905382000007.jpg84161

[0165] result Tissue rhGAA and glycogen Figures 15–25 show GAA activity 3 days after the final treatment dose and glycogen levels 14 days after the final treatment dose in tissue samples taken from animals that received one of the three treatments. For most of the tissues tested, the co-prescription of rhGAA with 1-DNJ significantly increased rhGAA uptake and activity in the test tissues compared to administration of rhGAA alone. In addition, rhGAA activity was elevated at the SQ injection sites in the ventral skin and forelimb muscles 3 days after treatment with rhGAA or the co-prescription of rhGAA and 1-DNJ. Except for the forelimbs, rhGAA activity was higher in the liver than in all the muscles tested.

[0166] Furthermore, as shown in Table 3, the rhGAA levels achieved using the 1-DNJ and rhGAA (Lumizyme®) co-formulation administered via SQ were the same as or higher than the levels achieved with intravenously administered hGAA (Myozyme®) alone (in most tissues).

[0167] JPEG0007905382000008.jpg179154

[0168] Regarding glycogen levels detected in tissue samples, the magnitude of glycogen depletion correlated with rhGAA uptake in some tissues. The heart, tongue, and ventral skin showed a greater correlation between improved rhGAA uptake and improved glycogen depletion with rhGAA+1-DNJ co-formulation than with rhGAA alone. While not bound by any theory, high rhGAA enzyme activity in some tissue lysates may originate from enzymes present in the tissue (e.g., fat, lymph, blood vessels) but not yet taken up into cells and lysosomes. In addition, while not bound by any theory, it is possible that a large amount of detectable cytoplasmic glycogen is present in whole-cell lysates, even if lysosomal enzymes are not utilized.

[0169] plasma rhGAA To determine whether 1-DNJ present in plasma samples from animals treated with the co-formulation inhibited rhGAA in the samples, rhGAA from plasma samples was incubated with a GAA substrate for 1 hour, and rhGAA activity was determined by determining enzyme activity based on substrate metabolism. Subsequently, the samples were incubated with the substrate for 3 hours, and these samples were re-analyzed by reversing any enzyme inhibition caused by 1-DNJ by dissociating 1-DNJ from the enzyme. No substantial change was observed between the results of the two assay formats.

[0170] Figures 26-28 show plasma rhGAA activity and protein concentrations in samples collected 2 and 4 hours after the final treatment dose. Two hours after the final SQ treatment dose, when 1-DNJ was administered with rhGAA in the co-formulation, higher rhGAA activity and protein concentrations were detected in plasma compared to when rhGAA was administered alone (i.e., by activity assay and Western blot, respectively). Four hours after the final SQ treatment dose, plasma rhGAA levels remained high in mice treated with the co-formulation, while plasma rhGAA levels from mice treated with rhGAA alone increased compared to the levels in the samples at 2 hours.

[0171] The present invention should not be limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and the accompanying drawings, in addition to those described herein. These modifications are included within the scope of the appended claims.

[0172] Patents, patent applications, literature, product descriptions, GenBank accession numbers, and protocols are referenced throughout this application, and these disclosures are incorporated herein by reference for all purposes.

Claims

1. A first composition and a second composition for use in the treatment of Pompe disease in the subject, (i) The first composition comprises n-butyl-deoxynojirimycin or a pharmaceutically acceptable salt thereof, (ii) The second composition comprises acid α-glucosidase; The first and second compositions for use are formulated with the acid α-glucosidase of the second composition at a concentration of approximately 15 mg / mL.

2. The first and second compositions for use according to claim 1, wherein the acid α-glucosidase is administered in a dose of about 5 mg / kg to about 50 mg / kg.

3. The first and second compositions for use according to claim 1 or 2, wherein the acid α-glucosidase is administered in a dose of about 10 mg / kg to about 40 mg / kg.

4. The first and second compositions for use according to any one of claims 1 to 3, wherein the acid α-glucosidase is administered in a dose of about 15 mg / kg to about 25 mg / kg.

5. The first and second compositions for use according to any one of claims 1 to 4, wherein alpha-acid glucosidase is administered at a dose of approximately 20 mg / kg.

6. The first and second compositions for use according to any one of claims 1 to 5, further comprising a buffer solution as the second composition.

7. The buffer is selected from the group consisting of citrate buffer, acetate buffer, bicarbonate buffer, phosphate buffer, and combinations thereof, the first composition and the second composition for use according to claim 6.

8. A first composition and a second composition for use according to any one of claims 1 to 7, wherein at least one of the first composition and the second composition further comprises an excipient.

9. A first composition and a second composition for use according to any one of claims 1 to 8, wherein the first composition is administered orally and the second composition is administered intravenously.

10. A first composition and a second composition for use in the treatment of Pompe disease in the subject, (i) The first composition comprises n-butyl-deoxynojirimycin or a pharmaceutically acceptable salt thereof, (ii) The second composition comprises acid α-glucosidase formulated at a concentration of about 15 mg / mL; Acid α-glucosidase is administered in amounts of approximately 5 mg / kg to approximately 50 mg / kg in a first and second composition for use.

11. The first and second compositions for use according to claim 10, wherein the acid α-glucosidase is administered in an amount of about 10 mg / kg to about 40 mg / kg.

12. The first and second compositions for use according to claim 10 or 11, wherein the acid α-glucosidase is administered in an amount of about 15 mg / kg to about 25 mg / kg.

13. The first and second compositions for use according to any one of claims 10 to 12, wherein alpha-acid glucosidase is administered in an amount of about 20 mg / kg.

14. The first and second compositions for use according to any one of claims 10 to 13, further comprising a buffer solution as the second composition.

15. The buffer is selected from the group consisting of citrate buffer, acetate buffer, bicarbonate buffer, phosphate buffer, and combinations thereof, the first composition and the second composition for use according to claim 14.

16. A first composition and a second composition for use according to any one of claims 10 to 15, wherein at least one of the first composition and the second composition further comprises an excipient.

17. A first composition and a second composition for use according to any one of claims 10 to 16, wherein the first composition is administered orally and the second composition is administered intravenously.

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