Isofagomine salts, methods of use and formulations thereof

Abstract

To fulfill a need for additional forms of IFG, including various salts and polymorphic forms of IFG.SOLUTION: The present invention relates generally to the field of pharmaceuticals, and specifically relates to isofagomine (IFG), novel salts thereof and preparation methods and uses of these, for example, in formulating pharmaceutical compositions for the treatment of Gaucher disease. Also provided are novel crystalline forms of isofagomine salts, methods for preparing the crystalline forms, and their use in formulating pharmaceutical compositions.SELECTED DRAWING: None

Description

Cross - reference to related applications

[0001] This international patent application claims the priority of U.S. Provisional Application No. 62 / 838,445, filed on April 25, 2019, the entire content of which is incorporated herein by reference.

Technical Field

[0002] The present invention relates to isophagomine, pharmaceutically acceptable salts of isophagomine, pharmaceutical compositions containing them, and their use in the formulation of compositions for the treatment of Gaucher disease.

Background Art

[0003] Gaucher disease is an autosomal recessive genetic disorder caused by mutations in the GBA gene, resulting in a deficiency of the lysosomal enzyme β - glucocerebrosidase (or GCase). GCase hydrolyzes glucocerebroside, a glycolipid formed after the degradation of sphingolipids in the membranes of white blood cells and red blood cells.

[0004] This accumulation causes various clinical symptoms, including splenomegaly, hepatomegaly, skeletal disorders, thrombocytopenia, and anemia (Non - Patent Document 1). In Gaucher disease, various forms of mutant GCase have reduced glucosylceramide cleavage activity, little activity, or no activity at all, depending on the mutated amino acid(s). The severity of this disease correlates with the relative level of residual enzyme activity and, as a result, the degree of substrate accumulation.

[0005] Certain GCase enzyme inhibitors can specifically bind to the enzyme during synthesis, stabilize protein folding in the endoplasmic reticulum (ER), and then dissociate from the enzyme at its original location within the lysosome, increasing the amount of enzyme that is processed without being degraded, thereby enhancing enzyme activity. Isophagomine ((3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine), known as IFG, is such a GCase enzyme inhibitor and has been shown to bind to the active sites of both wild-type and mutant GCase, stabilize the enzyme during synthesis and processing, and be effective in enhancing the activity of mutant GCase. In the absence of a "pharmacological chaperone", the mutant enzyme protein misfolds in the ER, maturation to the final product is retarded, and it is then degraded by the endoplasmic reticulum-associated degradation mechanism. In vitro, IFG has been shown to enhance the activity of mutant GCase in fibroblasts derived from Gaucher disease patients. The synthesis of this compound is described in Patent Document 1 by Sierks et al. and Patent Document 2 by Lundgren et al. [Chemical formula]

[0006] Also, IFG has been shown to enhance the stability of GCase pharmaceutical formulations such as VPRIV® (a formulation of velaglucerase alfa used in the treatment of Gaucher disease). In liquid compositions where high protein concentrations can promote aggregation, combining IFG (e.g., as an isophagomine salt) with GCase has been shown to improve the stability of GCase in vitro. In particular, liquid compositions with a molar ratio of at least 1:2.5 (GCase:IFG) provide formulations with substantially less GCase aggregation and degradation and higher GCase activity. See PCT / US18 / 57575, the contents of which are incorporated herein by reference.

[0007] The physicochemical properties of a pharmaceutical ingredient can be improved by selecting an appropriate salt form. Further, a single pharmaceutical ingredient or its salt can exist in multiple crystalline forms, i.e., polymorphs, including its hydrates and solvates. In this regard, alternative forms of a pharmaceutical ingredient can have widely different properties, such as enhanced thermodynamic stability, higher purity, or improved bioavailability (e.g., better absorption, dissolution pattern). Also, a particular form of a compound can facilitate the manufacture (e.g., improved flowability), handling, and storage (e.g., non-hygroscopic, long shelf life) of a compound formulation, or enable the use of a lower dose of a therapeutic agent, reducing its potential side effects and ensuring the equivalence of the pharmaceutical ingredient between batches. Thus, it is important to provide novel forms, including salts and polymorphs of a pharmaceutical ingredient, to offer the potential for improved properties for manufacture, formulation, storage, and pharmaceutical use.

[0008] To conduct pharmaceutical development of IFG and realize its potential, additional forms of IFG are needed that include various salts and polymorphs of IFG to facilitate the preparation of improved formulations of this pharmaceutical ingredient for use as an active pharmaceutical ingredient or as a stabilizer of GCase in a formulation. There remains a need for additional salts and crystalline polymorphs that can provide a broader range of solubility, stability, and physical properties to facilitate storage, subsequent processing, and improvement of the bioavailability of GCase.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0011] As a result of extensive research on various solid forms of isofagomine, surprisingly, some of its salts, including its crystalline, hydrate, and / or solvate forms, have been found and demonstrated to provide advantageous manufacturing, handling, storage, stability, solubility, and / or therapeutic properties.

[0012] Accordingly, in one aspect, the present disclosure relates to isofagomine salts, and their crystalline, hydrate or solvate forms, wherein the isofagomine salts are prepared from organic acids. In further embodiments, the isofagomine salts are selected from isofagomine cinchoninate, isofagomine malate, isofagomine fumarate, isofagomine oxalate, isofagomine malonate, isofagomine succinate, isofagomine D-tartrate, isofagomine cylamate, and isofagomine ascorbate. In another embodiment, the isofagomine salt is in crystalline form. In further embodiments, the crystalline isofagomine salts are selected from isofagomine cinchoninate, isofagomine fumarate, isofagomine oxalate, and isofagomine D-tartrate. Optionally, the crystalline forms of isofagomine are each characterized by three or more very strong, strong, and medium-strength XRPD peaks described in Tables 1, 3, 5, and 7, respectively.

[0013] In a further aspect, the present disclosure relates to a crystalline form of isophagomine quinate characterized by an X-ray diffraction pattern having one or more characteristic peaks at 2θ values of 9.5° ± 0.2°, 15.0° ± 0.2°, 17.4° ± 0.2°, 18.1° ± 0.2°, 20.3° ± 0.2°, 23.8° ± 0.2°, 24.8° ± 0.2° and 25.4° ± 0.2°.

[0014] In a further aspect, the present disclosure relates to a crystalline form of isophagomine fumarate characterized by an X-ray diffraction pattern having one or more characteristic peaks at 2θ values of 16.1° ± 0.2°, 18.3° ± 0.2°, 18.6° ± 0.2°, 21.9° ± 0.2°, 23.6° ± 0.2°, 23.8° ± 0.2°, and 25.5° ± 0.2°.

[0015] In a further aspect, the present disclosure relates to a crystalline form of isophagomine oxalate characterized by an X-ray diffraction pattern having one or more characteristic peaks at 2θ values of 27.8° ± 0.2°, 32.2° ± 0.2°, 35.3° ± 0.2°, 36.6° ± 0.2°, 37.4° ± 0.2°, 38.4° ± 0.2°, 18.5° ± 0.2°, 19.2° ± 0.2°, 21.4° ± 0.2°, 22.6° ± 0.2°, 24.5° ± 0.2°, 24.8° ± 0.2°, 26.8° ± 0.2°, 20.2° ± 0.2° and 23.7° ± 0.2°.

[0016] In a further aspect, the present disclosure relates to a crystalline form of isophagomine D-tartrate characterized by an X-ray diffraction pattern having one or more characteristic peaks at 2θ values of 9.8° ± 0.2°, 10.5° ± 0.2°, 15° ± 0.2°, 15.3° ± 0.2°, 15.8° ± 0.2°, 17.4° ± 0.2°, 17.9° ± 0.2°, 18.5° ± 0.2°, 18.9° ± 0.2°, 19.6° ± 0.2°, 21.1° ± 0.2°, 21.7° ± 0.2°, 22° ± 0.2°, 24.2° ± 0.2°, 24.8° ± 0.2°, 26.6° ± 0.2°, 27.1° ± 0.2°, 27.4° ± 0.2°, 33.8° ± 0.2°, 35.7° ± 0.2°, 36.5° ± 0.2° and 37.5° ± 0.2°.

[0017] Additional aspects of the present disclosure include pharmaceutical compositions comprising isophagomine (IFG) and at least one pharmaceutically acceptable carrier. In some embodiments of the pharmaceutical compositions of the present disclosure, IFG is a stabilizer of the GCase active ingredient. In certain embodiments, IFG is in the form of a free base or a salt (including its crystalline, hydrate, and solvate forms). In another aspect, isophagomine is present in an amount sufficient to reduce the degradation of the GCase active ingredient; that amount can be a significantly lower amount than the therapeutically effective amount of IFG alone or than when IFG is administered in a therapeutically effective amount in combination with the GCase active ingredient in a conventional combination therapy. In some embodiments, the composition comprises 60 - 180 mg / mL of glucocerebrosidase (GCase). In some embodiments, GCase is velaglucerase alfa. In a further aspect, compositions are provided that comprise GCase and IFG in a molar ratio of at least about 1:>2.5 (i.e., 1:x, where x is greater than 2.5), about 1:3, or about 1:2.5 - about 1:3.5. In additional embodiments, the composition further comprises sodium citrate buffer, sucrose, and a surfactant selected from PS20, PS80, or poloxamer 188. In additional embodiments of the pharmaceutical composition, the isophagomine salt is at least 95% pure.

[0018] In another aspect of the present disclosure, a method of preparing any of the pharmaceutical compositions described herein is provided, which includes the step of combining IFG and a pharmaceutically acceptable carrier. Some embodiments include the step of combining IFG, GCase, and a pharmaceutically acceptable carrier. Further embodiments of the methods provided herein include dissolving isophagomine in water, adjusting the pH to about 6.0, and adding glucocerebrosidase to obtain the composition.

[0019] In another aspect, provided is a method of treating a disorder associated with a dysfunction of the GCase pathway, or a method of preventing the onset of symptoms associated with a dysfunction of the GCase pathway, the method comprising administering a therapeutically effective amount of any one of the compositions described herein. In some embodiments, the composition is administered intravenously or subcutaneously. In some embodiments, the composition is administered subcutaneously, for example by subcutaneous injection. In some embodiments, the composition is administered twice a week, once a week, less than once a week, or once every other week.

[0020] Another aspect of the disclosure relates to a method of treating Gaucher disease, the method comprising administering to a patient in need thereof i) a therapeutically effective amount of an isophagomine salt or a pharmaceutical composition thereof as defined above, or ii) a therapeutically effective amount of a composition comprising isophagomine and GCase, wherein isophagomine is a stabilizing excipient of GCase. The treatment can be carried out as monotherapy or combination therapy. In another aspect, the disclosure relates to an isophagomine salt for use as a medicament, preferably for use in the treatment of Gaucher disease.

[0021] In another aspect, the disclosure relates to a method for producing an isophagomine salt, comprising the steps of: i) dissolving an organic acid in a polar protic solvent to prepare Solution 1; ii) dissolving isophagomine free base in a polar protic solvent to prepare Solution 2; iii) combining Solution 1 and Solution 2 to form a precipitate; iv) isolating the precipitate corresponding to the organic acid salt of isophagomine. In a further embodiment, the organic acid is selected from quinic acid, fumaric acid, oxalic acid, malonic acid, D-tartaric acid, L-tartaric acid, succinic acid, cyclamic acid, and ascorbic acid. In some embodiments, the isophagomine salt is in crystalline form. In an additional embodiment, the isophagomine free base has a purity of at least about 98%.

[0022] These aspects and their preferred embodiments are further defined in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

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Mode for Carrying Out the Invention

[0024] Definition The novel forms and formulations provide opportunities to improve the manufacture, formulation, and performance characteristics of pharmaceuticals. The present disclosure includes new forms of isophagomine having improved physicochemical properties such as improved stability, flowability, and purity.

[0025] The terms used herein generally have their ordinary meanings in the context of the present disclosure and in the particular context in which each term is used. Specific terms are described below or elsewhere in this specification to provide additional guidance to those skilled in the art in describing the compositions and methods of the present disclosure, as well as their methods of manufacture and use.

[0026] The terms “isophagomine” and “IFG” used interchangeably herein include references to isophagomine in its free base form, in addition to any salt forms disclosed herein, unless otherwise specified. The terms further include, unless otherwise designated, that the material may be in amorphous or crystalline form.

[0027] As used herein, the term "crystalline (or crystalline)" refers to a material that may be hydrated and / or solvated and has an order of chemical moieties sufficient to exhibit a diffraction pattern distinguishable by diffraction techniques such as XRPD. In many cases, crystalline materials obtained by direct crystallization of a compound dissolved in solution, or by interconversion of crystals obtained under different crystallization conditions, have crystals containing the solvent used for crystallization, which are referred to as crystal solvates. Also, the crystallization conditions, collectively, the specific solvent system and physical embodiments under which crystallization is carried out, result in crystalline materials having physical and chemical properties specific to the crystallization conditions, which are generally due to the orientation of the chemical sites of the compound relative to each other within the crystal, and / or the predominance of a particular polymorph of the compound in the crystalline material. Scientists in this field can understand that they can characterize the physical and chemical properties described herein, but experimental error depends on instrument conditions, sample preparation, and sample purity. In particular, scientists in this field are usually aware that X-ray diffraction patterns can vary with changes in experimental conditions. It is necessary to point out here that the relative intensities of X-ray diffraction patterns are likely to change with changes in experimental conditions; thus, the order of peak intensities may not be regarded as the only or decisive factor. Also, since the experimental error of the peak angle is generally less than 5%, such errors need to be considered, and the generally acceptable error is ±0.2° theta (θ). Furthermore, due to the influence of experimental factors including the height of the sample, the peak angle may shift overall; generally, a certain shift is acceptable. Thus, scientists in this field will be able to understand that the X-ray diffraction pattern of the crystalline form of the present disclosure need not be exactly the same as the X-ray diffraction pattern of the examples shown herein. Any crystalline form having the same or similar characteristic peaks in the X-ray diffraction pattern should be within the scope of the present disclosure. Scientists in this field can compare the patterns shown in the present disclosure with the patterns of unknown crystalline forms to identify whether these two groups of patterns reflect the same or different crystalline forms.

[0028] As used herein, the terms "crystal form", "polymorph", and other related terms refer to a solid compound in a state of a crystal form with a specific crystal structure. Differences in the physical and chemical properties of polymorphs can be embodied in storage stability, compressibility, density, dissolution rate, etc. At the extreme, differences in solubility and dissolution rate can result in an ineffective drug or even toxicity.

[0029] As used herein, the term "amorphous" refers to a composition containing a compound with too little crystalline content of the compound to obtain a pattern distinguishable by diffraction techniques such as XRPD. Glassy materials are a type of amorphous material. Glassy materials do not have a true crystal lattice and are technically similar to a very viscous amorphous liquid. Glass may be better described as a quasi-solid amorphous material rather than a true solid.

[0030] As used herein, the term "volume(s) of solvent" means the number of milliliters (ml) of solvent used per gram of the material to be dissolved. For example, dissolving 1 g of tartaric acid in 8 volumes of solvent means dissolving it in 8 milliliters of solvent.

[0031] "Gaucher disease" includes types 1, 2, and 3 (including 3a, 3b, and 3c), as well as intermediate forms and subgroups thereof based on the manifestation of phenotypes.

[0032] The terms "effective amount" and "an effective amount" mean an amount sufficient to bring about a therapeutic response. A therapeutic response may be any response that a user (e.g., a clinician) recognizes as an effective response to treatment and includes improvement of one or more symptoms and surrogate clinical markers. Thus, a therapeutic response in a subject with Gaucher disease is generally improvement of one or more symptoms of Gaucher disease. A "therapeutically effective amount" will vary depending on the formulation used, the type and severity of Gaucher disease, and the age, weight, health, responsiveness, etc. of the mammal being treated. A therapeutic response may also be improvement of one or more symptoms of another synucleinopathy, such as Parkinson's disease or Lewy body dementia, for which treatment with the compositions described herein is contemplated.

[0033] "Pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce adverse reactions at levels that are normally unacceptable when administered to a human. Preferably, the term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0034] As used herein, the term "about" refers to up to ±10% of the value modified by this term. For example, about 50 mM means 50 mM ± 5 mM, and about 4% means 4% ± 0.4%.

[0035] As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, "a carrier" includes one or more carriers.

[0036] As used herein, the terms "ambient temperature" and "room temperature" are understood in the art and generally refer to a temperature of the order of the temperature of the room in which the reaction is carried out (e.g., the reaction temperature), e.g., typically a temperature of about 20-22 °C (68-72 °F). More specifically, the material or reaction mixture is neither heated nor cooled.

[0037] "Subject" refers to any mammal classified as a mammal, including but not limited to humans, non-human primates, primates, baboons, chimpanzees, monkeys, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc. The term "subject" may be used interchangeably with the term "patient".

[0038] As used herein, the expressions "parenteral administration", "administered parenterally", "administer parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous (IV), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous (SC), subepidermal, intra-articular, subcapsular, subdural, intraspinal, epidural, intrasternal injection and infusion.

[0039] "Therapeutically effective dose" and "therapeutically effective amount" refer to the amount of a compound that results in the prevention of a symptom, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% prevention of a symptom (e.g., the symptoms of Gaucher's disease in a subject diagnosed with Gaucher's disease), delay in the onset of a symptom, or improvement of the symptoms of Gaucher's disease. A therapeutically effective amount is an amount sufficient to treat, prevent, reduce the severity of, delay the onset of, and / or reduce the risk of occurrence of one or more symptoms of a disorder associated with Gaucher's disease. The effective amount can be determined by the methods described in subsequent sections of this specification well known in the art.

[0040] The terms "treatment" and "therapeutic method" refer to the treatment of existing disorders and / or prophylactic / preventive measures to prevent or reduce the occurrence of symptoms associated with a disorder. Those in need of treatment include not only individuals who already have a specific medical disorder such as a dysfunction of the GCase pathway, but also individuals who are at risk of having a disorder or who may ultimately acquire a disorder. The need for treatment is evaluated, for example, by the presence of one or more risk factors associated with the onset of the disorder, the presence or progression of the disorder, or the receptiveness of a subject having the disorder to treatment. Treatment includes slowing or reversing the progression of the disorder or its symptoms.

[0041] "Treating" means treating in an amount, method, and / or manner effective to improve or prevent a condition, symptom, or parameter associated with a disorder (e.g., a disorder described herein), or to prevent the onset, progression, or worsening of a disorder, to a statistically significant degree or to a degree detectable by one of ordinary skill in the art. Thus, by treating, a therapeutic benefit can be achieved. The effective amount, method, or manner can vary depending on the subject and can be adjusted according to the subject. In certain embodiments, treatment of a disorder associated with a dysfunction of the GCase pathway (e.g., Gaucher disease) is, for example, in a subject not receiving treatment for the dysfunction of the GCase pathway, a treatment that results in an increase in hemoglobin concentration, an increase in platelet level, a decrease in liver volume, a decrease in spleen volume, or a change in skeletal parameters (e.g., an increase in bone density). In certain embodiments, treatment of a disorder associated with a dysfunction of the GCase pathway (e.g., Gaucher disease) is, for example, in a subject receiving treatment for the dysfunction of the GCase pathway, a treatment that results in an increase in hemoglobin concentration, an increase in platelet level, a decrease in liver volume, a decrease in spleen volume, or a change in skeletal parameters (e.g., an increase in bone density), or the maintenance of one or more of these parameters.

[0042] "Combination" refers to treating the same patient using two or more agents or therapies, where the use or action of the agents or therapies overlaps in time. The agents or therapies can be administered simultaneously (e.g., as a single formulation administered to the patient, or as two separate formulations administered simultaneously), or sequentially in any order.

[0043] As used herein, the terms "sustained release", "sustained release delivery", and "sustained release drug delivery" mean that the effective concentration in the blood is maintained for a long period of time, e.g., 12 hours or more, following a single drug administration. For example, common routes of administration of polypeptides are subcutaneous, intramuscular, or intravenous (IV) injection.

[0044] The terms "individual", "subject", or "patient" can be used interchangeably and refer to any mammal, including, but not limited to, humans, non-human primates, primates, baboons, chimpanzees, monkeys, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc., and any animal classified therein.

[0045] "An individual in need of treatment" refers to an individual who has developed or is at risk of developing a lysosomal storage disorder such as Gaucher disease, or an α-synucleinopathy such as Parkinson's disease. In one embodiment, the individual is a member of the Ashkenazi Jewish population diagnosed with Gaucher disease or identified as having a high risk of developing Gaucher disease due to a genetic mutation in the Gba gene. However, the term "individual" encompasses people worldwide who have Gaucher disease or are genetically at risk of developing it, or who have Parkinson's disease or are at risk of developing it, or other α-synucleinopathies.

[0046] When describing isophagomine salts in this specification, the expression "substantially pure" means that the isophagomine salt does not contain more than about 2% of other compounds. Preferably, a "substantially pure" isophagomine salt contains about 2% or less of other compounds, about 1.8% or less of other compounds, about 1.6% or less of other compounds, about 1.5% or less of other compounds, about 1.3% or less of other compounds. More preferably, a "substantially pure" isophagomine salt contains about 1% or less of other compounds, about 0.8% or less of other compounds, about 0.6% or less of other compounds, about 0.5% or less of other compounds, about 0.3% or less of other compounds.

[0047] Purity can be determined by those skilled in the art using, for example, HPLC analysis or analytical thin layer chromatography. Other methods are known to those skilled in the art and include chromatographic methods and spectrophotometric methods.

[0048] The term "unit" for GCase, velaglucerase, or velaglucerase alfa means the amount necessary to convert 1 micromole (μmol) of p-nitrophenyl β-D-glucopyranoside to p-nitrophenol or 4-methylumbelliferyl β-D-glucopyranoside to 4-methylumbelliferone per minute at 37°C.

[0049] According to the present disclosure, specific salt forms of isophagomine are provided. The provided isophagomine salts, and their crystalline forms, have improved properties compared to previously described forms of isophagomine, including improved synthetic manufacturability. For example, purification of the described organic acid salts of IFG in solvents such as water and ethanol can be facilitated. Also, some of the provided forms, for example, IFG-fumarate, have similar or greater stability than other known salt forms of isophagomine. Further, the IFG salts provided herein are particularly suitable for industrial scale manufacture, for example, the manufacture of products exceeding 1 kg.

[0050] In some embodiments of the present disclosure, the organic acid salts of isophagomine are prepared by the following general protocol.

[0051] Solution 1 is prepared as follows: Dissolve an organic acid in a polar protic organic solvent. The amount of the solvent is not particularly limited. Preferably, the organic acid is dissolved in the solvent. Typically, 2 to 10 volumes of the solvent, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 volumes of the solvent, or any amount therebetween can be used.

[0052] In one embodiment, the amount of the solvent used is the minimum amount necessary to achieve complete dissolution at room temperature. Typically, the solution is prepared at room temperature, but embodiments where the solvent is heated, for example, to facilitate dissolution or to create a supersaturated solution, are also contemplated. Before proceeding to the next step, the solution may be filtered to remove undissolved matter. Alternatively, a slurry method may be used where the material does not dissolve completely before combining with Solution 2 in the next step.

[0053] Solution 2 is prepared as follows: Dissolve an equimolar amount of isophagomine relative to the organic acid used in the preparation of Solution 1 in a polar protic organic solvent. The amount of the solvent is not particularly limited. Preferably, isophagomine is dissolved in the solvent. Typically, 2 to 10 volumes of the solvent, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 volumes of the solvent, or any amount therebetween can be used.

[0054] In one embodiment, the amount of the solvent used is the minimum amount necessary to achieve complete dissolution at room temperature. Typically, the solution is prepared at room temperature, but embodiments where the solvent is heated, for example, to facilitate dissolution or to create a supersaturated solution, are also contemplated. Before proceeding to the next step, the solution may be filtered to remove undissolved matter. Alternatively, a slurry method may be used where the material does not dissolve completely before combining with Solution 1 in the next step.

[0055] In certain embodiments, solution 1 is prepared using an organic acid. Organic acids suitable for use in this embodiment include quinic acid, maleic acid, fumaric acid, oxalic acid, malonic acid, D-tartaric acid, L-tartaric acid, succinic acid, cylamic acid, and ascorbic acid.

[0056] Solution 2 is then slowly added to solution 1, forming a precipitate. After stirring at room temperature, the resulting slurry is filtered and the resulting solid material is washed with an alcohol solvent, dewatered, and then dried in vacuum. The isolated yield is typically 40-80% of the theoretical yield.

[0057] In certain embodiments, the resulting isofagomine is selected from isofagomine quinate, isofagomine malate, isofagomine fumarate, isofagomine oxalate, isofagomine malonate, isofagomine succinate, isofagomine D-tartrate, isofagomine cyclamate, and isofagomine ascorbate. In certain embodiments, the resulting isofagomine salt is crystalline, as determined, for example, by measurement of diffraction peaks using X-ray powder diffraction techniques or other diffraction techniques known in the art.

[0058] In further embodiments, the isofagomine is a crystalline form selected from isofagomine quinate, isofagomine fumarate, isofagomine oxalate, isofagomine D-tartrate, characterized by three or more very intense, strong, and medium intensity XRPD peaks set forth in Tables 1, 3, 5, and 7, respectively.

[0059] In certain embodiments, the IFG is isophagomine quinate characterized in that the X-ray diffraction pattern has one or more, two or more, or three or more characteristic peaks at the 2θ values listed in Table 1, for example, having characteristic peaks at at least one, at least two, or at least three selected from 9.5° ± 0.2°, 15.0° ± 0.2°, 17.4° ± 0.2°, 18.1° ± 0.2°, 20.3° ± 0.2°, 23.8° ± 0.2°, 24.8° ± 0.2° and 25.4° ± 0.2°. In a further embodiment, the IFG is isophagomine quinate characterized in that the X-ray diffraction pattern has at least one, at least two, or at least three characteristic peaks at the 2θ values of 17.4° ± 0.2°, 15.0° ± 0.2°, 18.1° ± 0.2°, 20.3° ± 0.2° and 24.8° ± 0.2°; in certain embodiments, the IFG is isophagomine quinate characterized in that the X-ray diffraction pattern has at least one, at least two, or at least three characteristic peaks at the 2θ values of 15.0° ± 0.2°, 17.4° ± 0.2°, 18.1° ± 0.2° and 20.3° ± 0.2°.

[0060] In certain embodiments, the IFG is isophagomine fumarate characterized in that the X-ray diffraction pattern has one or more, two or more, or three or more characteristic peaks at the 2θ values listed in Table 3, for example having characteristic peaks at at least one, at least two, or at least three selected from 16.1° ± 0.2°, 18.3° ± 0.2°, 18.6° ± 0.2°, 21.9° ± 0.2°, 23.6° ± 0.2°, 23.8° ± 0.2°, and 25.5° ± 0.2°. In certain embodiments, the IFG is isophagomine fumarate characterized in that the X-ray diffraction pattern has at least one, at least two, or at least three characteristic peaks at the 2θ values of 23.6° ± 0.2°, 23.8° ± 0.2° and 25.5° ± 0.2°.

[0061] In certain embodiments, the IFG is isophagomine oxalate characterized in that the X-ray diffraction pattern has one or more, two or more, or three or more characteristic peaks at the 2θ values listed in Table 5, for example, 27.8° ± 0.2°, 32.2° ± 0.2°, 35.3° ± 0.2°, 36.6° ± 0.2°, 37.4° ± 0.2°, 38.4° ± 0.2°, 18.5° ± 0.2°, 19.2° ± 0.2°, 21.4° ± 0.2°, 22.6° ± 0.2°, 24.5° ± 0.2°, 24.8° ± 0.2°, 26.8° ± 0.2°, 20.2° ± 0.2° and 23.7° ± 0.2°, and has at least one, at least two, or at least three characteristic peaks. In certain embodiments, the IFG is isophagomine oxalate characterized in that the X-ray diffraction pattern has at least one, at least two, or at least three characteristic peaks at the 2θ values of 18.5° ± 0.2°, 19.2° ± 0.2°, 21.4° ± 0.2°, 22.6° ± 0.2°, 24.5° ± 0.2°, 24.8° ± 0.2°, 26.8° ± 0.2°, 20.2° ± 0.2° and 23.7° ± 0.2°. In certain embodiments, the IFG is isophagomine oxalate characterized in that the X-ray diffraction pattern has at least one, at least two, or at least three characteristic peaks at the 2θ values of 20.2° ± 0.2° and 23.7° ± 0.2°.

[0062] In certain embodiments, the IFG is isophagomine D-tartrate characterized by an X-ray diffraction pattern having one or more, two or more, or three or more characteristic peaks at the 2θ values set forth in Table 5, for example, 9.8° ± 0.2°, 10.5° ± 0.2°, 15.0° ± 0.2°, 15.3° ± 0.2°, 15.8° ± 0.2°, 17.4° ± 0.2°, 17.9° ± 0.2°, 18.5° ± 0.2°, 18.9° ± 0.2°, 19.6° ± 0.2°, 21.1° ± 0.2°, 21.7° ± 0.2°, 22° ± 0.2°, 24.2° ± 0.2°, 24.8° ± 0.2°, 26.6° ± 0.2°, 27.1° ± 0.2°, 27.4° ± 0.2°, 33.8° ± 0.2°, 35.7° ± 0.2°, 36.5° ± 0.2° and 37.5° ± 0.2°, having at least one, at least two, or at least three characteristic peaks selected therefrom. In a further embodiment, the IFG is isophagomine D-tartrate characterized in that the X-ray diffraction pattern has at least one, at least two, or at least three characteristic peaks at the 2θ values of 10.5° ± 0.2°, 15.0° ± 0.2°, 15.3° ± 0.2°, 18.5° ± 0.2°, 26.6° ± 0.2°, 21.1° ± 0.2°, 21.7° ± 0.2° and 24.2° ± 0.2°. In certain embodiments, the IFG is isophagomine D-tartrate characterized in that the X-ray diffraction pattern has at least one, at least two, or at least three characteristic peaks at the 2θ values of 21.1° ± 0.2°, 21.7° ± 0.2° and 24.2° ± 0.2°.

[0063] The polar protic organic solvents for use in the present disclosure typically include one or more lower aliphatic alcohols such as methanol, ethanol, n-propanol, isopropanol, etc., and may also include water. Preferably, methanol and / or ethanol are used.

[0064] In one embodiment, for example, to obtain a higher yield or to effect crystal growth more rapidly, the acid solution (Solution 1) is seeded (introduced as seed crystals) with crystalline material of the desired isophagomine salt obtained previously.

[0065] In certain embodiments, the isophagomine free base is purified before being dissolved for use in Solution 2. The purification techniques are those known in the art and include chromatography. In one embodiment, the purification of isophagomine includes chromatography on silica gel or chromatography via an ion exchange resin system. These chromatography methods remove various impurities such as intermediates formed during the synthesis of isophagomine, such as during the step of manufacturing isophagomine by hydrogenating (3R,4R,5S,6S)-6-(benzyloxy)-4,5-dihydroxytetrahydro-2H-pyran-3-carbonitrile. Some of the impurities removed by the purification of the isophagomine free base include dimer amine species, cyclic imine intermediates, and other unidentified intermediates or by-products. Specific impurities removed by the purification of the isophagomine free base include those identified below.

[0066] In one embodiment, the IFG free base for use in the method of manufacturing the IFG salts described herein contains less than 2%, less than 1.8%, less than 1.6%, less than 1.4%, less than 1.2%, less than 1%, less than 0.8%, less than 0.6%, less than 0.4%, less than 0.2% impurities. More specifically, the IFG free base for use in the method of manufacturing the IFG salts described herein contains less than 2%, less than 1.8%, less than 1.6%, less than 1.4%, less than 1.2%, less than 1%, less than 0.8%, less than 0.6%, less than 0.4%, less than 0.2% of any one of the impurities SRD006961, SRD006927, SRD006987, and SRD006925. In a further embodiment, the IFG free base does not contain one or more of SRD006961, SRD006927, SRD006987, and SRD006925.

[0067] In a further embodiment, the free IFG base for use in the method for producing the IFG salts described herein has a purity of at least 90%, 95%, 96%, 97%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 100% (w / w).

[0068] In another embodiment, the produced IFG salts have a purity of at least 95%, 96%, 97%, 98%, 98.2%, 98.4%, 98.6, 98.8%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% (w / w).

[0069] Glucocerebrosidase Veliglucerase is human β-glucocerebrosidase (GCase) produced by activating a gene in a human cell line, such as by targeted recombination using a promoter that activates the endogenous β-glucocerebrosidase gene in a selected human cell line. Veliglucerase is secreted as a monomeric glycoprotein of approximately 63 kDa. Veliglucerase is composed of 497 amino acids and has the same sequence as the native human protein. See Zimran et al., Blood Cells Mol Dis, 2007, 39: 115-118.

[0070] The glycosylation of velaglucerase alfa can be altered to produce a secreted protein that mainly contains high-mannose type glycans having 6 to 9 mannose units per sugar chain (glycan), such as by using kifunensine, a mannosidase I inhibitor, during cell culture, as described in detail in WO 2013 / 130963.

[0071] Imiglucerase (Cerezyme®) is another form of recombinant human β-glucocerebrosidase. Imiglucerase is recombinantly produced in Chinese hamster ovary (CHO) cells.

[0072] Taliglucerase alfa (Elelyso® or Uplyso®) is a recombinant glucocerebrosidase (prGCase) expressed in plant cells. The plant recombinant glucocerebrosidase can be obtained, at least, by the methods described in U.S. Patent Application Publication Nos. 2009 / 0208477, 2008 / 0038232, International Publication Nos. 2004 / 096978, and 2008 / 132743.

[0073] Any of the recombinant GCase can be produced using bioreactors and synthetic methods of production scale known in the art. Also, any number of purification systems of production scale can be used.

[0074] Pharmaceutical composition In certain embodiments, a pharmaceutical composition comprising isophagomine (IFG) and at least one pharmaceutically acceptable carrier is provided. In some embodiments, IFG is in the form of an organic acid salt. In further embodiments, IFG is selected from isophagomine quinate, isophagomine formate, isophagomine malate, isophagomine oxalate, isophagomine malonate, isophagomine succinate, isophagomine cyclamate, isophagomine D-tartrate, and isophagomine ascorbate. In additional embodiments, isophagomine is selected from isophagomine quinate, isophagomine fumarate, isophagomine oxalate, isophagomine D-tartrate and is in crystalline form. In further embodiments, the crystalline form is characterized by three or more very strong, strong, and medium strength XRPD peaks described in Tables 1, 3, 5, and 7, respectively.

[0075] In certain embodiments, a composition comprising an IFG is provided, wherein the IFG is effective as a novel excipient to stabilize the glucocerebrosidase (GCase) enzyme in the injectable formulations described herein, and further stabilizes the GCase enzyme at the injection site and in vivo, i.e., the IFG is a stabilizer of the GCase active ingredient. In some embodiments, the composition is an aqueous solution. In some embodiments, the composition is a lyophilized product. In another aspect, the IFG is present in an amount sufficient to reduce the degradation of the GCase active ingredient; in certain embodiments, the amount of the IFG can be a significantly lower amount than the therapeutically effective amount of the IFG alone, or when the IFG is administered in combination with the GCase active ingredient in a conventional combination therapy. In certain embodiments, the IFG is at least about 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5-fold molar excess over the GCase. In further embodiments, the IFG in the composition is present in an amount that does not increase the endogenous serum GCase activity when administered as a single active substance. In certain embodiments, the IFG is present in an amount sufficient to maintain the stability of the GCase in the composition. In some embodiments, the IFG is present in an amount sufficient to maintain the stability of the GCase in the composition for at least 3 days at 0 to 50 °C. In some embodiments, the IFG is present in an amount sufficient to maintain the stability of the GCase in the composition for at least 6 months at 0 to 40 °C.

[0076] In a further aspect, a composition is provided that comprises glucocerebrosidase (GCase) and IFG in a molar ratio of from about 1:2.5 to about 1:5 or about 1:>2.5 (i.e., 1:x, where x is greater than 2.5). In a further embodiment, x is greater than 2.5 and less than the amount that increases endogenous serum GCase activity when administered as a single active agent. In some embodiments, GCase is velaglucerase alfa. In some embodiments, the molar ratio of GCase to IFG is from about 1:1 to about 1:30. In some embodiments of the IFG / GCase compositions described herein, the IFG is an IFG salt as described herein.

[0077] In some embodiments of the IFG / GCase compositions described herein, the composition comprises GCase at 0.5 to 5.0 mg / kg body weight and is in a dosage form further comprising IFG (including the free base, salts, and crystalline forms described herein), for example, IFG is at least about 1, 1.25, 1.5, 2, 2.5, 3, 4, or 5 molar excess relative to GCase. In some embodiments, the IFG in the composition is present in an amount that does not increase endogenous serum GCase activity when administered as a single agent. In some embodiments, the composition comprises GCase at 0.8 to 4.0 mg / kg. In some embodiments, the composition comprises GCase at 1.0 to 3.0 mg / kg. In some embodiments, the composition comprises GCase at 1.2 to 2.0 mg / kg. In some embodiments, the composition comprises about 1.5 mg / kg of GCase. In some embodiments, the composition comprises 1.5 mg / kg of GCase. In some embodiments, the composition comprises GCase at 2.0 to 5.0 mg / kg. In some embodiments, the composition comprises GCase at 2.25 to 4.5 mg / kg. In some embodiments, the composition comprises GCase at 2.25 to 3.75 mg / kg. In some embodiments, the composition comprises GCase at 3.5 to 5.0 mg / kg. In some embodiments, the IFG is in a molar ratio of 1 to 5 or 1 to 10 relative to GCase. In some embodiments, the IFG is in a molar ratio of 2 to 10 relative to GCase. In some embodiments, the IFG is in a molar ratio of 10 to 30 relative to GCase. In some embodiments, the IFG is in a molar ratio of 30 to 100 relative to GCase. In some embodiments, the IFG is in a molar ratio of 2.5 to 3.5 relative to GCase. In some embodiments, the IFG is in a molar ratio of 3 relative to GCase.

[0078] For example, the concentration of GCase in any of the provided compositions, such as the lyophilized composition when reconstituted, may be from about 60 mg / ml to about 180 mg / ml, from about 65 mg / ml to about 175 mg / ml, from about 70 mg / ml to about 170 mg / ml, from about 75 mg / ml to about 165 mg / ml, from about 80 mg / ml to about 160 mg / ml, from about 85 mg / ml to about 155 mg / ml, from about 90 mg / ml to about 150 mg / ml, from about 95 mg / ml to about 145 mg / ml, from about 100 mg / ml to about 140 mg / ml, from about 105 mg / ml to about 135 mg / ml, from about 110 mg / ml to about 130 mg / ml, or from about 115 mg / ml to about 125 mg / ml. In alternative embodiments, the concentration of GCase in any of the provided compositions may be in the range between any two concentrations selected from 60 mg / ml, 63 mg / ml, 66 mg / ml, 69 mg / ml, 72 mg / ml, 75 mg / ml, 78 mg / ml, 81 mg / ml, 84 mg / ml, 87 mg / ml, 90 mg / ml, 93 mg / ml, 96 mg / ml, 99 mg / ml, 102 mg / ml, 105 mg / ml, 108 mg / ml, 111 mg / ml, 114 mg / ml, 117 mg / ml, 120 mg / ml, 123 mg / ml, 126 mg / ml, 129 mg / ml, 132 mg / ml, 135 mg / ml, 138 mg / ml, 141 mg / ml, 144 mg / ml, 147 mg / ml, 150 mg / ml, 153 mg / ml, 156 mg / ml, 159 mg / ml, 162 mg / ml, 165 mg / ml, 168 mg / ml, 171 mg / ml, 174 mg / ml, 177 mg / ml, and 180 mg / ml. In certain embodiments, the concentration of GCase in any of the provided compositions is from about 120 mg / ml to about 160 mg / ml, from about 125 mg / ml to about 155 mg / ml, from about 130 mg / ml to about 150 mg / ml, from about 135 mg / ml to about 145 mg / ml, or about 140 mg / ml.

[0079] In alternative embodiments, the concentration of GCase in any of the compositions disclosed herein, such as, for example, a lyophilized composition when reconstituted, can be from about 30 mg / ml to about 200 mg / ml, about 40 to about 180 mg / ml, about 40 mg / ml to about 180 mg / ml, about 50 mg / ml to about 160 mg / ml, about 55 mg / ml to about 140 mg / ml, about 60 to about 120 mg / ml, about 0.5 to about 10 mg / ml, about 5 to about 15 mg / ml, about 10 to about 20 mg / ml, about 15 to about 25 mg / ml, about 20 to about 30 mg / ml, about 25 to about 35 mg / ml, about 30 to about 40 mg / ml, about 2 to about 8 mg / ml, about 5 to about 11 mg / ml, about 8 to about 14 mg / ml, about 11 to about 17 mg / ml, about 14 to about 20 mg / ml, about 17 to about 23 mg / ml, about 20 to about 26 mg / ml, about 23 to about 29 mg / ml, about 26 to about 32 mg / ml, about 29 to about 35 mg / ml, about 32 to about 38 mg / ml, about 2 to about 5 mg / ml, about 5 to about 8 mg / ml, about 8 to about 11 mg / ml, about 11 to about 14 mg / ml, about 14 to about 17 mg / ml, about 17 to about 20 mg / ml, about 20 to about 23 mg / ml, about 23 to about 26 mg / ml, about 26 to about 29 mg / ml, about 29 to about 32 mg / ml, about 32 to about 35 mg / ml, about 35 to about 38 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, about 30 mg / ml, about 31 mg / ml, about 32 mg / ml, about 33 mg / ml, about 34 mg / ml, about 35 mg / ml, about 36 mg / ml, about 37 mg / ml, about 38 mg / ml, about 39 mg / ml, or about 40 mg / ml.

[0080] The concentration of GCase may be 50 units / ml to 200 units / ml, 70 units / ml to 160 units / ml, 80 units / ml to 175 units / ml, 90 units / ml to 190 units / ml, 60 units / ml to 145 units / ml, 50 units / ml to 130 units / ml, 80 units / ml to 140 units / ml, 70 units / ml to 120 units / ml, 60 units / ml to 100 units / ml, 50 units / ml to 85 units / ml, 90 units / ml to 160 units / ml, 100 units / ml to 180 units / ml, 120 units / ml to 200 units / ml, 90 units / ml to 125 units / ml, 80 units / ml to 105 units / ml, 70 units / ml to 100 units / ml, 60 units / ml to 90 units / ml, 50 units / ml to 80 units / ml, 100 units / ml to 140 units / ml, 115 units / ml to 160 units / ml, 130 units / ml to 180 units / ml, 145 units / ml to 200 units / ml, 100 units / ml to 115 units / ml, 90 units / ml to 105 units / ml, 80 units / ml to 95 units / ml, 70 units / ml to 85 units / ml, 60 units / ml to 75 units / ml, 50 units / ml to 65 units / ml, 110 units / ml to 125 units / ml, 120 units / ml to 135 units / ml, 130 units / ml to 145 units / ml, 140 units / ml to 160 units / ml, 160 units / ml to 180 units / ml, 180 units / ml to 200 units / ml, about 50 units / ml, about 60 units / ml, about 70 units / ml, about 80 units / ml, about 90 units / ml, about 100 units / ml, about 110 units / ml, about 120 units / ml, about 130 units / ml, about 140 units / ml, about 150 units / ml, about 160 units / ml, about 170 units / ml, about 180 units / ml, about 190 units / ml, about 200 units / ml, 50 units / ml, 60 units / ml, 70 units / ml, 80 units / ml, 90 units / ml, 100 units / ml, 110 units / ml, 120 units / ml, 130 units / ml, 140 units / ml, 150 units / ml, 160 units / ml, 170 units / ml, 180 units / ml, 190 units / ml, or 200 units / ml.

[0081] In various embodiments, the composition comprises glucocerebrosidase (GCase) and isophagomine (IFG), and the IFG may be the free base, salt, or crystalline form provided herein, and the IFG is present in a molar ratio of at least about 1:1, 1:1.5, 1:2, or 1:2.5 (GCase:IFG). The molar ratio of GCase to IFG is 1:1, 1:1.5, 1:2, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6.0, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7.0, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8.0, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9.0, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10.0, 1:10.1, 1:10.2, 1:10.3, 1:10.4, 1:10.5, 1:10.6, 1:10.7, 1:10.8, 1:10.9, 1:11.0, 1:11.1, 1:11.2, 1:11.3, 1:11.4, 1:11.5, 1:11.6, 1:11.7, 1:11.8, 1:11.9, 1:12.0, 1:12.1, 1:12.2, 1:12.3, 1:12.4, 1:12.5, 1:12.6, 1:12.7, 1:12.8, 1:12.9, 1:13.0, 1:13.1, 1:13.2, 1:13.3, 1:13.4, 1:13.5, 1:13.6, 1:13.7, 1:13.8, 1:13.9, 1:14.0, 1:14.1, 1:14.2, 1:14.3, 1:14.4, 1:14.5, 1:14.6, 1:14.7, 1:14.8, 1:14.9, 1:15.0, 1:15.1, 1:15.2, 1:15.3、1:15.4、1:15.5、1:15.6、1:15.7、1:15.8、1:15.9、1:16.0、1:16.1、1:16.2、1:16.3、1:16.4、1:16.5、1:16.6、1:16.7、1:16.8、1:16.9、1:17.0、1:17.1、1:17.2、1:17.3、1:17.4、1:17.5、1:17.6、1:17.7、1:17.8、1:17.9、1:18.0、1:18.1、1:18.2、1:18.3、1:18.4、1:18.5、1:18.6、1:18.7、1:18.8、1:18.9、1:19.0、1:19.1、1:19.2、1:19.3、1:19.4、1:19.5、1:19.6、1:19.7、1:19.8、1:19.9、1:20.0、1:20.1、1:20.2、1:20.3、1:20.4、1:20.5、1:20.6、1:20.7、1:20.8、1:20.9、1:21.0、1:21.1、1:21.2、1:21.3、1:21.4、1:21.5、1:21.6、1:21.7、1:21.8、1:21.9、1:22.0、1:22.1、1:22.2、1:22.3、1:22.4、1:22.5、1:22.6、1:22.7、1:22.8、1:22.9、1:23.0、1:23.1、1:23.2、1:23.3、1:23.4、1:23.5、1:23.6、1:23.7、1:23.8、1:23.9、1:23.9、1:24.0、1:24.1、1:24.2、1:24.3、1:24.4、1:24.5、1:24.6、1:24.7、1:24.8、1:24.9、1:25.0、1:25.1、1:25.2、1:25.3、1:25.4、1:25.5、1:25.6、1:25.7、1:25.8、1:25.9、1:26.0、1:26.1、1:26.2、1:26.3、1:26.4、1:26.5、1:26.6、1:26.7、1:26.8、1:26.9、1:27.0、1:27.1、1:27.2、1:27.3、1:27.4、1:27.5、1:27.6、1:27.7、1:27.8、1:27.9、1:28.0、1:28.1、1:28.2、1:28.3、1:28.4、1:28.5、1:28.6、1:28.7、1:28.8、1:28.9、1:29.0、1:29.1、1:29.2、1:29.3、1:29.4、1:29.It may be in the range between any two values selected from 5, 1:29.6, 1:29.7, 1:29.8, 1:29.9, or 1:30.0.

[0082] The molar ratio of GCase to IFG can be 1:2.5 to 1:3.5, 1:2.6 to 1:3.4, 1:2.7 to 1:3.5, 1:2.7 to 1:3.4, 1:2.5 to 1:3.3, 1:2.8 to 1:3.5, 1:2.8 to 1:3.3, 1:2.7 to 1:3.2, 1:2.6 to 1:3.1, 1:2.5 to 1:3.0, 1:2.9 to 1:3.3, 1:2.8 to 1:3.2, 1:2.7 to 1:3.1, 1:2.6 to 1:3.0, 1:2.5 to 1:2.9, 1:3.0 to 1:3.4, or 1:3.1 to 1:3.5.

[0083] The molar ratio of GCase to IFG can be 1:7 to 1:33, 1:8 to 1:32, 1:9 to 1:33, 1:7 to 1:31, 1:9 to 1:31, 1:8 to 1:30, 1:7 to 1:29, 1:10 to 1:32, 1:11 to 1:33, 1:7 to 1:29, 1:10 to 1:30, 1:9 to 1:29, 1:8 to 1:28, 1:7 to 1:27, 1:11 to 1:31, 1:12 to 1:32, 1:13 to 1:33, 1:11 to 1:29, 1:10 to 1:28, 1:9 to 1:27, 1:8 to 1:26, 1:7 to 1:25, 1:12 to 1:30, 1:13 to 1:31, 1:14 to 1:32, 1:15 to 1:33, 1:13 to 1:29, 1:12 to 1:28, 1:11 to 1:27, 1:10 to 1:26, 1:9 to 1:25, 1:8 to 1:24, 1:7 to 1:23, 1:14 to 1:30, 1:15 to 1:31, 1:16 to 1:32, 1:17 to 1:33, 1:14 to 1:28, 1:13 to 1:27, 1:12 to 1:26, 1:11 to 1:25, 1:10 to 1:24, 1:9 to 1:23, 1:8 to 1:22, 1:7 to 1:21, 1:15 to 1:29, 1:16 to 1:30, 1:17 to 1:31, 1:18 to 1:32, 1:19 to 1:33, 1:15 to 1:27, 1:14 to 1:26, 1:13 to 1:25, 1:12 to 1:24, 1:11 to 1:23, 1:10 to 1:22, 1:9 to 1:21, 1:8 to 1:20, 1:7 to 1:19, 1:16 to 1:28, 1:17 to 1:29, 1:18 to 1:30, 1:19 to 1:31, 1:20 to 1:32, or 1:21 to 1:33.

[0084] The molar ratio of GCase to IFG can be 1:16 to 1:26, 1:15 to 1:25, 1:14 to 1:24, 1:13 to 1:23, 1:12 to 1:22, 1:11 to 1:31, 1:10 to 1:30, 1:9 to 1:29, 1:8 to 1:28, 1:7 to 1:27, 1:17 to 1:27, 1:18 to 1:28, 1:19 to 1:29, 1:20 to 1:30, 1:21 to 1:31, 1:22 to 1:32, 1:23 to 1:33, 1:17 to 1:25, 1:14 to 1:24, 1:13 to 1:23, 1:12 to 1:22, 1:11 to 1:21, 1:10 to 1:20, 1:9 to 1:19, 1:18 to 1:26, 1:19 to 1:27, 1:20 to 1:28, 1:21 to 1:29, 1:22 to 1:30, 1:23 to 1:31, 1:18 to 1:24, 1:17 to 1:23, 1:16 to 1:22, 1:15 to 1:21, 1:14 to 1:20, 1:13 to 1:19, 1:12 to 1:18, 1:11 to 1:17, 1:19 to 1:25, 1:20 to 1:26, 1:21 to 1:27, 1:22 to 1:28, 1:23 to 1:29, 1:24 to 1:30, 1:19 to 1:23, 1:17 to 1:21, 1:15 to 1:19, 1:13 to 1:17, 1:11 to 1:15, 1:9 to 1:13, 1:7 to 1:11, 1:21 to 1:25, 1:23 to 1:27, 1:25 to 1:29, 1:27 to 1:31, 1:29 to 1:33, 1:20 to 1:23, 1:18 to 1:21, 1:16 to 1:19, 1:14 to 1:17, 1:12 to 1:15, 1:10 to 1:13, 1:8 to 1:11, 1:22 to 1:25, 1:24 to 1:27, 1:26 to 1:29, 1:28 to 1:31, or 1:30 to 1:33.

[0085] The molar ratio of GCase to IFG can be 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:35, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, or 1:100.

[0086] The molar ratio of GCase to IFG can be 1:30 to 1:100, 1:30 to 1:80, 1:40 to 1:90, 1:50 to 1:100, 1:30 to 1:60, 1:40 to 1:70, 1:50 to 1:80, 1:60 to 1:90, 1:70 to 1:100, 1:30 to 1:50, 1:40 to 1:60, 1:50 to 1:70, 1:60 to 1:80, 1:70 to 1:90, 1:80 to 1:100, 1:30 to 1:40, 1:40 to 1:50, 1:50 to 1:60, 1:60 to 1:70, 1:70 to 1:80, 1:80 to 1:90, or 1:90 to 1:100.

[0087] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCase) and IFG fumarate in a molar ratio of 1:2.5 to 1:3.5.

[0088] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCase) and IFG quinolate in a molar ratio of 1:2.5 to 1:3.5.

[0089] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCase) and IFG oxalate in a molar ratio of 1:2.5 to 1:3.5.

[0090] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCase) and IFG succinate in a molar ratio of 1:2.5 to 1:3.5.

[0091] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCase) and IFG cyclamate in a molar ratio of 1:2.5 to 1:3.5.

[0092] In another aspect, provided is a method of preparing any of the IFG or IFG / GCase compositions described herein. In one aspect, the method comprises dissolving IFG, or a salt thereof described herein, in a solvent (e.g., water), adjusting the pH to about 6.0, and adding glucocerebrosidase (GCase) to obtain the composition. In some embodiments, the method further comprises lyophilizing the IFG before adding the GCase. In some embodiments, the method further comprises adding polysorbate 20 up to 0.01%. In some embodiments, the method further comprises filtering the composition through a 0.22 μm membrane. In some embodiments, the IFG is present in an amount sufficient to maintain the stability of GCase in the composition. In some embodiments, the IFG is present in an amount sufficient to maintain the stability of GCase in the composition for at least 3 days at 0 - 50°C. In some embodiments, the IFG is present in an amount sufficient to maintain the stability of GCase in the composition for at least 6 months at 0 - 40°C.

[0093] In some embodiments, the composition comprises 45 - 120 mg / mL of velaglucerase alfa and 0.2 - 1.8 mg / mL of crystalline IFG D-tartrate. In some embodiments, the composition comprises 60 mg / mL of velaglucerase alfa and 0.9 mg / mL of crystalline IFG D-tartrate.

[0094] In some embodiments, the composition comprises velaglucerase alfa and IFG at 60 - 180 mg / mL in a molar ratio as described above, for example, from about 1:2.5 to about 1:3.5, or about 1:3.3. In certain embodiments, the composition further comprises a buffer (e.g., sodium citrate or citric acid or a combination thereof) at about 5 mM to about 15 mM or about 10 mM, a carbohydrate (e.g., sucrose) in an amount of about 200 mM to about 300 mM or about 250 mM, and a surfactant (e.g., PS20, PS80 or poloxamer 188) in an amount of about 0.05% to about 0.5% or about 0.1%. In certain embodiments, IFG is in the free base form. In alternative embodiments, IFG is in the form of a salt, and the salt is prepared from an organic acid such as quinic acid, maleic acid, fumaric acid, oxalic acid, malonic acid, D-tartaric acid, L-tartaric acid, succinic acid, cyclamic acid or ascorbic acid. In another embodiment, the IFG salt is in crystalline form.

[0095] In any of the above embodiments, the isophagomine compound used for the stabilization of GCase in solution is isophagomine tartrate. In any of the above embodiments, the isophagomine compound used for the stabilization of GCase in solution is isophagomine fumarate. In certain embodiments, GCase is velaglucerase alfa.

[0096] In a further embodiment of the composition described herein, GCase is stable for 18, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 months when stored at ≦ -65°C. In certain embodiments, GCase is stable for 18 months when stored under long-term storage conditions of ≦ -65°C. In certain embodiments, GCase is stable for 30 months when stored under long-term storage conditions of ≦ -65°C. In certain embodiments, GCase is stable for 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 months when stored at -20 ± 5°C. In certain embodiments, GCase is stable for 1, 2, 3, 4, 5, or 6 months when stored at 5 ± 3°.

[0097] Pharmaceutical carrier The pharmaceutical compositions of the present disclosure can include one or more pharmaceutically acceptable carriers. As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, excipients, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Pharmaceutical formulations are well-established techniques and are further described, for example, in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X). Such media can be used in the compositions of the present disclosure, except in cases where any conventional media or agents are incompatible with the active compound. Supplementary active compounds can also be incorporated into the compositions.

[0098] Sterile injectable solutions can be prepared by placing IFG, and optionally other active ingredients such as GCase, in a suitable solvent, along with one or a combination of the ingredients described above as necessary, and then filter sterilizing. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients such as those described above. In the case of powders for sterile injectable compositions, the preferred methods of making are vacuum drying and freeze drying (e.g., lyophilization), by which powders of the active ingredient plus any additional desired ingredients are obtained from a previously sterile filtered solution.

[0099] Compounds (e.g., the IFG and IFG / GCase compositions described herein) can be prepared using carriers that protect the compound against rapid excretion from the body, such as controlled release formulations, which include implants and microencapsulation delivery systems. For example, biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes that target infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0100] For intravenous (IV) administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate-buffered saline (PBS). In each case, the composition must be sterile and must have a fluidity that allows easy passage through a hypodermic needle. The composition must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintenance of the required particle size in the case of dispersions, or by the use of surfactants such as nonionic poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO) copolymers (e.g., poloxamers 68, 88, 98, 108, 124, 188, 237, 338, 407) or polysorbate-type nonionic surfactants formed by ethoxylating sorbitan before adding lauric acid (e.g., polysorbate 20, known as polyoxyethylene (20) sorbitan monolaurate). Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal. In many cases, it is preferable to include in the composition isotonic agents such as, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, sodium chloride, etc. Long-term stability of injectable compositions can be brought about, for example, by including agents that delay absorption such as aluminum monostearate, human serum albumin, gelatin, etc.

[0101] Antioxidants and other stabilizers The IFG and GCase / IFG compositions described herein may further contain an antioxidant. One suitable antioxidant is cysteine. Cysteine may be present at 0.030% - 0.100%, 0.050% - 0.080%, 0.040% - 0.070%, 0.030% - 0.060%, 0.060% - 0.090%, 0.070% - 0.100%, 0.065% - 0.080%, 0.060% - 0.075%, 0.055% - 0.070%, 0.050% - 0.065%, 0.070% - 0.085%, 0.075% - 0.090%, about 0.065%, about 0.070%, about 0.075%, about 0.080%, 0.065%, 0.070%, 0.075%, or 0.080%. Without wishing to be bound by theory, cysteine may further stabilize GCase.

[0102] The IFG and GCase / IFG compositions described herein may further contain a carbohydrate such as sucrose or trehalose. The carbohydrate, e.g., sucrose or trehalose, may be present at 12% - 19%, 13% - 18%, 14% - 17%, 12% - 15%, 13% - 16%, 15% - 17%, about 16%, or 16%. Without wishing to be bound by theory, sucrose or trehalose may further stabilize GCase by reducing the availability of thiol (-SH) groups.

[0103] The IFG and GCase / IFG compositions herein may further contain a surfactant. The surfactant may be polysorbate 20 or any number of poloxamer-based compounds.

[0104] In certain embodiments, the stability of GCase is at least 5 - 80% higher (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% higher) under pre - selected conditions than the stability of GCase in different compositions lacking carbohydrates (sucrose or trehalose), antioxidants, or both carbohydrates and antioxidants.

[0105] IFG and GCase / IFG compositions may be purged of oxygen prior to storage in a container. Further, the container is preferably airtight so as to prevent the ingress of oxygen. GCase in the compositions described herein (e.g., liquid compositions containing GCase) can have long - term stability. For example, under pre - selected conditions, e.g., in an airtight container, at a temperature of 2 - 8 °C, when stored for up to 3, 6, 9, 12, or 24 months (or more in some embodiments), the GCase in the composition retains at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% of the stability it had prior to storage.

[0106] A suitable protein concentration provides a composition containing 0.075% cysteine, 16% sucrose, adjusts the pH to 5.7, adjusts GCase to the candidate concentration, and further purges the composition with O 2 can be tested. For example, the stability of GCase in a GCase / IFG composition, e.g., a GCase / IFG composition at a candidate concentration, as measured by the aggregation rate or degradation rate over a given time, is compared to one or more standards. The stability of GCase at each concentration is compared. Suitability can be indicated by candidate concentrations that have an effect on comparable or better stability compared to the concentrations described herein.

[0107] The stability of GCase can be measured by any of the methods described throughout this application, for example, by measuring protein aggregation or protein degradation. Protein aggregation can be determined, for example, by size exclusion chromatography, non-denaturing PAGE, or other methods for determining size. Protein degradation can be determined, for example, by reverse phase HPLC, non-denaturing PAGE, ion exchange chromatography, SEC, SEC HPLC, peptide mapping, or similar methods.

[0108] pH can affect the stability of GCase in the various GCase / IFG compositions described herein. pH can affect the conformation and / or aggregation and / or degradation and / or reactivity of GCase. Buffers that can be used to adjust the pH of the protein composition include salt solutions of histidine, citrate, phosphate, glycine, succinate, acetate, glutamate, Tris, tartrate, aspartate, maleate, and lactate. In certain embodiments, the IFG / GCase formulation comprises sodium citrate buffer.

[0109] Surfactant The IFG and GCase / IFG compositions described herein may further comprise one or more surfactants. Without wishing to be bound by theory, surfactants can enhance protein stability, such as by providing an air / liquid interface that can reduce protein degradation during agitation or shipping. Surfactants can be selected that enhance protein stability, such as not causing protein degradation in a particular liquid composition. Suitable surfactants include nonionic poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO) copolymers (e.g., poloxamers 68, 88, 98, 108, 124, 188, 237, 338, 407), and polysorbate-type nonionic surfactants formed by ethoxylating sorbitan prior to adding an unsaturated fatty acid such as lauric acid or oleic acid (e.g., polysorbate 20, also known as polyoxyethylene(20)sorbitan monolaurate, and polysorbate 80, also known as polyoxyethylene(80)sorbitan monooleate). Exemplary surfactants are poloxamer 188, PS20, PS80 and / or Pluronic® F68. The surfactant can be present in an amount between about 0.005% and about 5%, such as between about 0.01% and about 1%, such as between about 0.025% and about 0.5%, such as between about 0.03% and about 0.25%, such as between about 0.04 and about 0.1%, such as between about 0.05% and about 0.075%, such as 0.05%. The ideal surfactant or combination thereof is one that is not modified or cleaved by GCase.

[0110] For example, a candidate surfactant provides a composition comprising 2 mg / ml of GCase, an amount of IFG, 0.075% cysteine, 16% sucrose, then adjusts the pH to 5.7, then adds the candidate surfactant, and further adds O to the composition 2It can be tested by purging. The stability of the GCase / IFG composition containing the candidate surfactant is measured as the rate of aggregation or degradation, for example, at a given time compared to one or more standards. For example, a suitable standard is a composition similar to the test conditions, except that no surfactant is added to the composition. The stability of the treated (surfactant-containing) composition and the untreated (surfactant-free) composition can be compared under conditions that simulate "real-world" scenarios such as storage and shipping. The standard can be a composition similar to the test composition, except that a different surfactant is used instead of poloxamer 188. Next, poloxamer 188 serves as the standard for the comparison reference. Compatibility can be shown by the candidate surfactant having an effect on stability equal to or greater than that of the surfactants described herein. If the candidate surfactant is determined to be suitable (e.g., improves the stability of the composition compared to one of the standards), the concentration of the candidate surfactant can be improved. For example, the concentration can be increased or decreased over a range of values to determine which concentration causes the greatest increase in stability compared to the standard and other concentrations during the test.

[0111] Alternatively, in the compositions described herein, combinations of two or more surfactants are used. The suitability of the combination can be tested as described above by comparing the stability of the GCase / IFG composition containing the test combination of surfactants to the stability of the GCase / IFG composition containing poloxamer 188.

[0112] Packaging and delivery The IFG and IFG / GCase compositions described herein can be administered using a variety of medical devices. For example, the compositions described herein can be administered with a needleless subcutaneous injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present disclosure include those disclosed below: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dosing at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses an implantable infusion device with variable flow rate for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chamber compartment; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Of course, many other such implants, delivery systems, and modules are also known.

[0113] The IFG and GCase / IFG compositions described herein can be packaged in a two-chamber syringe. For example, the lyophilized form of the IFG and GCase / IFG compositions can be contained in the first syringe chamber, and a liquid can be included in the second syringe chamber (see, e.g., U.S. Patent Application Publication No. 2004-0249339).

[0114] The IFG and GCase / IFG compositions described herein can be packaged in a needleless syringe (see, e.g., U.S. Patent Nos. 6,406,455 and 6,939,324). Briefly, as an example, an injection device includes a gas chamber containing a gas or a gas source; a port capable of allowing the release of the gas from the gas chamber; a plunger capable of causing the movement of at least a first piston when the gas is released from the gas chamber; a first piston; a second piston; a first chamber, e.g., a chamber useful for storing and mixing a drug; a piston housing in which the first piston, the second piston, and the first chamber are disposed; a displacement member (the displacement member may be the plunger or a separate member) capable of causing the movement of one or both of the first and second pistons independently of the power of the gas from the gas chamber; an orifice suitable for needleless injection communicating with the first chamber; and the first and second pistons are slidably disposed within the piston housing, and the displacement member, the gas source, and the plunger are arranged as follows: at a first position of the piston, a second chamber, e.g., a fluid reservoir, is defined within the piston housing by the first piston, the piston housing, and the second piston, and the displacement member can move one or both of the pistons to a second position, at which time the first piston is in a position such that the second chamber, which can be the fluid reservoir, communicates with the first chamber, which can be the drug storage and mixing chamber, the second piston is moved in the direction of the first piston, thereby reducing the volume of the second chamber and allowing the movement of fluid from the second chamber to the first chamber, and the plunger moves the first piston to reduce the volume of the first chamber when the gas is released from the gas chamber, discharging a substance from the chamber, e.g., to a subject, through the orifice.

[0115] The needleless syringe can include separate modules for a first component, e.g., a dry or liquid component, and a second component, e.g., a liquid component. The modules may be provided as two separate components and can be assembled, for example, by the subject to whom the components are to be self-administered or by another person, e.g., a person providing or performing medical treatment. The modules can form all or part of the piston housing of the device described herein. The device can be used to provide any first and second components when it is desirable to store or provide the components separately and combine them prior to administration to a subject.

[0116] Treatment method In another aspect, a method of treating a disorder associated with a dysfunction of the GCase pathway is provided, comprising administering any of the compositions described herein. In another aspect, a composition comprising the disclosed GCase and IFG is provided for use in a method of treating a disorder associated with a dysfunction of the GCase pathway, comprising administering any of the compositions described herein. In another aspect, the use of a composition comprising the GCase and IFG provided herein in the manufacture of a medicament for a method of treating a disorder of the GCase pathway is provided. In some embodiments, the method is effective to treat a disorder in the GCase pathway. In certain embodiments, the disorder is treated when one or more symptoms associated with the dysfunction of the GCase pathway are improved or alleviated. In some embodiments, the composition is administered intravenously or subcutaneously. In some embodiments, the composition is administered subcutaneously, e.g., by subcutaneous injection. In some embodiments, the composition is administered twice a week, once a week, less than once a week, or once every other week.

[0117] In some embodiments, the disorder comprises a deficiency in GCase activity. In some embodiments, the deficiency in GCase activity comprises a decrease in enzyme activity. In some embodiments, the disorder comprises dysregulation of α-synuclein. In some embodiments, the disorder is a lysosomal storage disorder (lysosomal disease), such as Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidosis, or multiple system atrophy. In some embodiments, the disorder is a neurodegenerative disease, such as Parkinson's disease, Alzheimer's disease, or Lewy body dementia.

[0118] In another aspect, provided is a method of treating a dysfunction of the GCase pathway, comprising administering to a subject in need thereof any of the compositions described herein. In some embodiments, the subject is human.

[0119] In some embodiments, administering to a subject in need thereof any of the compositions comprising GCase and IFG described herein results in an increase in the exposure, activity, or bioavailability of GCase, for example, as compared to an equivalent exposure, activity, or bioavailability of GCase alone. In some embodiments, the exposure, activity, or bioavailability of GCase in the spleen is increased. In some embodiments, the exposure, activity, or bioavailability of GCase in the liver is increased. In some embodiments, the exposure, activity, or bioavailability of GCase in the serum is increased. In some embodiments, the composition is administered IV. In some embodiments, the composition is administered subcutaneously.

[0120] In another aspect, there is provided a method of treating a dysfunction of the GCase pathway, comprising administering to a subject a composition comprising a dosage of GCase of 0.5 to 5.0 mg / kg body weight and an IFG (including, but not limited to, the salts and crystal forms described herein), for example, the IFG is at least about 1, 1.25, 1.5, 2, 2.5, 3, 4, or 5-fold molar excess relative to GCase. In some embodiments, the composition is administered subcutaneously. In some embodiments, the IFG in the composition is administered in an amount that does not increase endogenous serum GCase activity when administered as a single agent (or particularly when administered without co-administration of GCase).

[0121] In another aspect, there is provided a composition for use in a method of treating a disorder associated with a dysfunction of the GCase pathway, the composition comprising a dosage of GCase of 0.5 to 5.0 mg / kg body weight and an IFG (including, but not limited to, the salts and crystal forms described herein), for example, the IFG is at least about 1, 1.25, 1.5, 2, 2.5, 3, 4, or 5-fold molar excess relative to GCase. In another aspect, there is provided the use of a composition comprising a dosage of GCase of 0.5 to 5.0 mg / kg body weight and an IFG (including, but not limited to, the salts and crystal forms described herein) in the manufacture of a medicament for a method of treating a dysfunction of the GCase pathway, for example, the IFG is at least about 1, 1.25, 1.5, 2, 2.5, 3, 4, or 5-fold molar excess relative to GCase. In some embodiments, the method is effective in treating a disorder associated with a dysfunction of the GCase pathway. In certain embodiments, the disorder is considered treated when one or more symptoms associated with the dysfunction of the GCase pathway are improved or alleviated. In some embodiments, the composition is administered intravenously or subcutaneously. In some embodiments, the composition is administered subcutaneously, for example, by subcutaneous injection. In some embodiments, the composition is administered twice a week, once a week, less frequently than once a week, or once every other week.

[0122] In some embodiments, the composition for use in the disclosed method comprises a dosage of GCase of 0.8 to 4.0 mg / kg body weight. In some embodiments, the composition comprises a dosage of GCase of 1.0 to 3.0 mg / kg. In some embodiments, the composition comprises a dosage of GCase of 1.2 to 2.0 mg / kg. In some embodiments, the composition comprises approximately 1.5 mg / kg of GCase. In some embodiments, the composition comprises 1.5 mg / kg of GCase. In some embodiments, the composition comprises 2.0 to 5.0 mg / kg of GCase. In some embodiments, the composition comprises 2.25 to 4.5 mg / kg of GCase. In some embodiments, the composition comprises 2.25 to 3.75 mg / kg of GCase. In some embodiments, the composition comprises 3.5 to 5.0 mg / kg of GCase.

[0123] In any of the foregoing embodiments, the isophagomine compound used for the stabilization of GCase in solution is isophagomine tartrate. In any of the foregoing embodiments, the isophagomine compound used for the stabilization of GCase in solution is isophagomine fumarate. In certain embodiments, GCase is velaglucerase alfa.

[0124] Any of the foregoing embodiments wherein the isophagomine compound used for the stabilization of GCase in solution is isophagomine tartrate. Any of the foregoing embodiments wherein the isophagomine compound used for the stabilization of GCase in solution is isophagomine fumarate. Certain embodiments wherein GCase is velaglucerase alfa.

[0125] In further embodiments of the compositions described herein, GCase is stable for 18, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 months when stored at ≤ -65°C. In certain embodiments, GCase is stable for 18 months when stored under long-term storage conditions of ≤ -65°C. In certain embodiments, GCase is stable for 30 months when stored under long-term storage conditions of ≤ -65°C. In certain embodiments, GCase is stable for 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 months when stored at -20 ± 5°C. In certain embodiments, GCase is stable for 1, 2, 3, 4, 5, or 6 months when stored at 5 ± 3°C.

[0126] In some embodiments of the compositions for use in the disclosed methods, IFG is in a molar ratio of 1 to 5 or 1 to 10 times that of GCase. In some embodiments, IFG is in a molar ratio of 2 to 10 times that of GCase. In some embodiments, IFG is in a molar ratio of 10 to 30 times that of GCase. In some embodiments, IFG is in a molar ratio of 30 to 100 times that of GCase. In some embodiments, IFG is in a molar ratio of 2.5 to 3.5 times that of GCase. In some embodiments, IFG is in a molar ratio of 3 times that of GCase.

[0127] Either the IFG and GCase / IFG formulations described herein can be administered to a patient. The dosage of GCase may be about 60 units / kg or 60 units / kg on alternate weeks. The dosage of GCase may be about 30 units / kg or 30 units / kg weekly. Alternatively, the dosage of GCase may range from 30 to 80 units / kg on alternate weeks, from 40 to 70 units / kg on alternate weeks, from 50 to 80 units / kg on alternate weeks, from 45 to 65 units / kg on alternate weeks, from 40 to 60 units / kg on alternate weeks, from 35 to 55 units / kg on alternate weeks, from 30 to 50 units / kg on alternate weeks, from 45 to 65 units / kg on alternate weeks, from 50 to 70 units / kg on alternate weeks, from 55 to 75 units / kg on alternate weeks, from 60 to 80 units / kg on alternate weeks, from 55 to 65 units / kg on alternate weeks, from 45 to 55 units / kg on alternate weeks, from 35 to 45 units / kg on alternate weeks, or from 65 to 75 units / kg on alternate weeks. Alternatively, the dosage of GCase may range from 15 to 40 units / kg weekly, from 20 to 35 units / kg weekly, from 25 to 40 units / kg weekly, from 22.5 to 32.5 units / kg weekly, from 20 to 30 units / kg weekly, from 17.5 to 22.5 units / kg weekly, from 15 to 25 units / kg weekly, from 22.5 to 32.5 units / kg weekly, from 25 to 35 units / kg weekly, from 22.5 to 37.5 units / kg weekly, from 30 to 40 units / kg weekly, from 27.5 to 32.5 units / kg weekly, from 22.5 to 27.5 units / kg weekly, from 17.5 to 22.5 units / kg weekly, or from 32.5 to 37.5 units / kg weekly. The dosage of GCase may be about 1.5 mg / kg or 1.5 mg / kg on alternate weeks. The dosage of GCase may be about 0.75 mg / kg or 0.75 mg / kg weekly.Alternatively, the dosage of GCase may be in the range of 0.75 to 2.0 mg / kg administered every other week, 1.0 to 1.75 mg / kg administered every other week, 1.25 to 2.0 mg / kg administered every other week, 1.125 to 1.625 mg / kg administered every other week, 1.0 to 1.5 mg / kg administered every other week, 0.875 to 1.375 mg / kg administered every other week, 0.75 to 1.25 mg / kg administered every other week, 1.215 to 1.625 mg / kg administered every other week, 1.25 to 1.75 mg / kg administered every other week, 1.375 to 1.875 mg / kg administered every other week, 1.5 to 2.0 mg / kg administered every other week, 1.375 to 1.625 mg / kg administered every other week, 1.125 to 1.375 mg / kg administered every other week, 0.875 to 1.125 mg / kg administered every other week, or 1.625 to 1.875 mg / kg administered every other week. Alternatively, the dosage of GCase may be in the range of 0.375 to 1.0 mg / kg administered weekly, 0.5 to 0.875 mg / kg administered weekly, 0.625 to 1.0 mg / kg administered weekly, 0.5625 to 0.8125 mg / kg administered weekly, 0.5 to 0.75 mg / kg administered weekly, 0.4375 to 0.5625 mg / kg administered weekly, 0.375 to 0.625 mg / kg administered weekly, 0.5625 to 0.8125 mg / kg administered weekly, 0.625 to 0.875 mg / kg administered weekly, 0.5625 to 0.9375 mg / kg administered weekly, 0.75 to 1.0 mg / kg administered weekly, 0.6875 to 0.8125 mg / kg administered weekly, 0.5625 to 0.6875 mg / kg administered weekly, 0.4375 to 0.5625 mg / kg administered weekly, or 0.8125 to 0.9375 mg / kg administered weekly.

[0128] Either the IFG and GCase / IFG formulations described herein can be administered to a patient. The dosage of GCase may be about 90-180 units / kg with bi-weekly administration. The GCase dosage may be about 90 units / kg or 90 units / kg with weekly administration. Alternatively, the GCase dosage may be in the range of 90-150 units / kg with bi-weekly administration, 110-160 units / kg with bi-weekly administration, 120-180 units / kg with bi-weekly administration, 120-150 units / kg with bi-weekly administration, 90-120 units / kg with bi-weekly administration, 100-130 units / kg with bi-weekly administration, 110-140 units / kg with bi-weekly administration, 120-150 units / kg with bi-weekly administration, 130-160 units / kg with bi-weekly administration, 140-170 units / kg with bi-weekly administration, or 150-180 units / kg with bi-weekly administration. Alternatively, the dosage of GCase may be in the range of 90-110 units / kg with bi-weekly administration, 100-120 units / kg with bi-weekly administration, 110-130 units / kg with bi-weekly administration, 120-140 units / kg with bi-weekly administration, 130-150 units / kg with bi-weekly administration, 140-160 units / kg with bi-weekly administration, 150-170 units / kg with bi-weekly administration, or 160-180 units / kg with bi-weekly administration. Also, the dosage of GCase may be about 2.25-4.5 mg / kg with bi-weekly administration. Alternatively, the dosage of GCase may be in the range of 2.25-3.75 mg / kg with bi-weekly administration, 2.75-4.0 mg / kg with bi-weekly administration, 3.0-4.5 mg / kg with bi-weekly administration, 3.0-3.75 mg / kg with bi-weekly administration, 2.25-3.0 mg / kg with bi-weekly administration, 2.5-3.25 mg / kg with bi-weekly administration, 2.75-3.5 mg / kg with bi-weekly administration, 3.0-3.75 mg / kg with bi-weekly administration, 3.25-4.0 mg / kg with bi-weekly administration, 3.5-4.25 mg / kg with bi-weekly administration, or 3.75-4.5 mg / kg with bi-weekly administration. Alternatively, the dosage of GCase may be in the range of 2.25-2.75 mg / kg with bi-weekly administration, 2.5-3.0 mg / kg with bi-weekly administration, 2.75-3.25 mg / kg with bi-weekly administration, 3.0-3.5 mg / kg with bi-weekly administration, 3.25-3.75 mg / kg with bi-weekly administration, 3.5-4.0 mg / kg with bi-weekly administration, 3.75-4.25 mg / kg with bi-weekly administration, 4.0-4.5 mg / kg with bi-weekly administration.

[0129] Administration of the IFG and GCase / IFG compositions can be carried out to treat disorders associated with dysfunction of the GCase pathway such as lysosomal storage diseases. Exemplary lysosomal storage diseases include Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidosis, and multiple system atrophy. The disorder may be a neurodegenerative disease such as Parkinson's disease, Alzheimer's disease, or Lewy body dementia. Alternatively, the disorder may be one associated with abnormal regulation of α-synuclein.

[0130] In the treatment of the disorder, the IFG and GCase / IFG compositions can be administered intravenously or subcutaneously. Subcutaneous administration includes subcutaneous injection. The compositions can be administered using various dosing schedules. For example, the compositions can be administered once a week, once every two weeks, or once a month. The compositions can be administered, for example, every three days, every four days, every five days, every six days, every eight days, every nine days, every ten days, every eleven days, every twelve days, every thirteen days, every fifteen days, or every sixteen days. The dosing frequency can be changed during the treatment period depending on various factors.

Examples

[0131] The present invention is also illustrated and demonstrated by the following examples. However, nowhere in the specification are these examples and other examples used to do more than illustrate and in no way limit the scope and meaning of the present invention or the terms exemplified. Similarly, the present invention is not limited to any of the embodiments described in the specification. Indeed, many modifications and variations of the present invention may become apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention should be limited only by the terms of the appended claims and the full scope of equivalents to which those claims are entitled.

[0132] The X-ray powder diffraction patterns in the present disclosure are obtained by a Stoe Stadi P X-ray powder diffractometer. The parameters of the X-ray powder diffraction method of the present disclosure are as follows.

[0133] Measurements with this apparatus were carried out by the transmission method at a tube voltage of 40 kV and a tube output of 40 mA. The curved Ge monochromator enables tests with Cu-Kα1 irradiation. The following parameters were set: a 2θ step size of 0.02°, a step time of 12 seconds (s), a 2θ scanning range of 1.5 - 50.5°, and a detector step of 1° in 2θ (detector mode in step scanning). As a typical sample preparation, about 10 mg of the sample was placed between two acetate foils and attached to a transmission sample holder from Stoe. The sample was rotated during measurement. All sample preparations and measurements were carried out in air.

[0134] Example A: Purification of isophagomine free base : Isophagomine free base was obtained by known methods, for example, according to the synthesis from D-(−)-arabinose reported by Danishefsky et al. in Tetrahedron Letters 1990; 31(16), 2229 or described in European Patent No. 1860101, or according to the synthesis from L-(−)-xylose (see Meloncelli, P.J. and Stick, R.V. Aust. J. Chem. 2006, vol. 59, pp 827 - 833). The obtained IFG free base was purified using a silica gel-packed column and eluted with a solvent gradient consisting of dichloromethane, methanol, and ammonium hydroxide, gradually increasing the polarity of the eluent until the IFG free base eluted. After analyzing the fractions by TLC, they were combined as necessary, volatile substances were removed in vacuo, and a purified IFG free base with impurities less than 2% as measured by HPLC was obtained.

[0135] Example 1: Isophagomine cinchonate Preparation of Solution 1: A solution of quinic acid was prepared at room temperature in about 4 volumes of methanol.

[0136] Preparation of Solution 2: A methanol solution of purified IFG (obtained according to Example A) was prepared at room temperature. An equimolar amount of IFG was used relative to the quinic acid used in the preparation of Solution 1.

[0137] Next, when Solution 2 was slowly added to Solution 1, a crystalline substance was formed. After stirring at room temperature, the slurry was filtered, and the obtained solid substance was washed with methanol, de-liquored, and then dried in vacuo. A crystalline sample SP245-QNC-P2 of the quinate salt of IFG was obtained.

[0138] Powder X-ray diffraction of the obtained isophagomine cinchonate The XRPD pattern of SP245-QNC-P2 obtained by the protocol described above in Example 1 is shown in Figure 1 and also presented in Table 1 below (vs = very strong, s = strong, m = medium, w = weak, vw = very weak intensity). The characteristic peaks are selected from very strong, strong, and medium diffraction peaks. [Table 1]

[0139] Raman spectroscopy of isophagomine cinchonate The Raman spectrum of SP245-QNC-P2 was recorded and is shown in Figure 2. The overview of the FT-Raman (FT-Raman) spectrum from 200 to 3500 cm -1 (Figure 2A), and the fingerprint region of the FT-Raman spectrum from 200 to 2000 cm -1 .

[0140] Of isophagomine cinchonate 1 H-NMR spectroscopy The 1 1H-NMR spectrum of SP245-QNC-P2 was recorded in DMSO-d6 and is shown in Figure 3. This spectrum is consistent with 1:1 salt formation.

[0141] TG-FTIR of isophagomine cinchonate TG-FTIR measurements were performed and showed a mass loss of less than 1% between 25 °C and 200 °C. The obtained crystalline form SP245-QNC-P2 is anhydrous / non-solvated. The TG-FTIR thermogram is shown in Figure 4 and shows a mass loss of 0.9%, which corresponds to water and trace amounts of methanol. Decomposition is observed above 200 °C.

[0142] Differential scanning calorimetry of isophagomine cinchonate Differential scanning calorimetry showed a melting peak temperature of 204.4 °C (see Figure 5). However, decomposition had already started before the end of melting, and it was not possible to determine the enthalpy of melting.

[0143] Dynamic water vapor adsorption measurement of isophagomine cinchonate The behavior of sample SP245-QNC-P2, which is an IFG quinolate salt, in the presence of variable water vapor pressure was investigated by DVS measurement. The results of the DVS test are shown in Figures 6A and 6B as the DVS isotherm of sample SP245-QNC-P2: the change in relative sample weight (red curve) and relative humidity (blue curve) as a function of time.

[0144] No significant weight loss was observed even when the relative humidity was decreased from 50% to 0%. Furthermore, no weight increase was observed even when the relative humidity (RH) was increased from 0% to 80%, and then a 20% weight increase was observed up to RH 95%, and a further 70% weight increase was observed when stored at 95%. The SP245-QNC-P2 salt prepared in Example 1 shows hygroscopicity at high relative humidity, i.e., RH above about 80%. When the sample recovered after DVS measurement was subjected to XRPD, no change in shape was observed, but an improvement in crystallinity was observed.

[0145] Identification of isophagomine cinchonate by elemental composition analysis The chemical identity of IFG quinolate SP245-QNC-P2 was verified by elemental composition analysis by CHNO content measurement and TG-FTIR of water and solvent content. The results obtained are summarized in Table 2, which provides the CHNO analysis results of SP245-QNC-P2 compared to the theoretical composition of a solvent- and water-free (1:1) salt with a molecular weight of 339.34 g / mol and the formula C13H25NO9. The results of the CHNO analysis are in very good agreement with the theoretical content of the quinolate salt without solvent and water.

Table 2

[0146] Example 2: Isophagomine fumarate Preparation of Solution 1: A solution of fumaric acid was prepared in approximately 4 volumes of methanol at room temperature.

[0147] Preparation of Solution 2: A solution of purified IFG (obtained according to Example A) in approximately 4 volumes of methanol was prepared at room temperature. An equimolar amount of IFG was used relative to the fumaric acid used in the preparation of Solution 1.

[0148] Next, Solution 2 was slowly added to Solution 1, and a crystalline substance was formed. After stirring at room temperature, the slurry was filtered, and the obtained solid substance was washed with a methanol solvent, drained, and then dried in vacuo. A crystalline sample SP245-FUM-P4 of the fumarate salt of IFG was obtained.

[0149] Powder X-ray diffraction of isophagomine fumarate The XRPD pattern of SP245-FUM-P4 obtained from the protocol described above in Example 2 is shown in Figure 7 and also presented in Table 3 below (vs = very strong, s = strong, m = medium, w = weak, vw = very weak intensity). The characteristic peaks are selected from very strong, strong, and medium diffraction peaks.

Table 3

[0150] Raman spectroscopy of isophagomine fumarate The Raman spectrum of SP245-FUM-P4 was recorded and is shown in Figure 8. The overview of the FT-Raman spectrum from 200 to 3500 cm -1 (Figure 8A), and the fingerprint region of the FT-Raman spectrum from 200 to 2000 cm -1 (Figure 8B).

[0151] Of isophagomine fumarate 1 H-NMR spectroscopy For SP245-FUM-P4 1 the 1H-NMR spectrum was recorded in D 2Recorded with O and shown in Fig. 9. This spectrum is consistent with 1:1 salt formation.

[0152] TG-FTIR of isophagomine fumarate The TG-FTIR thermogram of SP245-FUM-P4 is shown in Fig. 10. No mass loss was observed between 25 °C and 150 °C. This fumarate is in a form that does not contain solvents and water. Decomposition is observed above 150 °C.

[0153] Differential scanning calorimetry of isophagomine fumarate Differential scanning calorimetry of SP245-FUM-P4 showed a melting peak temperature of 168.6 °C, a melting onset temperature of 165.6 °C, and a melting enthalpy of 157 J / g (see Fig. 11).

[0154] Dynamic water vapor adsorption measurement of isophagomine fumarate Using DVS, the effect of changes in relative humidity on the IFG fumarate sample SP245-FUM-P4 was investigated. Fig. 12 shows the DVS isotherm of sample SP245-FUM-P4: changes in relative sample weight (red curve) and relative humidity (blue curve) as a function of time (Fig. 12A), and changes in relative sample weight as a function of relative humidity (Fig. 12B).

[0155] Even when the relative humidity was lowered from 50% to 0% RH, no significant weight loss was observed (<1 wt%), and no significant increase in sample mass was observed when the humidity was then increased from 0% to 80% RH. Scanning up to RH 95% showed an 11% weight increase, and storing at RH 95% for 5 hours showed a further 47% weight increase. During the second cycle, a 5% weight increase was observed at 95% RH compared to the first cycle. The IFG fumarate shows hygroscopicity at relative humidities above about 80%.

[0156] Analysis of the crystalline material recovered after DVS measurement by XRPD was consistent with the sample SP245-FUM-P4 used as the starting material for the DVS test.

[0157] Identification of isophagomine fumarate by elemental composition analysis The chemical identity of IFG fumarate SP245-FUM-P4 was verified by elemental composition analysis by CHNO content measurement and by TG-FTIR of water and solvent content. The obtained results were summarized in the CHNO content analysis of sample SP245-FUM-P4 - compared with the theoretical composition of the solvent- and water-free (1:1) salt with a molecular weight of 263.35 g / mol and the formula C10H17NO7. The results of the CHNO analysis are in very good agreement with the theoretical content of the fumarate salt without solvent and water.

Table 4

[0158] Example 3: Isophagomine oxalate Preparation of Solution 1: A solution of oxalic acid was prepared in approximately 2.5 volumes of methanol at room temperature.

[0159] Preparation of Solution 2: A solution of purified IFG (obtained according to Example A) in methanol was prepared in approximately 2.6 volumes of methanol at room temperature. An equimolar amount of IFG was used relative to the oxalic acid used in the preparation of Solution 1.

[0160] Next, Solution 2 was slowly added to Solution 1, and a crystalline substance was formed. After stirring at room temperature, the slurry was filtered, the obtained solid material was washed with methanol, drained, and then dried in vacuo. A crystalline sample SP245-OXA-P1 of IFG oxalate was obtained.

[0161] Powder X-ray diffraction of isophagomine oxalate The XRPD pattern of SP245-OXA-P1 obtained from the protocol described above in Example 1 is shown in Figure 13 and also presented in Table 5 below (vs = very strong, s = strong, m = medium, w = weak, vw = very weak intensity). The characteristic peaks are selected from very strong, strong, and medium diffraction peaks.

Table 5

[0162] Raman spectroscopy of isophagomine oxalate The Raman spectrum of SP245 - OXA - P1 was recorded and is shown in Figure 14. 200 - 3500 cm -1 The overview of the FT - Raman spectrum (Figure 14A), and the fingerprint region of the FT - Raman spectrum from 200 - 2000 cm -1 (Figure 14B).

[0163] Of isophagomine oxalate 1 H-NMR spectroscopy The 1 1H - NMR spectrum of SP245 - OXA - P1 was recorded in D 2 2O and is shown in Figure 15. This spectrum is consistent with a 1:1 salt formation.

[0164] TG-FTIR of isophagomine oxalate The TG - FTIR thermogram of SP245 - OXA - P1 is shown in Figure 16, and no mass loss worthy of consideration is observed between 25 °C and 150 °C. This oxalate is in a form that does not contain solvent and water.

[0165] Differential scanning calorimetry of isophagomine oxalate Differential scanning calorimetry of SP245 - OXA - P1 showed a melting peak temperature of 143.9 °C, a melting onset temperature of 140.6 °C, and a related melting enthalpy of 158.7 J / g (Figure 17).

[0166] Dynamic water vapor adsorption measurement of isophagomine oxalate Using DVS, the effect of changes in relative humidity on the IFG oxalate sample SP245 - OXA - P1 was investigated. Figure 18 shows the DVS isotherm: the change in relative sample weight (red curve) and relative humidity (blue curve) as a function of time (Figure 18A), and the change in relative sample weight as a function of relative humidity (Figure 18B).

[0167] Even when the relative humidity was decreased from 50% to 0%, no significant weight loss was observed. Furthermore, no weight increase was seen even when the relative humidity was increased from 0 to 75%. Subsequently, a 30% weight increase was observed up to 95%RH, and a further 90% weight increase was seen when stored at 95%. The oxalate shows hygroscopicity above 80%RH. When the sample recovered after DVS measurement was subjected to XRPD, the obtained XRPD pattern matched the starting material.

[0168] Identification of isophagomine oxalate by elemental composition analysis The chemical identity of IFG fumarate SP245 - OXA - P1 was verified by elemental composition analysis by CHNO content measurement and TG - FTIR of moisture and solvent content. The obtained results were summarized in the CHNO content analysis of sample SP245 - OXA - P1, which was compared with the theoretical composition of the solvent - and water - free (1:1) salt with a molecular weight of 237.21 g / mol and the formula C8H15NO7. The results of the CHNO analysis are in very good agreement with the theoretical content of the oxalate without solvent and water.

Table 6

[0169] Example 4: Crystalline isophagomine tartrate Both D - tartaric acid and L - tartaric acid were prepared according to the following protocol.

[0170] Preparation of Solution 1: A solution of tartaric acid was prepared in approximately 2 volumes of methanol at room temperature.

[0171] Preparation of Solution 2: A solution of purified IFG (obtained according to Example A) in approximately 2 volumes of methanol was prepared at room temperature. An equimolar amount of IFG was used relative to the tartaric acid used in the preparation of Solution 1.

[0172] Next, when Solution 2 was slowly added to Solution 1, a crystalline substance was formed. After stirring at room temperature, the slurry was filtered, the obtained solid material was washed with methanol, drained, and then dried in vacuo.

[0173] The crystalline IFG-D-(-)-tartrate sample SP245-DTA-P3 was obtained according to the above protocol using D-tartaric acid in Solution 1. The crystalline IFG L-(+)-tartrate sample SP245-LTA-P5 was obtained according to the above protocol using L-tartaric acid in Solution 1.

[0174] D-tartrate is produced on various scales from milligrams to 600+ grams, and typical yields are about 40 - 60%.

[0175] Powder X-ray diffraction of isophagomine D-(-)-tartrate The XRPD pattern of SP245-DTA-P3 obtained by the protocol described above in Example 4 is shown in Figure 19 and is also presented in Table 7 below (vs = very strong, s = strong, m = medium, w = weak, vw = very weak intensity). Characteristic peaks are selected from very strong, strong, and medium diffraction peaks.

Table 7

[0176] Raman spectroscopy of isophagomine D-(-)-tartrate The Raman spectrum of SP245-DTA-P3 between 200 and 3500 cm -1 was recorded and is shown in Figure 21.

[0177] Of isophagomine D-(-)-tartrate 1 H-NMR spectroscopy The 1 1H-NMR spectrum of SP245-DTA-P3 was recorded in D 2 2O and is shown in Figure 22. This spectrum is consistent with 1:1 salt formation.

[0178] TG-FTIR of isophagomine D-(-)-tartrate The TG-FTIR thermogram of SP245-DTA-P3 is shown in Figure 23, and no mass loss worthy of consideration is observed between 25°C and 150°C. This tartrate is in a form that does not contain solvent and water.

[0179] Differential scanning calorimetry of isophagomine D-(-)-tartrate Differential scanning calorimetry of SP245-DTA-P3 shows a melting peak temperature of 146.6 °C, a melting onset temperature of about 132 °C, and an associated melting enthalpy of 158.7 J / g (Figure 24).

[0180] Dynamic water vapor adsorption measurement of isophagomine D-(-)-tartrate Using DVS, the effect of changes in relative humidity on the crystalline IFG tartrate salt sample SP245-DTA-P3 was investigated. Figure 25 shows the DVS isotherm: the change in the relative weight (red curve) and relative humidity (blue curve) of the sample as a function of time. No mass change was observed when the relative humidity (RH) was increased from 0% to 70%, indicating that this salt is non-hygroscopic up to RH 70%. However, when the RH was changed from 70% to 95%, a 32% mass increase was observed, and a further 70% mass increase was observed when held at 95% RH.

[0181] Identification of isophagomine D-tartrate by elemental composition analysis The chemical identity of IFG D-tartrate SP245-DTA-P3 was verified by elemental composition analysis by CHNO content measurement and TG-FTIR of the moisture and solvent content. The results obtained were summarized in the CHNO content analysis of sample SP245-DTA-P3, compared with the theoretical composition of the solvent- and water-free (1:1) salt with a molecular weight of 237.21 g / mol and the formula C8H15NO7. The results of the CHNO analysis are in very good agreement with the theoretical content of the monotartrate salt without solvent and water.

Table 8

[0182] Example 5: Stability test of isophagomine salts To obtain insights into the thermal stability of four types of crystalline IFG salts, a storage stress test was conducted at 80 °C for several days. The free base sample (SP245-FB-P4), fumarate sample (SP245-FUM-P5), D-tartrate sample (SP245-DTA-P7), L-tartrate sample (SP245-LTA-P5), and oxalate sample (SP245-OXA-P2) were placed in sealed vials and stored at 80 °C for 1 day, 3 days, and 7 days. The samples were examined by CAD and 1 1H-NMR.

[0183] To identify possible adducts and thermal decomposition products that may occur when stored under certain conditions, CAD-HPLC was performed. All samples were examined by CAD-HPLC, but no significant changes were observed between the starting material and the stored samples. The results are shown in Table 9, suggesting that the IFG salts are stable under these conditions. Furthermore, XRPD was performed on the crystalline salt samples stored for 7 days, and the XRPD patterns before and after storage were essentially the same.

[0184] All salts were significantly more stable than the free base after storage at 80 °C for 7 days. NMR spectroscopy was performed on the samples stored for 7 days. In the free base sample, some additional NMR signals were observed after storage compared to the sample that was not stressed, confirming the decrease in purity observed by HPLC. No significant changes were observed in the NMR samples of the salts.

Table 9

[0185] The IFG used in Formulations A, B, and C above is selected from any of the following: IFG free base, IFG kinatate, IFG malate, IFG fumarate, IFG oxalate, IFG malonate, IFG succinate, IFG D-tartrate, IFG cyclamate, or IFG ascorbate.

[0186] Example 7: Stability test of tartrate and fumarate IFG / GCase formulations This report summarizes data collected for the stability testing of velaglucerase alfa in formulations containing isofagomine tartrate or isofagomine fumarate. The objective of this study was to evaluate whether isofagomine fumarate has an equivalent stabilizing effect to isofagomine tartrate and whether isofagomine fumarate can be an alternative salt to isofagomine tartrate.

[0187] In this study, the stability of a ~140 mg / mL velaglucerase alfa formulation containing two different IFG salt forms, isofagomine tartrate and isofagomine fumarate, to storage and mechanical stress was evaluated. The results are as follows: 1) In terms of appearance, pH, protein concentration, osmotic pressure, P188 concentration, subvisible particles, RP-HPLC, SDS-PAGE, activity, intact mass in reduced and non-reduced states, and peptide mapping, there were no significant differences between the two formulations after preparation up to 1 month at T0, 40 °C, and -20 °C, and after shaking for 24 hours. Slight differences in the A320 values were observed at T0, after 1 month at -20 °C, and after shaking at 200 rpm for 24 hours. 2) The isofagomine content in the fumarate formulation was slightly higher than that in the tartrate formulation both at T0 and after storage at 40 °C for 1 month. The viscosity of the fumarate formulation was slightly higher than that of the tartrate formulation. 3) Aggregation data by two different SE-HPLC methods indicated that the fumarate formulation was less stable than the tartrate formulation during storage at 40 °C. No significant differences were observed after storage at -20 °C or after shaking for 24 hours. 4) The DSC results showed that the Tm value of velaglucerase alfa was slightly higher in the tartrate formulation than in the fumarate formulation.

[0188] Overall, these results indicate that the two formulations prepared at time point T0 were equivalent in all assays, except that the isophagomine content was slightly higher in the tartrate formulation than in the fumarate formulation. Both formulations were stable during shaking, storage at -20°C, and storage at -40°C. However, the fumarate formulation had relatively lower stability during storage at -40°C as measured by SEC, and the viscosity of the fumarate formulation was slightly higher than that of the tartrate formulation.

[0189] This dataset suggests that isophagomine fumarate could be an alternative to isophagomine tartrate with some compromise in stability and viscosity.

[0190] 1. Sample Preparation Veliglucerase alfa was thawed at room temperature. The thawed veliglucerase alfa was concentrated at room temperature using a Millipore Labscale® TFF system with a Pellicon XL cassette (catalog number: PXB010A50, Biomax® 10KDa, Chemistry polyethersulfone). The veliglucerase alfa was concentrated to approximately 160 mg / mL. Next, the concentrated veliglucerase alfa (47.5 mL) was loaded into a Slide-A-Lyzer® dialysis cassette (10K MWCO) and dialyzed twice at 2 - 8°C against 2.0 L of formulation buffer containing either 7.5 mM isophagomine fumarate or isophagomine tartrate.

[0191] Table 10 shows the composition of the two formulation buffers. The pH of the isophagomine tartrate formulation and the isophagomine fumarate formulation was adjusted to 6.0 from 5.61 and 4.48, respectively, using 1.0 N NaOH.

Table 10

[0192] Each solution of the concentrated protein preparation was filtered (using Millipore Steriflip-GV®), aseptically filled into type I glass vials of the 6R type, stoppered with a 20 mm gray rubber serum stopper, and crimped with an aluminum seal.

[0193] 1.1 Stress Conditions 1.1.1 Storage at 40 °C The set of filled vials was stored in an inverted position at 40 °C for 17 days (0.5 months) and 32 days (1 month).

[0194] 1.1.2 Storage at 20 °C The filled vials were stored upright at -20 °C for 32 days (1 month).

[0195] 1.1.3 Stirring Stirring was carried out horizontally at room temperature for 24 hours at a speed of 200 rpm using a rotary shaker.

[0196] 1.2 Test Methods 1.2.1 Normal Assay Method Table 11 shows a list of test methods.

Table 11

[0197] 2. Data Analysis The stability trends of the velaglucerase alfa preparation containing IFG-fumarate (hereinafter referred to as the fumarate preparation) were compared with those of the velaglucerase alfa preparation containing IFG-tartrate (hereinafter referred to as the tartrate preparation), and the stability at 40 °C and -20 °C, as well as the stability under shaking stress, were evaluated.

[0198] 2.1 Appearance The appearance was observed in an intact vial under normal light. Both the tartrate and fumarate formulations were clear to slightly opalescent, colorless, and essentially free of visible particles after storage at 40 °C for 0.5 and 1 month, storage at -20 °C for 1 month, or shaking at 200 rpm for 24 hours.

[0199] 2.2 pH The pH results are shown in Table 12. There was no change in pH for either formulation at 40 °C.

Table 12

[0200] 2.3 Protein Concentration The protein concentration was measured using SoloVPE by varying the pathlength and accurately measuring the absorbance of the undiluted drug substance at 280 nm as a function of pathlength. The absorbance at 280 nm was corrected with the absorbance at 320 nm, which serves as an indicator of light scattering. The slope of the data (absorbance units / mm) was directly proportional to the protein concentration according to Beer's law. The protein concentration of velaglucerase alfa was calculated using the slope of the linear plot of absorbance vs. pathlength and the extinction coefficient of velaglucerase alfa of 1.63 (mg / mL) -1 (cm) -1 and was expressed in mg / mL. The concentration results are shown in Table 13. There was no significant change after storage at 40 °C for 1 month.

Table 13

[0201] 2.4 P188 Concentration Table 14 shows the results of the P188 concentration. There was no significant difference in the P188 concentration between the two formulations.

Table 14

[0202] 2.5 IFG Concentration The vela-glucerase alpha bulk drug was formulated in a buffer containing 3 mM isofagomine (IFG). The IFG concentration in the vela-glucerase alpha bulk drug was analyzed by reversed-phase ultra-high performance liquid chromatography (UPLC). In this method, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reacted quantitatively with IFG, converted IFG into a UV-detectable form, and then separated it by UPLC. The detection wavelength was 260 nm. A standard curve was created by measuring standard samples of IFG. Linear regression was performed on the data of the IFG standard samples, and the obtained parameters were used to calculate the IFG concentration in the samples. The results are reported in mM.

[0203] Table 15 shows the results of the IFG concentration. There was no change during storage at 40 °C for 1 month. However, the fumarate formulation contained a slightly higher IFG concentration than the tartrate formulation.

Table 15

[0204] 2.6 Osmolality Table 16 shows the results of the osmolality. There was no significant difference in the osmolality, and the results were higher than the optimal osmolality but consistent with the results of other studies using the same method.

Table 16

[0205] 2.7 Sub-visible Particle Table 17 shows the results of the subvisible particles. There was no significant difference between these two formulations. However, during storage at 40 °C and -20 °C, and after shaking at 200 rpm for 24 hours, the number of subvisible particles increased in both formulations.

Table 17

[0206] 2.8 Viscosity Viscosity was measured using a 500 μL syringe with an m-VROC viscometer. Each sample was tested at 20 °C at flow rates of 50, 100, and 200 μL / min, and the average results were reported. The samples for which viscosity was measured had been stored at 2 - 8 °C for 3 months before measurement, and no obvious change in appearance was observed.

[0207] Table 18 shows the viscosities of these two formulations. The viscosity of velaglucerase alfa in the tartrate formulation was slightly lower than that in the fumarate formulation.

Table 18

[0208] 2.9 A320 Data for A320 was collected using a plate reader. Samples (each well) were set in a UV-Star® Microplate (96 wells, μClear®, clear), and data was collected at 320 nm.

[0209] Table 19 shows the A320 values of velaglucerase alfa formulations under different treatment conditions. The A320 value of the fumarate formulation was slightly higher than that of the tartrate formulation after T0, 1 month at -20 °C, and after shaking for 24 hours. After storage at 40 °C for 0.5 months and 1 month, no significant difference was observed between these two formulations.

Table 19

[0210] 2.10 Activity The in vitro activity was determined by measuring the rate at which the substrate p-nitrophenyl β-D-glucopyranoside was hydrolyzed by veraglucerase alfa into p-nitrophenol and β-D-glucopyranoside. The reaction was stopped by the addition of glycine carbonate buffer. The absorbance of the characteristic yellow p-nitrophenol product was measured at 405 nm. The enzyme activity of veraglucerase alfa was determined by interpolation on the standard curve prepared in the assay. One unit (U) of enzyme activity was defined as the amount of veraglucerase alfa required to convert 1 micromole (μmol) of p-nitrophenyl β-D-glucopyranoside per minute under the specified assay conditions. The reportable activity value for the veraglucerase alfa sample was U / mL.

[0211] The results of the activity are shown in Table 20. There were no significant changes after storage at 40 °C and -20 °C for up to 1 month and after shaking at 200 rpm for 24 hours.

Table 20

[0212] 2.11 SE-HPLC Size-exclusion HPLC (SE HPLC) was used to evaluate the size homogeneity of velaglucerase alfa. This method separates proteins by hydrodynamic size. Larger species (e.g., aggregates, dimers, oligomers) elute earlier than the monomer main species of velaglucerase alfa as a pre-peak, and smaller species (e.g., degradation products and fragments) elute later as a post-peak. The eluted peaks are detected at an absorbance of 214 nm. In this method, the percentage of the main peak (%) and the percentage of high molecular weight (HMW) species (%) are reported. All samples were tested by two SE-HPLC methods, RE-PMP-0010 and RE-PMP-0013. RE-PMP-0010 is the method for commercial VPRIV (registered trademark), and the velaglucerase alfa monomer molecule elutes after the buffer peak, presumably due to the interaction between the velaglucerase alfa molecule and the stationary phase. RE-PMP-0013 is the method developed for the VPRIV-SubQ formulation, and in this method, 10% acetonitrile is used as a component of the elution buffer. In this study, both methods were used to characterize velaglucerase alfa in these two formulations.

[0213] 2.11.1 VPRIV Method (RE-PMP-0010) The SE-HPLC chromatograms of velaglucerase alfa in both formulations were evaluated at T0, at 40 °C for 0.5 and 1 month, at -20 °C for 1 month, and with 24-hour horizontal shaking, respectively.

[0214] Table 21 shows the SE-HPLC retention times of velaglucerase alfa after treatment under different conditions. 40C17D corresponds to the storage condition at 40 °C for 17 days (or 0.5 months), 40C1M corresponds to the storage condition at 40 °C for 1 month, and N20C1M corresponds to the storage condition at -20 °C for 1 month. The monomer peak eluted at 34 minutes, and the aggregates eluted between 12.8 and 23.9 minutes. The buffer peak eluted between 25 and 27 minutes. However, the distribution of aggregate species after storage at 40 °C for 0.5 and 1 months was different from that of velaglucerase alfa at T0, or after storage at -20 °C for 1 month or after shaking for 24 hours. However, there was no obvious difference in the peak positions of these species under all treatment conditions between the tartrate formulation and the fumarate formulation.

Table 21

[0215] Table 22 shows the SE-HPLC relative peak areas of various species of velaglucerase alfa after treatment under different conditions. There was no obvious difference between these two formulations at T0, after storage at 40 °C for 0.5 months, after storage at -20 °C for 1 month, or after shaking for 24 hours. When stored at 40 °C for 1 month, the fumarate formulation showed a higher level of aggregation than the tartrate formulation.

Table 22

[0216] 2.11.2 SE-HPLC Method (RE-PMP-0013) Using the method adapted for the VPRIV SubQ formulation, the SE-HPLC chromatograms of velaglucerase alfa were evaluated in both formulations at T0, at 40 °C for 0.5 and 1 months, at -20 °C for 1 month, and with 24-hour horizontal shaking.

[0217] Table 23 shows the retention times of the SE-HPLC peaks of velaglucerase alfa. For both formulations, after storage at T0, -20°C for 0.5 months and after shaking for 24 hours, there was an aggregate species at approximately 16.4 minutes and a monomer peak at 19.6 minutes. Also, after storage at 40°C for 0.5 months and 1.0 months, an additional aggregate peak (unknown 1) appeared. However, there were no obvious differences in retention time and species distribution between these two formulations under the same treatment conditions.

Table 23

[0218] Table 24 shows the relative peak areas of the SE-HPLC peaks of both formulations. After storage at T0, -20°C and after shaking for 24 hours, there were no significant differences between these two formulations. The total amount of HMWS (unknown 1 + aggregates) in the fumarate formulation was higher than that in the tartrate formulation.

Table 24

[0219] 2.12 RP-HPLC Reverse-phase high-performance liquid chromatography (HPLC) is performed to measure the relative amounts of potential process-related and product-related impurities in velaglucerase alfa. This method separates the degradation products of velaglucerase alfa by a reverse-phase column with a gradient that increases the organic content. This method detects other peaks in addition to the main peak of velaglucerase alfa, both before and after the main peak at 214 nm. Purity is reported as the relative percent area of the main peak.

[0220] The RP-HPLC chromatograms of velaglucerase alfa were evaluated for storage at T0, 40°C for 0.5 months and 1 month, -20°C for 1 month, and horizontal shaking for 24 hours, respectively. After storage at 40°C for 0.5 months and 1.0 months, there were slight differences in the chromatograms between these two formulations.

[0221] Table 25 shows the retention times of the RP-HPLC peaks of velaglucerase alfa. After storage at -20 °C for 1 month and after shaking for 24 hours at T0, there was no significant difference in the retention time of velaglucerase alfa between these two formulations. The relative peak elution times of velaglucerase alfa stored at 40 °C for 0.5 months and 1.0 months were slightly different between these two formulations.

Table 25

[0222] Table 26 shows the relative RP-HPLC peak areas of both formulations. After storage at -20 °C and after shaking for 24 hours at T0, there was no significant difference between these two formulations. There were slight differences in the relative peak areas of peak C and the main peak between these two formulations stored at 40 °C for 0.5 months and 1.0 months.

Table 26

[0223] 2.13 SDS-PAGE The purity of velaglucerase alfa was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) under denaturing and reducing conditions. The test samples of velaglucerase alfa, reference standards, and two assay controls were diluted with sample dilution buffer, mixed with a reducing agent (dithiothreitol), denatured by heat, and loaded onto a Tris-glycine gradient gel. Molecular weight markers were also tested on the gel. After electrophoresis, the gel was stained with Coomassie blue and developed. The test samples were evaluated for the presence of fragments or non-reducing aggregates. The purity was evaluated by comparison with the reference standards and two assay controls at different impurity levels (1.0% and 3.0%).

[0224] The results of SDS-PAGE of veraglucerase alfa in the tartrate and fumarate formulations were evaluated respectively. In both formulations, no significant degradation of veraglucerase alfa was observed after storage at -20 °C for 1 month and after shaking for 24 hours. However, obvious degradation of veraglucerase alfa into smaller MW species was seen in both formulations stored at 40 °C for 0.5 months and 1.0 months. However, there was no obvious difference between these two formulations under all treatment conditions.

[0225] 2.14 DSC DSC data were collected using Micro-Cal VP DSC software at a ramping rate of 1 °C / min from 10 °C to 95 °C. DSC thermograms were collected after diluting the formulation to approximately 4.67 mg / mL. Also, data for each buffer were collected. The thermogram of each buffer was corrected against the water-water thermogram. The thermograms of each active formulation were corrected against their respective buffers and evaluated.

[0226] Both buffers had a phase transition at approximately 52 °C. In the fumaric acid buffer, the heat transfer event of the phase transition was more prominent. Veraglucerase alfa in both buffers showed prominent peaks, and the Tm of the peaks is summarized in Table 27. The Tm values of veraglucerase alfa in these two formulations were equivalent.

Table 27

[0227] 2.15 Mass Spectrometry 2.15.1 Intact Mass Intact masses were collected for both non-reduced and reduced samples.

[0228] The non-reduced samples were prepared by mixing 1 μl of each sample with 400 μl of 0.1% TFA, and 10 μl of the resulting mixture was injected into the LC-MS system. The reduced samples were prepared by the following procedure: each sample was denatured with 6 M guanidine, reduced with DTT, and alkylated with IAA. After desalting, 10 μl of each sample (in 100 mM Tris, pH 8.5) at approximately 0.5 mg / ml was injected into the 6600.

[0229] HPLC conditions: Column: ACE 5 Phenyl-300, 1.0 × 50 mm, 5 μm, P / N ACE-225-0501M; Mobile phase A: 0.1% trifluoroacetic acid in water; and Mobile phase B: 0.085% trifluoroacetic acid in acetonitrile; The gradient elution conditions are shown in Table 28.

Table 28

[0230] The non-reduced and reduced mass chromatograms of the velaglucerase alfa formulation were evaluated. No significant differences in retention time and mass were observed between these two formulations under each condition.

[0231] 2.16 Peptide Map Samples of velaglucerase alfa were denatured, reduced, and alkylated. Additional DTT was added to quench the excess alkylation, and the reaction mixture was desalted using a NAP-5 desalting column. Endoproteinase Lys-C was added to the desalted sample and reacted at 37 °C for 5 hours, then N-Glycanse-PLUS was added to the digested sample and reacted at 37 °C for 1 hour. The resulting deglycosylated peptides were separated on an ACE C18 column (150 × 2.1 mm, 3 μ, 300 Å) maintained at 40 °C.

[0232] The peptides were separated using a curve gradient ranging from 2% to 43% acetonitrile in water containing trifluoroacetic acid and a flow rate of 0.25 mL / min.

[0233] The results of peptide mapping of velaglucerase alfa in tartrate and fumarate formulations were evaluated after shaking at 200 rpm for 24 hours at T0, after storage at 40°C for 1 month, and after storage at -20°C for 1 month. The sequences and abundances of the identified peptides were evaluated. There were no significant differences in the results of retention time and mass of each peptide between the formulations.

[0234] 3. Conclusion Velaglucerase alfa formulations containing 140 mg / mL of velaglucerase alfa and isofagomine tartrate or isofagomine fumarate were similarly prepared and evaluated under storage conditions up to 1 month at 40°C and -20°C, and after shaking at 200 rpm for 24 hours.

[0235] At T0, there were no significant differences between the two formulations with respect to appearance, pH, protein concentration, osmotic pressure, P188 concentration, subvisible particles, RP-HPLC, SE-HPLC, SDS-PAGE, activity, intact mass in the reduced and non-reduced states, and peptide mapping. The isofagomine content of the fumarate formulation was higher than that of the tartrate formulation, and the A320 value was slightly higher than that of the tartrate formulation. The Tm value of the tartrate formulation was slightly higher than that of the fumarate formulation. Also, the viscosity results of the fumarate formulation were slightly higher than those of the tartrate formulation.

[0236] During storage at 40°C for up to 1 month, the results of the quality characteristics of these two formulations were equivalent as determined by appearance, pH, protein concentration, subvisible particles, RP-HPLC, SDS-PAGE, activity, A320, intact mass in the reduced and non-reduced states, and peptide mapping. However, the results of SE-HPLC indicated that the fumarate formulation was slightly less stable than the tartrate formulation in terms of the level of aggregation. Furthermore, the isofagomine content of the fumarate formulation was slightly higher than that of the tartrate formulation, consistent with the results at T0.

[0237] After storing these two formulations at -20°C for one month and shaking them at 200 rpm for 24 hours, the results of the quality characteristics of these two formulations were equivalent in terms of appearance, subvisible particles, RP-HPLC, SE-HPLC, SDS-PAGE, activity, intact mass in the reduced and non-reduced states, and peptide mapping. The A320 value of the fumarate formulation was slightly higher than that of the tartrate formulation.

[0238] In summary, isophagomine fumarate has a stabilization effect comparable to that of isophagomine tartrate during storage and mechanical stress. However, isophagomine fumarate was expected to have a slightly higher level of aggregation and a slightly higher viscosity during storage.

[0239] Example 8: Stability Test of Vela Glucerase Alpha Formulation B-IFG Tartrate under Various Storage Conditions To obtain findings regarding the long-term storage stability of the pharmaceutical velaglucerase alfa-IFG tartrate formulation B (see Example 6 above) formulated with isophagomine tartrate, a long-term storage stress test was conducted. This formulation has a high protein concentration suitable for subcutaneous administration.

[0240] The isophagomine compound used for stabilizing velaglucerase alfa in solution is isophagomine tartrate. The formulation buffer contains 3 mM of isophagomine (free isophagomine), and the protein is bound to isophagomine in a 1:1 molar ratio. At a protein concentration of 60 mg / mL, the molar concentration of the protein is approximately 1 mM (the molecular weight of glycosylated velaglucerase alfa is 63 kDa). Approximately 1 mM of isophagomine binds to the protein during the UFDF process in which the virus filtration pool is concentrated and the buffer is exchanged. The total content of isophagomine in the drug substance is the sum of free and bound isophagomine and is monitored for velaglucerase alfa-IFG tartrate formulation B.

[0241] Under long-term storage conditions (≤ -65°C), the results of purity tests up to 18 months showed an increase in high molecular weight species by SE-HPLC, but no changes were shown by RP-HPLC and SDS-PAGE (Coomassie). The results of protein content, pH, and potency did not show any trends up to 18 months. These procedures are as described in Example 7.

[0242] All results under long-term storage conditions met the specifications. Visible particles were observed, but these were due to the non-controlled environment in which the material was handled and were not related to the formulation or stability of the protein. The appearance criterion for the velaglucerase alfa formulation B-IFG tartrate is "essentially particle-free". Visible particles are not expected under GMP conditions. The tests for color and clarity are in compliance with the specifications at all time points and storage conditions.

[0243] Based on real-time stability data, the velaglucerase alfa drug substance is stable for 18 months when stored at ≤ -65°C. See Table 29 below. The available data and the trend charts generated therefrom support the prediction that the velaglucerase alfa formulation will remain within the specification acceptance criteria over 30 months when stored under long-term storage conditions of ≤ -65°C.

[0244] Data for 12 months and 3 months were generated under accelerated conditions (-20 ± 5°C) and stress conditions (5 ± 3°C) respectively. See Tables 30 and 31 below respectively. Under accelerated storage conditions (-20 ± 5°C), a trend equivalent to that of long-term storage conditions was observed. Under stress storage conditions (5 ± 3°C), the velaglucerase alfa drug substance is stable up to 3 months.

[0245] 3.1 Appearance The appearance results of the pharmaceutical product at a long-term storage temperature of ≤ -65°C met the acceptance criteria. No obvious changes were observed in all test samples.

[0246] 3.2 pH The pH results of the velaglucerase alfa formulation at a long-term storage temperature of ≤ -65°C met the acceptance criteria. No significant changes or trends were observed.

[0247] 3.3 Protein Concentration (A280) The protein concentration was determined by measuring the absorbance at 280 nm (A280). The protein concentration results of the velaglucerase alfa formulation at a long-term storage temperature of ≤ -65°C were within the acceptance criteria for all test samples. Also, no obvious changes or trends were observed.

[0248] 3.4 SE-HPLC The available SE-HPLC results of the 60 mg / mL velaglucerase alfa formulation at a long-term storage temperature of ≤ -65°C were within the acceptance criteria for all test samples. Also, no obvious changes or trends were observed.

[0249] 3.5 Specific Activity The available specific activity results of the velaglucerase alfa formulation at a long-term storage temperature of ≤ -65°C were within the acceptance criteria for all test samples. No obvious changes or trends were observed.

[0250] 3.6 SDS-PAGE The SDS-PAGE results of the velaglucerase alfa formulation at a long-term storage temperature of ≤ -65°C were within the acceptance criteria.

[0251] 3.7 Sub-visible Particles The available results of the velaglucerase alfa formulation at a long-term storage temperature of ≤ -65°C were within the acceptance criteria for all test samples.

[0252] 3.8 Cell Uptake Bioassay (CUB) The CUB results of the velaglucerase alfa formulation at a long-term storage temperature of ≤ -65°C were within the acceptance criteria for all test samples. A slight increase in the relative uptake amount was observed.

[0253] 3.9 Isophagomine (IFG) Content The results of the total isophagomine concentration available for the velaglucerase alfa preparation at a long-term storage temperature of ≤ -65°C are within the acceptance criteria for all samples tested. A slight decrease in the IFG content was observed. If the concentration of isophagomine falls below the amount required to maintain the stability of glucocerebrosidase, visible and / or subvisible particulates, as well as an increase in SEC-HPLC HMWS, are expected. No statistically significant trends in these characteristics were observed.

[0254] 3.10 Conclusion The stability results support that the shelf life of velaglucerase alfa was extended from 12 months to 30 months under long-term storage conditions of ≤ -65°C.

[0255] [Table 29]

[0256] [Table 30]

[0257] [Table 31]

[0258] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. Further, it is to be understood that all values are approximate and provided for illustrative purposes only.

[0259] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Also, the materials, methods, and examples are illustrative only and not intended to be limiting. Finally, the preferred embodiments of the present invention are described item by item.

[0260] [Embodiment 1] A salt of isophagomine, wherein the salt is selected from cinchoninate, maleate, fumarate, oxalate, malonate, succinate, cyclamate, and ascorbate, a salt of isophagomine.

[0261] [Embodiment 2] The salt according to Embodiment 1, wherein the salt is isophagomine cinchoninate.

[0262] [Embodiment 3] The salt according to Embodiment 1, wherein the salt is isophagomine fumarate.

[0263] [Embodiment 4] The salt according to Embodiment 1, wherein the salt is isophagomine oxalate.

[0264] [Embodiment 5] The salt according to Embodiment 1, wherein the salt is isophagomine succinate.

[0265] [Embodiment 6] The salt according to Embodiment 1, wherein the salt is isophagomine cyclamate.

[0266] [Embodiment 7] The salt according to Embodiment 2, wherein the isophagomine cinchoninate is a crystalline form characterized by an X-ray diffraction pattern having three or more characteristic peaks at 2θ values selected from 9.5° ± 0.2°, 15.0° ± 0.2°, 17.4° ± 0.2°, 18.1° ± 0.2°, 20.3° ± 0.2°, 23.8° ± 0.2°, 24.8° ± 0.2°, and 25.4° ± 0.2°.

[0267] [Embodiment 8] The salt according to Embodiment 3, wherein the isophagomine fumarate is a crystal form characterized by an X-ray diffraction pattern having three or more characteristic peaks at 2θ values selected from 16.1° ± 0.2°, 18.3° ± 0.2°, 18.6° ± 0.2°, 21.9° ± 0.2°, 23.6° ± 0.2°, 23.8° ± 0.2°, and 25.5° ± 0.2°.

[0268] [Embodiment 9] The salt according to Embodiment 4, wherein the isophagomine oxalate is a crystal form characterized by an X-ray diffraction pattern having three or more characteristic peaks at 2θ values selected from 27.8° ± 0.2°, 32.2° ± 0.2°, 35.3° ± 0.2°, 36.6° ± 0.2°, 37.4° ± 0.2°, 38.4° ± 0.2°, 18.5° ± 0.2°, 19.2° ± 0.2°, 21.4° ± 0.2°, 22.6° ± 0.2°, 24.5° ± 0.2°, 24.8° ± 0.2°, 26.8° ± 0.2°, 20.2° ± 0.2°, and 23.7° ± 0.2°.

[0269] [Embodiment 10] The salt according to Embodiment 5, wherein the isophagomine succinate is a crystal form characterized by having XRPD peaks.

[0270] [Embodiment 11] The salt according to Embodiment 6, wherein the isophagomine cyclamate is a crystal form characterized by having XRPD peaks.

[0271] [Embodiment 12] A salt of isophagomine, which is a crystalline form of isophagomine D-tartrate characterized by an X-ray diffraction pattern having three or more characteristic peaks at 2θ values selected from 9.8° ± 0.2°, 10.5° ± 0.2°, 15° ± 0.2°, 15.3° ± 0.2°, 15.8° ± 0.2°, 17.4° ± 0.2°, 17.9° ± 0.2°, 18.5° ± 0.2°, 18.9° ± 0.2°, 19.6° ± 0.2°, 21.1° ± 0.2°, 21.7° ± 0.2°, 22° ± 0.2°, 24.2° ± 0.2°, 24.8° ± 0.2°, 26.6° ± 0.2°, 27.1° ± 0.2°, 27.4° ± 0.2°, 33.8° ± 0.2°, 35.7° ± 0.2°, 36.5° ± 0.2°, and 37.5° ± 0.2°.

[0272] [Embodiment 13] A pharmaceutical composition comprising at least one isophagomine salt according to any one of Embodiments 1 to 12 and a pharmaceutically acceptable carrier.

[0273] [Embodiment 14] The pharmaceutical composition according to Embodiment 13, further comprising glucocerebrosidase and wherein isophagomine is present in an amount sufficient to stabilize glucocerebrosidase.

[0274] [Embodiment 15] The pharmaceutical composition according to Embodiment 13 or 14, wherein the glucocerebrosidase is velaglucerase alfa.

[0275] [Embodiment 16] The pharmaceutical composition according to any one of Embodiments 13 to 15, wherein the molar ratio of glucocerebrosidase to isophagomine is from about 1:2.5 to about 1:3.5.

[0276] [Embodiment 17] The pharmaceutical composition according to Embodiment 16, comprising 60 to 180 mg / mL of glucocerebrosidase and wherein isophagomine is present in at least about 3-fold molar excess relative to glucocerebrosidase.

[0277] [Embodiment 18] A pharmaceutical composition comprising glucocerebrosidase and isofagomine in a molar ratio of about 1:3, further comprising sodium citrate buffer, sucrose, and one or more surfactants selected from PS20, PS80, and poloxamer 188.

[0278] [Embodiment 19] A method for preparing a pharmaceutical composition according to any one of Embodiments 13 to 18, comprising combining isofagomine with a pharmaceutically acceptable carrier.

[0279] [Embodiment 20] A method for preparing a pharmaceutical composition according to any one of Embodiments 14 to 18, comprising dissolving isofagomine in water, adjusting the pH to about 6.0, and further adding glucocerebrosidase to obtain the composition.

[0280] [Embodiment 21] A method for treating a disorder associated with dysfunction of the GCase pathway, comprising administering to a patient in need thereof a composition according to any one of Embodiments 13 to 18.

[0281] [Embodiment 22] The method according to Embodiment 21, which is effective for treating the disorder.

[0282] [Embodiment 23] The method according to Embodiment 21, wherein the composition is administered intravenously or subcutaneously.

[0283] [Embodiment 24] The method according to Embodiment 23, wherein the composition is administered subcutaneously.

[0284] [Embodiment 25] A method for treating a disorder associated with dysfunction of the GCase pathway, comprising administering to a patient in need thereof a composition comprising 0.5 to 5.0 mg / kg of glucocerebrosidase and at least about 3-fold molar excess of isofagomine relative to glucocerebrosidase, wherein the composition is administered subcutaneously.

[0285] [Embodiment 26] A method for treating Gaucher's disease, comprising administering to a patient in need of such treatment: i) a therapeutically effective amount of an isophagomine salt or a formulation thereof according to any one of Embodiments 1 to 12; or ii) a therapeutically effective amount of a pharmaceutical composition according to any one of Embodiments 13 to 18.

[0286] [Embodiment 27] A method for producing an isophagomine salt, comprising: i) dissolving an organic acid in a polar protic solvent to prepare Solution 1; ii) dissolving isophagomine free base in a polar protic solvent to prepare Solution 2; iii) combining Solution 1 and Solution 2 to form a precipitate; iv) isolating the precipitate corresponding to the organic acid salt of isophagomine; wherein the organic acid is selected from quinic acid, fumaric acid, oxalic acid, malonic acid, D-tartaric acid, L-tartaric acid, succinic acid, cyclamic acid and ascorbic acid.

[0287] [Embodiment 28] The method for producing an isophagomine salt according to Embodiment 26, wherein the isophagomine salt is in crystalline form.

[0288] [Embodiment 29] The method for producing an isophagomine salt according to Embodiment 26, wherein the isophagomine free base has a purity of at least about 98%.

[0289] [Embodiment 30] A composition comprising an isophagomine salt according to any one of Embodiments 1 to 13, wherein the isophagomine salt has a purity of at least about 95%.

[0290] [Embodiment 31] The composition according to Embodiment 30, wherein the isophagomine salt has a purity of at least about 96%.

[0291] [Embodiment 32] The composition according to Embodiment 30, wherein the isophagomine salt has a purity of at least about 97%.

[0292] [Embodiment 33] The composition according to Embodiment 30, wherein the isophagomine salt has a purity of at least about 98%.

[0293] [Embodiment 34] The composition according to Embodiment 30, wherein the isophagomine salt has a purity of at least about 99%.

Claims

1. A salt of isofagomine, said salt being selected from quinate, maleate, fumarate, oxalate, malonate, succinate, cyclamate and ascorbate salts.

2. The salt of claim 1 , wherein the salt is isofagomine quinate.

3. The salt of claim 1 , wherein the salt is isofagomine fumarate.

4. The salt of claim 1 , wherein the salt is isofagomine oxalate.

5. The salt of claim 1 , wherein the salt is isofagomine succinate.

6. The salt of claim 1 , wherein the salt is isofagomine cyclamate.

7. 3. The salt of claim 2, wherein isofagomine quinate is in a crystalline form characterized by an X-ray diffraction pattern having three or more characteristic peaks at 2θ values ​​selected from 9.5°±0.2°, 15.0°±0.2°, 17.4°±0.2°, 18.1°±0.2°, 20.3°±0.2°, 23.8°±0.2°, 24.8°±0.2° and 25.4°±0.2°.

8. 4. The salt of claim 3, wherein isofagomine fumarate is in a crystalline form characterized by an X-ray diffraction pattern having three or more characteristic peaks at 2θ values ​​selected from 16.1°±0.2°, 18.3°±0.2°, 18.6°±0.2°, 21.9°±0.2°, 23.6°±0.2°, 23.8°±0.2° and 25.5°±0.2°.

9. 5. The salt of claim 4, wherein isofagomine oxalate is in a crystalline form characterized by an X-ray diffraction pattern having three or more characteristic peaks at 2θ values ​​selected from 27.8°±0.2°, 32.2°±0.2°, 35.3°±0.2°, 36.6°±0.2°, 37.4°±0.2°, 38.4°±0.2°, 18.5°±0.2°, 19.2°±0.2°, 21.4°±0.2°, 22.6°±0.2°, 24.5°±0.2°, 24.8°±0.2°, 26.8°±0.2°, 20.2°±0.2° and 23.7°±0.2°.

10. 6. The salt of claim 5, wherein the isofagomine succinate is in a crystalline form characterized by having XRPD peaks:

11. 7. The salt of claim 6, wherein the isofagomine cyclamate is in a crystalline form characterized by having XRPD peaks.

12. 9.8°±0.2°, 10.5°±0.2°, 15°±0.2°, 15.3°±0.2°, 15.8°±0.2°, 17.4°±0.2°, 17.9°±0.2°, 18.5°±0.2°, 18.9°±0.2°, 19.6°±0.2°, 21.1°±0.2°, 21.7°±0.2°, 22°±0.2°, 24.2°±0.2°, 24.8°±0.

20. A salt of isofagomine, which is a crystalline form of isofagomine D-tartrate characterized by an X-ray diffraction pattern having three or more characteristic peaks at 2θ values ​​selected from: 26.6°±0.2°, 27.1°±0.2°, 27.4°±0.2°, 33.8°±0.2°, 35.7°±0.2°, 36.5°±0.2° and 37.5°±0.2°.

13. A pharmaceutical composition comprising at least one isofagomine salt according to any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

14. 14. The pharmaceutical composition of claim 13, further comprising glucocerebrosidase, wherein the isofagomine is present in an amount sufficient to stabilize the glucocerebrosidase.

15. 15. The pharmaceutical composition of claim 13 or 14, wherein the glucocerebrosidase is velaglucerase alfa.

16. 16. The pharmaceutical composition of any one of claims 13 to 15, wherein glucocerebrosidase and isofagomine are in a molar ratio of about 1:2.5 to about 1:3.

5.

17. 17. The pharmaceutical composition of claim 16, comprising 60-180 mg / mL glucocerebrosidase, wherein isofagomine is present in at least about a 3-fold molar excess over glucocerebrosidase.

18. A pharmaceutical composition comprising glucocerebrosidase and isofagomine in a molar ratio of about 1:3, further comprising sodium citrate buffer, sucrose, and one or more surfactants selected from PS20, PS80, and poloxamer 188.

19. A process for preparing a pharmaceutical composition according to any one of claims 13 to 18, comprising combining isofagomine with a pharma- ceutically acceptable carrier.

20. A process for preparing the pharmaceutical composition according to any one of claims 14 to 18, comprising dissolving isofagomine in water, adjusting the pH to about 6.0 and adding glucocerebrosidase to obtain the composition.

21. A method for treating a disorder associated with dysfunction of the GCase pathway, comprising administering to a patient in need thereof a composition according to any one of claims 13 to 18.

22. 22. The method of claim 21, which is effective in treating the disorder.

23. 22. The method of claim 21, wherein the composition is administered intravenously or subcutaneously.

24. 24. The method of claim 23, wherein the composition is administered subcutaneously.

25. 1. A method for treating a disorder associated with dysfunction of the GCase pathway, comprising administering to a patient in need thereof a composition comprising 0.5-5.0 mg / kg glucocerebrosidase and at least about a 3-fold molar excess of isofagomine relative to glucocerebrosidase, wherein the composition is administered subcutaneously.

26. A method for treating Gaucher's disease, comprising administering to a patient in need of such treatment: i) a therapeutically effective amount of an isofagomine salt or a formulation thereof according to any one of claims 1 to 12; or ii) a therapeutically effective amount of a pharmaceutical composition according to any one of claims 13 to 18.

27. 1. A method for producing an isofagomine salt comprising the steps of: i) dissolving an organic acid in a polar protic solvent to form solution 1; ii) dissolving isofagomine free base in a polar protic solvent to form solution 2; iii) combining solution 1 and solution 2 to form a precipitate; iv) isolating the precipitate corresponding to the organic acid salt of isofagomine; wherein the organic acid is selected from quinic acid, fumaric acid, oxalic acid, malonic acid, D-tartaric acid, L-tartaric acid, succinic acid, cyclamic acid and ascorbic acid.

28. 27. The method of claim 26, wherein the isofagomine salt is in crystalline form.

29. 27. The method of claim 26, wherein the isofagomine free base is at least about 98% pure.

30. 14. A composition comprising an isofagomine salt according to any one of claims 1 to 13, wherein the isofagomine salt is at least about 95% pure.

31. 31. The composition of claim 30, wherein the isofagomine salt is at least about 96% pure.

32. 31. The composition of claim 30, wherein the isofagomine salt is at least about 97% pure.

33. 31. The composition of claim 30, wherein the isofagomine salt is at least about 98% pure.

34. 31. The composition of claim 30, wherein the isofagomine salt is at least about 99% pure.

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