RU2790262C2 - Compositions containing glucocerebrosidase and isofagomine - Google Patents

Abstract

FIELD: medicine; therapy.

SUBSTANCE: group of inventions relates to the field of medicine, namely to therapy; it is intended for the treatment of GCase path disfunction (glucocerebrosidase enzyme). A composition for the treatment of GCase path disfunction contains glucocerebrosidase (hereinafter – GCB) and isofagomine (IFM) at a molar ratio of at least about 1:2.5. The composition is intended for subcutaneous injection. In other embodiments, a method for the production of the specified composition, methods for the treatment of GCase path disfunction, and the use of the specified composition in the production of a drug, as well as a container containing the specified composition are presented.

EFFECT: use of the group of inventions allows for an increase in the activity and bioavailability, which reduces a number of subcutaneous injections, which are necessary to be injected to a subject, as well as provides improved stability allowing for maintenance of high concentrations of circulating GCB, which leads to greater absorption of GCB by monocytes, macrophages, and tissue histiocytes.

73 cl, 21 dwg, 13 tbl, 19 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority over U.S. Provisional Application No. 62/577,429, filed October 26, 2017, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Glucocerebrosidase (GCB) is a protein drug that can be used to treat Gaucher's disease, an autosomal recessive lysosomal deficient storage disease (GCB).

[0003] Gaucher's disease is an autosomal recessive disorder caused by mutations in the GBA gene that result in deficiency of the lysosomal enzyme beta-glucocerebrosidase. Glucocerebrosidase catalyzes the conversion of sphingolipid glucocerebroside to glucose and ceramide. Enzyme deficiency causes accumulation of glucocerebroside primarily in the lysosomal compartment of macrophages, leading to the formation of foam cells or "Gaucher cells". In Gaucher disease, various forms of mutant GCase have reduced, negligible, or no glucosylceramide cleavage activity, depending on the mutated amino acid or amino acids. The severity of this disorder correlates with the relative levels of residual enzyme activity and the resulting degree of substrate accumulation.

[0004] GCB is a lysosomal enzyme that hydrolyzes the glycolipid glucocerebroside, which is formed after the degradation of glycosphingolipids in the membranes of leukocytes and erythrocytes. Deficiency of this enzyme causes the accumulation of glucocerebroside in large quantities in the lysosomes of phagocytic cells located in the liver, spleen and bone marrow of patients with Gaucher disease. Accumulation of these molecules causes a range of clinical manifestations, including splenomegaly, hepatomegaly, skeletal dysfunction, thrombocytopenia, and anemia. (Beutler et al. "Gaucher disease" The Metabolic and Molecular Bases of Inherited Disease (McGraw-Hill, Inc, New York, 1995, pp. 2625-2639.)

[0005] Velaglucerase alpha is a form of GCB used to treat Gaucher's disease. VPRIV is a composition containing velaglucerase alpha. Velaglucerase alpha catalyzes the hydrolysis of glucocerebroside, reducing the amount of accumulated glucocerebroside. In clinical studies, VPRIV reduced the size of the spleen and liver and improved anemia and thrombocytopenia.

[0006] VPRIV and velaglucerase alfa and other similar drugs that contain protein are stored in liquid or lyophilized, that is, freeze-dried form. The lyophilized drug is often reconstituted by adding a suitable diluent for administration immediately prior to use by the patient. As a result of physical instability, there is a decrease in the amount of alpha velaglucerase or GCB in liquid or lyophilized form, including denaturation and aggregation, as well as chemical instability, including, for example, hydrolysis, deamidation and oxidation.

[0007] There is a need for improved compositions with improved stability of GCB, VPRIV, or velaglucerase alfa, especially those suitable for subcutaneous (SC) administration. GCB has a solubility limit of less than 30 mg/mL at room temperature for 24 hours. A convenient volume for an injectable PC product is typically 2.5 ml or less. This requires a composition that can be concentrated to a high enough level to deliver a therapeutically adequate dose. In addition, the compositions should ideally have adequate storage stability at room temperature or under refrigeration conditions.

[0008] There is also a need for subcutaneous formulations that have improved bioavailability of GCB, VPRIV, or velaglucerase alfa. The current composition of VPRIV, which is administered intravenously (IV), provides approximately 1% serum bioavailability. Subcutaneous (SC) administration is unlikely to provide the same tissue exposure as intravenous administration. GCB has a serum half-life of less than 15 minutes as an intravenous drug. Improved serum stability would allow more HCB administered by PC to be dispersed from the PC compartment into the systemic circulation. Improved serum stability will also allow high concentrations of circulating GCB to be maintained, allowing more GCB to be taken up by monocytes, macrophages, and tissue histiocytes.

SUMMARY OF THE INVENTION

[0009] In one aspect, a composition is provided comprising glucocerebrosidase (GCB) and isophagomine (IPM) in a molar ratio of 1:1, or at least about 1:>1 (eg, 1:x, where x is greater than 1). In some embodiments, the GCB is alpha-velaglucerase. Velaglucerase alpha is a recombinantly produced enzyme with the same amino acid sequence as natural human GCB produced in a human cell line and is a particularly suitable form of GCB for practicing the invention. In some embodiments, the pH of the composition is about 6.0. In some embodiments, the pH of the composition is about 6.5. In some embodiments, the pH of the composition is about 7.0. In some embodiments, the molar ratio of GCB to IFM is from about 1:1 to about 1:30. In some embodiments, the molar ratio of GCB to IFM is from about 1:1 to about 1:10. In some embodiments, the molar ratio of GCB to IFM is from about 1:1 to about 1:5. In some embodiments, the molar ratio of GCB to IFM is from about 1:2 to about 1:10. In some embodiments, the molar ratio of GCB to IFM is from about 1:2.5 to about 1:10. In some embodiments, the molar ratio of GCB to IFM is from about 1:2.5 to about 1:5. In some embodiments, the molar ratio of GCB to IFM is from about 1:10 to about 1:30. In some embodiments, the molar ratio of GCB to IFM is from about 1:30 to about 1:100. In some embodiments, the molar ratio of GB to IPM is from about 1:2.5 to about 1:3.5. In some embodiments, the molar ratio of GCB to IFM is about 1:3.0. In some embodiments, the molar ratio of GCB to IFM is about 1:3.0, which is particularly suitable for practicing the invention.

[0010] In some embodiments, the composition is at a temperature of at least 20°C. In some embodiments, the composition is at a temperature of 0°C to 20°C. In some embodiments, the composition is at a temperature of less than 0°C. In some embodiments, the composition is an aqueous solution. In some embodiments, the composition is a lyophilisate.

[0011] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient, a pharmaceutically acceptable salt, or both a pharmaceutically acceptable excipient and a pharmaceutically acceptable salt.

[0012] In some embodiments, IFM is isophagomine tartrate (IFMT). In some embodiments, the isophagomine tartrate is isophagomine D-tartrate. IPMT, and in particular isophagomine D-tartrate, is a particularly suitable salt of IPMT for the practice of the invention. Isophagomine tartrate can advantageously increase serum GCB activity above the upper limit usually achieved with a subcutaneous dose of 2.5 mg/kg. Accordingly, HCB combined with IFMT can provide serum bioavailability that allows for subcutaneous administration, in particular at a molar ratio of at least 1:3.0 HCB:IFMT. The combination of IPMT also increases the overall activity of the GCB enzyme. In some embodiments, IFM is different from isophagomine tartrate. In some embodiments, the composition is a liquid. In some embodiments, the composition further comprises an antioxidant. In some embodiments, the composition further comprises a carbohydrate. In some embodiments, the composition further comprises a surfactant. In some embodiments, the composition contains 45-120 mg/ml velaglucerase alfa and 0.2-1.8 mg/ml isophagomine D-tartrate. In some embodiments, the composition contains 60 mg/ml velaglucerase alfa and 0.9 mg/ml isophagomine D-tartrate.

[0013] In some embodiments, the composition further comprises citrate or phosphate and polysorbate 20 (eg, 50 mmol sodium citrate or sodium phosphate and 0.01% polysorbate 20). In some embodiments, the composition further comprises 5-20 mmol sodium citrate and 0.01% polysorbate-20. In some embodiments, the composition further comprises 10 mmol sodium citrate and 0.01% polysorbate-20. In some embodiments, the composition further comprises 5-20 mmol sodium phosphate and 0.01% polysorbate-20. In some embodiments, the composition further comprises 10 mmol sodium phosphate and 0.01% polysorbate-20. In some embodiments, the composition further comprises 5-20 mmol sodium citrate and 0.01% (w/v) polysorbate-20. In some embodiments, the composition further comprises 10 mmol sodium citrate and 0.01% (w/v) polysorbate-20. In some embodiments, the composition further comprises 5-20 mmol sodium phosphate and 0.01% (w/v) polysorbate-20. In some embodiments, the composition further comprises 10 mmol sodium phosphate and 0.01% (w/v) polysorbate-20. In some embodiments, the composition further comprises 5-20 mmol sodium citrate and 0.01% (v/v) polysorbate-20. In some embodiments, the composition further comprises 10 mmol sodium citrate and 0.01% (v/v) polysorbate-20. In some embodiments, the composition further comprises 5-20 mmol sodium phosphate and 0.01% (v/v) polysorbate-20. In some embodiments, the composition further comprises 10 mmol sodium phosphate and 0.01% (v/v) polysorbate-20. In some embodiments, the composition has a pH of about 6.0. In some embodiments, the composition has a pH of 6.0.

[0014] In another aspect, a container is provided that contains any of the compositions described herein. In some embodiments, the container is selected from the group consisting of a pre-filled syringe, vial, or ampoule.

[0015] In another aspect, a method for preparing any of the compositions described herein is provided. The method includes dissolving the IFM (eg, in water), adjusting the pH to about 6.0, and adding HCB to form the composition. In some embodiments, the method further comprises lyophilizing the IFM prior to the addition of GCB. In some embodiments, the method further comprises adding 0.01% polysorbate 20. In some embodiments, the method further comprises adding 0.01% (w/v) polysorbate 20. In some embodiments, the method further comprises adding 0.01% ( v/v) polysorbate 20. In some embodiments, the method further comprises filtering the composition through a 0.22 micron membrane. In some embodiments, the IFM is present in an amount sufficient to maintain the stability of the GCB in the composition. In some embodiments, the IFM is present in an amount sufficient to maintain the stability of the GCB in the composition for at least three days at 0-50°C. In some embodiments, the IFM is present in an amount sufficient to maintain the stability of the GCB in the composition for at least 6 months at 0-40°C.

[0016] In another aspect, a method for treating a disorder associated with dysfunction of the GCase pathway is provided, comprising administering any of the compositions described herein. In some embodiments, the method is effective for treating the disorder. In some embodiments, the composition is administered intravenously or subcutaneously. In some embodiments, the composition is administered subcutaneously, eg, by subcutaneous injection, which is particularly suitable for the practice of the invention. In some embodiments, the composition is administered twice a week, once a week, less than once a week, or once a week. Typically, the compositions described herein are administered by subcutaneous injection either once or twice a week or once every two weeks. The compositions described herein (particularly those with IFMT) administered subcutaneously can provide significantly greater serum exposure compared to comparable intravenous doses of GCB alone. The increased bioavailability in serum advantageously allows for a reduction in the number of subcutaneous injections that need to be administered to a subject. For example, fewer injections need to be administered to achieve a therapeutically effective amount and/or the time interval between subcutaneous injections can be increased.

[0017] In another aspect, the compositions described herein are for use in therapy. In one embodiment, the compositions described herein are for use in a method for treating a disorder associated with dysfunction of the GCase pathway as described herein. In another embodiment, the compositions described herein are for use in the manufacture of a medicament for the treatment of a disorder associated with dysfunction of the GCase pathway, for example, by the methods 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 a week. Typically, the compositions described herein are administered by subcutaneous injection once or twice a week or once every two weeks.

[0018] In some embodiments, the disorder includes a defect in GCase activity. In some embodiments, a defect in GCase activity includes reduced enzymatic activity. In some embodiments, the disorder includes dysregulation of alpha-synuclein. In some embodiments, the disorder is a lysosomal storage disease, such as Gaucher's disease, Fabry's disease, Pompe's disease, mucopolysaccharidosis, or multiple system atrophy. The compositions described herein are particularly suitable for the treatment of Gaucher's disease. In some embodiments, the disorder is a neurodegenerative disorder, such as Parkinson's disease, Alzheimer's disease, or Lewy body dementia.

[0019] In another aspect, a method for treating dysfunction of the GCase pathway is provided, comprising administering to a subject in need thereof any of the compositions described herein. In some embodiments, the subject is a human.

[0020] In another aspect, a method is provided for treating dysfunction of the GCase pathway, comprising administering to a subject a composition comprising 0.5 to 5.0 mg/kg of GCB and IFM, for example, wherein the IFM is at least about 1, 1.25, 1.5, 2, 2.5, 3, 4, or 5-fold molar excess relative to HCB, while the composition is administered subcutaneously. In another aspect, a composition is provided comprising from 0.5 to 5.0 mg/kg GCB and IFM, for example, where IFM is at least about 1, 1.25, 1.5, 2, 2.5, 3, 4 or 5-fold molar excess relative to GCB, for use in a method for treating GCase pathway dysfunction, wherein the composition is administered subcutaneously. In another aspect, the use of a composition containing from 0.5 to 5.0 mg/kg GCB and IFM is provided, for example, where IFM is at least about 1, 1.25, 1.5, 2, 2.5, 3 , 4 or 5-fold molar excess with respect to GCB in the manufacture of a drug for a method for treating dysfunction of the GCA pathway. In some embodiments, the IFM in the composition is administered in an amount that does not increase the activity of endogenous GCB in serum. In some embodiments, the composition contains from 0.8 to 4.0 mg/kg GCB. In some embodiments, the composition contains from 1.0 to 3.0 mg/kg GCB. In some embodiments, the composition contains from 1.2 to 2.0 mg/kg GCB. In some embodiments, the composition contains about 1.5 mg/kg GCB. In some embodiments, the composition contains 1.5 mg/kg GCB. In some embodiments, the composition contains from 2.0 to 5.0 mg/kg GCB. In some embodiments, the composition contains from 2.25 to 4.5 mg/kg GCB. In some embodiments, the composition contains from 2.25 to 3.75 mg/kg GCB. In some embodiments, the composition contains from 3.5 to 5.0 mg/kg GCB. In some embodiments, the IFM is in a 1-5 or 1-10 times molar ratio to the GCB. In some embodiments, the IFM is in a 2-10 fold molar ratio to the GCB. In some embodiments, the IFM is in a 10-30 fold molar ratio to the GCB. In some embodiments, the IFM is in a 30-100-fold molar ratio to the GCB. In some embodiments, the IFM is in a 2.5-3.5-fold molar ratio to the GCB. In some embodiments, the IFM is in a 3-fold molar ratio to the GCB. In some embodiments, the exposure, activity, or bioavailability of GCB is increased, for example, compared to the exposure, activity, or bioavailability of an equivalent amount of GCB administered intravenously, alone. In some embodiments, the exposure, activity, or bioavailability of GCB in the spleen is increased. In some embodiments, the exposure, activity, or bioavailability of GCB in the liver is increased. In some embodiments, serum exposure, activity, or bioavailability of GCB is increased.

BRIEF DESCRIPTION OF GRAPHICS

FIG. 1 is a flow chart illustrating the process for preparing a composition of glucocerebrosidase (GCB) and isophagomine (IPM).

FIG. 2A and 2B show SDS-EPAG testing of GB samples on the first day after the addition of IPM (2A) and two weeks after the addition of IPM (2B). IFM pH adjustment was not carried out.

FIG. 3 shows Eppendorf tubes containing pH-adjusted lyophilized solutions of isophagomine tartrate (IPMT).

FIG. 4A and 4B show SDS-EPAG testing of GB samples on the same day that IFM was added (4A) and after three days of storage (4B). The pH of the IFM was adjusted.

FIG. 5 shows the results of size exclusion chromatography (SEC) analysis of pH adjusted IPMT added to GCB.

FIG. 6A and 6B show the results of size exclusion chromatography (SEC) analysis of pH adjusted IFM added to GCB.

FIG. 7A-7D show the results of surface plasmon resonance studies of IPM binding to GCB.

FIG. 8 shows the results of a nano-differential scanning fluorimetry (nano-DSF) analysis evaluating changes in the melting point of GCB with various IPM molar ratios ranging from 1:3 to 1:100 GCB:IPM.

FIG. 9A-9C show the results of enzymatic activity reactions carried out with velaglucerase alfa preincubated with IPMT. FIG. 9A shows the inhibition curve with the pNP-GPS synthetic colorimetric substrate. FIG. 9B shows the inhibition curve with 4MU-GPS synthetic fluorometric substrate. FIG. 9C shows inhibition by natural glycosphingolipid substrate C12-GluCer.

FIG. 10A shows the appearance of GB/IFMT samples stored for three weeks at 40°C. FIG. 10B shows SDS-EPAG analysis of GB/IFMT samples stored for three weeks. Solutions of Groups 1-3 (G1, G2, G3) appear clear. Group 4 (G4) solution appears cloudy.

FIG. 11 shows negative and positive controls for GCB immunohistochemistry (IGI) from a pharmacokinetic study of intravenous GCB and subcutaneous GCB with IFM in cynomolgus monkey.

FIG. 12 shows staining of GCB in the liver at 2-fold magnification at various time points after subcutaneous injection of GCB (upper panels) and subcutaneous injection of GCB (lower panels).

FIG. 13 shows staining of GCB in the liver at 20x magnification at various time points after subcutaneous injection of GCB (upper panels) and intravenous injection of GCB (lower panels).

FIG. 14 shows staining of GCB in the spleen at 2x magnification at different time points after subcutaneous injection of GCB (upper panels) and intravenous injection of GCB (lower panels).

FIG. 15 shows staining of GCB in the spleen at 20x magnification at different time points after subcutaneous administration of GCB (upper panels) and intravenous administration of GCB (lower panels).

FIG. 16A and 16B show the results of analysis of protein and enzyme activity levels of velaglucerase alfa in liver and spleen homogenates after administration of velaglucerase alfa (16A) and velaglucerase alfa with IPMT in a 1:3 molar ratio (16B).

FIG. 16C shows the results of an analysis of serum GCB activity levels in cynomolgus monkeys after subcutaneous administration of velaglucerase alfa with IPMT.

FIG. 17A and 17B show the results of an ECL ELISA assay for serum GCB (17A) bioavailability and GCB activity (17B) assay after subcutaneous administration of 4mg/kg velaglucerase alfa and IFM at various molar ratios ranging from (1:3 - 1:100) .

FIG. 18A and 18B show the results of an ECL ELISA analysis of the hepatic (18A) and spleen (18B) GCB profile following intravenous administration of velaglucerase alfa 10 mg/kg or velaglucerase alfa 4 mg/kg subcutaneous administration and IFM in a molar ratio of 1:100.

FIG. 19A and 19B show the results of the ECL ELISA assay for GCB content in the liver (19A) and spleen (19B) after subcutaneous administration of 4 mg/kg velaglucerase alfa and IFM in a 1:3 molar ratio.

FIG. 20A and 20B show the results of the ECL ELISA assay for serum bioavailability of GCB (20A) and the activity of GCB (20B) after subcutaneous administration of 1.5 mg/kg velaglucerase alfa and IFM at various molar ratios in the range (1:1-1: thirty).

FIG. 21 shows the results of the analysis of VPRIV activity in human serum incubated at 37°C without IFM, 3 nmol IFM, 10 nmol IFM, 30 nmol IFM, 100 nmol IFM, 300 nmol IFM and 1000 nmol IFM

DETAILED DESCRIPTION OF THE INVENTION Overview of the invention

[0021] Compositions containing glucocerebrosidase (GCB) may benefit from increased stability, for example, when the compositions are liquids. The three open free thiol groups in GCB can enter into reactions that lead to a decrease in stability, for example, by aggregation of GCB molecules. For example, in buffer at pH 6, typically 1-2% of the protein aggregates after one month of storage and about 15% aggregates after 6 months of storage. While not wishing to be strictly bound by theory or mechanism, protein stability depends on a number of factors.

[0022] The addition of isophagomine (IFM), eg, isophagomine tartrate (IFMT), improves the in vitro stability of GCB, particularly when the IFM, eg, IFMT, is adjusted to pH 6.0 prior to addition to GCB. IFM has the following structure:

[0023] While not wishing to be bound by theory, IFM can interact with amino acids adjacent to the active site to lock GCB into a conformation that provides increased stability. See Shen, J.S. et al., Biochem. Biophys. Res. Comm., 2008, 369:1071-1075. The IFM can also prevent GB aggregation, since the IFM can associate with the GB to make the GB more compact and thermally more stable.

[0024] The authors of the present invention have shown that the molar ratio of IFM to GCB is critical for the stabilization of GCB in liquid compositions. As described in more detail in this application, compositions with a molar ratio of at least 1:2.5 (GCB:IFM) (i.e. 1:x (where x is at least 2.5)), can have a significantly lower aggregation and decomposition of government securities. Significantly greater aggregation and degradation of GS with molar ratios well below 1:2.5 may occur.

[0025] The present inventors have also shown that compositions with an IPM/IFMT to GCB molar ratio of at least 1:2.5 (GCB:IFM) have improved bioavailability, GCB, potency, tissue exposure, and systemic exposure when administered subcutaneously. Improved bioavailability can be detected by one or more of increased HCB tissue staining in the liver, increased HCB tissue staining in the spleen, increased serum HCB concentration, and increased serum HCB activity. Improved systemic exposure can be analyzed by measuring the concentration of GCB protein or serum GCB enzyme activity. The addition of an IFM, such as IFMT, to GCB in a molar ratio of at least 1:2.5 (HCB:IFM) may allow the bioavailability, potency, tissue exposure, or systemic exposure of GCB in a subcutaneous formulation to be similar to or greater than the bioavailability, potency, tissue exposure or systemic exposure in intravenous formulations, especially formulations without IFM.

Definitions

[0026] The term "subject" refers to any mammal, including, but not limited to, any animal classified as such, including 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" can be used interchangeably with the term "patient".

[0027] The term "isolated" refers to a molecule that is essentially free from its natural environment. For example, an isolated protein is essentially free of cellular material or other proteins from the cells or tissues of the source from which it is derived. Isolated protein formulations are pure enough to be administered as a therapeutic composition, or at least 70% to 80% (w/w) pure, more preferably at least 80% to 90% (w/w). ./wt.) pure, even more preferably 90 to 95% pure; and most preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% (w/w) pure.

[0028] As used herein, the term "about" refers to up to +/-10% of the value qualified by this term. For example, about 50 mmol refers to 50 mmol +/- 5 mmol; about 4% refers to 4% +/- 0.4%.

[0029] As used herein, the phrases "parenteral administration", "introduced parenterally" and "introduced parenterally" refer to methods of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous (IV), intramuscular , intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous (PC), subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrawall injections and infusions.

[0030] The terms "therapeutically effective dose" and "therapeutically effective amount" refer to that amount of a compound that results in the prevention of symptoms, for example, prevention by 80%, 85%, 90%, 95%, 96%, 97%, 98 % or 99% of the symptoms, e.g. symptoms of Gaucher disease in a subject diagnosed with Gaucher disease), delaying the onset of symptoms, or alleviating the symptoms of Gaucher disease. A therapeutically effective amount will, for example, be sufficient to treat, prevent, ameliorate, delay onset and/or reduce the risk of one or more symptoms of a Gaucher disorder. An effective amount can be determined using methods well known in the art and as described in the following sections of this description.

[0031] The terms "treatment" and "therapeutic method" refer to the treatment of an existing disorder and/or prophylactic/preventive measures. Those in need of treatment may include those who already have a particular medical disorder, as well as those who are at risk or who may eventually become ill. The need for treatment is assessed, for example, in the presence of one or more risk factors associated with the development of the disorder, the presence or progression of the disorder, or the likely responsiveness to treatment of the subject having the disorder. Treatment may include slowing or reversing the progression of the disorder.

[0032] The term "treatment" refers to the administration of a therapy in an amount, order, and/or regimen effective to ameliorate or prevent a condition, symptom, or parameter associated with a disorder (e.g., the disorder described herein), or to prevent the onset, progression or exacerbation of the disorder, either to a statistically significant extent, or to an extent determined by a person skilled in the art. Accordingly, treatment may achieve therapeutic and/or prophylactic benefits. The effective amount, order, or regimen may vary depending on the subject and may be adapted to the subject. In some embodiments, the treatment for disorders associated with GCase pathway dysfunction (e.g., Gaucher disease) is a treatment that results in one or more of: an increase in hemoglobin concentration, an increase in platelet levels, a decrease in liver volume, a decrease in spleen volume, or a change in skeletal parameter (eg, increase in bone mineral density), for example, in a subject who has not been treated for GCase pathway dysfunction. In some embodiments, the treatment for a disorder associated with GCase pathway dysfunction (e.g., Gaucher disease) is a treatment that results in one or more of: an increase in hemoglobin concentration, an increase in platelet levels, a decrease in liver volume, a decrease in spleen volume, or a change in skeletal parameter (eg, increase in bone mineral density), or maintenance of one or more of these parameters, for example, in a subject who was treated for dysfunction of the GCase pathway.

[0033] the Term "combination" refers to the use of two or more agents or therapy for the treatment of the same patient, in which the use or effect of the agents or therapy overlap in time. The agents or therapies may be administered simultaneously (eg, as a single composition administered to a patient or as two separate compositions administered simultaneously) or sequentially in any order.

[0034] The terms “sustained release”, “sustained release delivery” and “sustained release drug delivery” in the context of the present description mean that a single administration of the drug maintains the effective concentration of the drug in the blood for a long period, for example, 12 hours or longer. For example, a common route of administration for polypeptides is subcutaneous, intramuscular, or intravenous (IV) injection.

[0035] The term "salts" embraces addition salts of free acids or free bases. The term "pharmaceutically acceptable salt" refers to salts that have toxicity profiles within the range that provides for use in pharmaceutical applications. Salts that are not pharmaceutically acceptable salts may still be useful in synthesis, purification or formulation due to properties such as high crystallinity.

[0036] The term "unit" in relation to GCB, velaglucerase, or velaglucerase alfa refers to the amount required to convert one micromol of p-nitrophenyl-beta-D-glucopyranoside to p-nitrophenol or 4-methylumbelliferone beta-D-glucopyranoside to 4-methylumbelliferone, per minute at 37°C.

Glucocerebrosidase

[0037] Velaglucerase is a human β-glucocerebrosidase produced by gene activation in a human cell line, for example, by directed recombination with a promoter that activates the endogenous β-glucocerebrosidase gene in a selected human cell line. Velaglucerase is secreted as a monomeric glycoprotein of approximately 63 kDa. Velaglucerase consists of 497 amino acids with a sequence identical to the natural human protein. See Zimran et al., Blood Cells Mol. Dis., 2007, 39:115-118.

[0038] Glycosylation of velaglucerase alfa can be altered by using kyfunesin, a mannosidase I inhibitor, during cell culture to produce a secreted protein containing primarily high-mannose type glycans having 6-9 mannose units per glycan, as in more detail described in WO 2013/130963.

[0039] Imiglucerase (Cerezyme®) is another form of recombinant human β-glucocerebrosidase. Imiglucerase is recombinantly produced in Chinese Hamster Ovary (Chamster Ovary) cells.

[0040] Alpha-taliglucerase (Elelyso® or Uplyso®) is a recombinant glucocerebrosidase (prGCB) expressed in plant cells. Plant recombinant glucocerebrosidase can be produced by the methods described in at least US Patent Publications 2009/0208477 and 2008/0038232 and PCT Publications WO 2004/096978 and WO 2008/132743.

[0041] Any of the recombinant GCB can be produced using bioreactors and industrial scale synthesis methods known in the art. Any number of industrial scale cleaning systems can be used.

Isophagomine

[0042] Various alternative forms of isophagomine can be used. These include any of isophagomine tartrate, isophagomine HCl, isophagomine free base, and isophagomine citrate. In some embodiments, isophagomine includes one or more of isophagomine HCl, isophagomine free base, and isophagomine citrate. In some embodiments, isophagomine includes isophagomine tartrate.

[0043] Isophagomine HCl, described in US Pat. Nos. 5,844,102 and 7,501,439. Isophagomine HCl is a yellow solid with a low melting point. Isophagomine free base can be obtained by converting isophagomine HCl to the free base form.

[0044] In any of the aspects and embodiments described herein, isophagomine may not be in the form of isophagomine tartrate, or the GCB/IFM composition may not contain isophagomine tartrate.

Isophagomine tartrate

[0045] Isophagomine tartrate (IFMT) is a specific form of isophagomine (IFM) that can be used in the various embodiments described herein and is particularly suited to the practice of the invention. IFMT has the following formula:

[0046] IFMT has improved characteristics compared to IFM, which include improved synthetic processability. For example, it may be easier to clean the IFMT in solvents such as water and ethanol. IPMT is more stable than other known salt forms of isophagomine IPMT is also particularly suitable for industrial scale production, e.g. production of more than 1 kg of product

[0047] A composition containing GS and IFM is sometimes referred to in this application as a GS/IFM composition. A composition containing GSW and IFMT is sometimes referred to in this application as a GSB/IFMT composition.

Molar ratios of GS to IPM IFMT

[0048] In various embodiments, the composition comprises glucocerebrosidase (GCB) and isophagomine (IFM), e.g., isophagomine tartrate (IFMT), in a molar ratio of at least about 1:1, 1:1.5, 1:2, or 1 :2.5 (GS:IFM). The molar ratio of GB to IFM, for example, GB to IFMT, can be 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, 1:2 7.3, 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, 1:29.5, 1:29.6, 1:29.7, 1:29 .8, 1:29.9 or 1:30.0.

[0049] The molar ratio of GCB to IFM, e.g. GCB to IFM, can be from 1:2.5 to 1:3.5, from 1:2.6 to 1:3.4, from 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, from 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.

[0050] The molar ratio of GCB to IFM, e.g., GCB to IFM, may 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, from 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, from 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, from 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, from 1:11 to 1:23, from 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.

[0051] The molar ratio of GCB to IFM, e.g., GCB to IFM, can be from 1:16 to 1:26, from 1:15 to 1:25, from 1:14 to 1:24, from 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, from 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, from 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, from 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, from 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, from 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.

[0052] The molar ratio of GCB to IFM, for example, GCB to IFM, 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

[0053] The molar ratio of GCB to IFM, e.g., GCB to IFM, can be from 1:30 to 1:100, from 1:30 to 1:80, from 1:40 to 1:90, from 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.

[0054] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCB) and isophagomine tartrate (IFMT) in a molar ratio of at least about 1:2.5.

[0055] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCB) and isophagomine citrate in a molar ratio of at least about 1:2.5.

[0056] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCB) and isophagomine HCl in a molar ratio of at least about 1:2.5.

[0057] In various other embodiments described herein, the composition comprises glucocerebrosidase (GCB) and isophagomine free base in a molar ratio of at least about 1:2.5.

[0058] In various other embodiments described herein, the composition contains glucocerebrosidase (GCB) and isophagomine, which does not contain IPMT, in a molar ratio of at least about 1:2.5.

GS concentration

[0059] The concentration of HCB in any of the compositions can be from about OD to about 40 mg/ml, from about 0.5 to about 10 mg/ml, from about 5 to about 15 mg/ml, from 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 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 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.

[0060] The concentration of HCB can be from 50 units/ml to 200 units/ml, from 70 units/ml to 160 units/ml, from 80 units/ml to 175 units/ml, from 90 units/ml to 190 units/ml , 60 U/mL to 145 U/mL, 50 U/mL to 130 U/mL, 80 U/mL to 140 U/mL, 70 U/mL to 120 U/mL, 60 U/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, from 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 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, from 90 units/ml to 105 units/ml, 80 units/ml to 95 units/ml, from 70 units/ml to 85 units/ml, from 60 units/ml to 75 units/ml, from 50 units /ml up to 65 units/ml, from 110 units/ml to 125 units ic/ml, 120 units/ml up to 135 units/ml, 130 units/ml up to 145 units/ml, 140 units/ml up to 160 units/ml, 160 units/ml up to 180 units/ml, 180 units/ml up 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 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.

Pharmaceutical compositions

[0061] The pharmaceutical composition may include a "therapeutically effective amount" of GCB/IFM, such as GCB/IFMT, of the composition described herein. Such effective amounts may be determined based on the effect of the administered composition. A therapeutically effective amount of a GCB/IFM composition, e.g., GCB/IFMT, may also vary depending on factors such as disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual, such as improvement in at least one symptom. , conditions or disorders, such as glucocerebrosidase deficiency, such as Gaucher's disease. A therapeutically effective amount is also one in which, when used, the therapeutically beneficial effects outweigh any toxic or detrimental effects of the composition.

[0062] The composition of GB/IPM may not contain IPMT.

[0063] The pharmaceutical composition of the invention may be formulated to be compatible with the intended route of administration. For example, the composition of GCB/IFM, for example, GCB/IFMT, can be administered parenterally, for example, intravenous, subcutaneous, intraperitoneal or intramuscular injection. In various embodiments, the route of administration is intravenous. In various embodiments, the route of administration is subcutaneous. Solutions or suspensions used for parenteral use may include the following components: a sterile diluent such as water for injection, saline, fatty oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonic agents such as sodium chloride or dextrose. The pH of pharmaceutical compositions can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

pH

[0064] pH can affect the stability of GB in the various GB/IFM and GB/IFMT formulations described herein. The pH can affect conformation and/or aggregation and/or degradation and/or reactivity of GCB. For example, at higher pH, oxygen may be more reactive. the pH is preferably less than 7.0, more preferably in the range from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.0, and more preferably from about 5.5 to about 5.8, more preferably about 5.7. With GCB, aggregation can reach undesirable levels above pH 7.0 and degradation (eg, fragmentation) can reach undesirable levels at pH 4.5 or 5.0 or above pH 6.5 or 7.0.

[0065] A suitable pH can be verified by providing a GCB/IFM composition, such as GCB/IFMT, for the test, adjusting the composition to a suitable pH, and purging the composition with oxygen. The stability of the GCB in the composition at a suitable pH can be measured, for example, as a percentage of aggregation or degradation at a predetermined time. The measured stability can be compared to one or more standards. For example, a suitable standard would be a composition similar to the tested composition, except that the pH of the composition was not adjusted. The stability of compositions with and without pH adjusted can then be compared. A GB/IFM composition, such as GB/IFMT, may be more appropriate if the GB is more stable than a standard reference composition. Suitability can be demonstrated by test processing increasing stability over this standard. For example, if a reference standard GBP/IFM formulation has a pH of 5.5, but increased stability of GBP is observed when the GBP/IPM formulation has a pH of 6.3, then a formulation with a pH of 6.3 is more appropriate because GBP is more stable at pH 6. .3 than at pH 5.5.

[0066] Buffers that can be used to adjust the pH of the protein composition include histidine, citrate, phosphate, glycine, succinate, acetate, glutamate, Tris, tartrate, aspartate, maleate, and lactate.

GS stability analyzes

[0067] The stability of proteins can be measured by measuring protein aggregation or protein degradation. Protein aggregation can be determined using various methods, which include, for example, size exclusion chromatography (SEC), non-denaturing EPAG, or other sizing methods, etc. Protein degradation can be determined, for example, by reverse phase HPLC, non-denaturing EPAG, ion exchange chromatography, peptide mapping, or the like.

[0068] Stability, as used herein, includes parameters such as protein structure (e.g., minimizing or preventing changes in protein structure, e.g., protein aggregation or protein degradation (e.g., fragmentation)) and/or protein biological activity, e.g. , the ability to convert a substrate into a product.

[0069] The stability of GCB can be measured, for example, by measuring protein aggregation, protein degradation, or GCB biological activity levels. GB aggregation can be determined in a variety of ways, including size exclusion chromatography, non-denaturing EPAG, and other sizing methods. For example, the composition may have less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase in GCB protein aggregation (e.g., as measured by size exclusion chromatography) compared to the amount protein aggregation that was in the composition prior to storage (eg, storage at 2-8° C. for up to 3, 6.9, 12, or 24 months (or longer)).

[0070] Protein degradation can be determined by various methods, including reverse phase HPLC, non-denaturing EPAG, ion exchange chromatography, peptide mapping, or the like. By way of example, the composition may have less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% increase in HCB degradation (e.g. as measured by reverse phase HPLC)) compared to the amount degradation of HCB that was in the composition prior to storage (eg, storage at 2-8°C for up to 3, 6, 9, 12, or 24 months (or longer))). The biological activity of GCB can be measured, for example, by in vitro or in vivo assays, for example, ELISA (for example, to measure binding or enzymatic activity) and other enzymatic assays (for example, spectrophotometric, fluorimetric, calorimetric, chemiluminescent, radiometric or chromatographic assays ), kinase assays, and so on. For example, the composition may have less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% reduction in GCB biological activity (e.g., enzymatic activity, e.g., as measured by an in vitro assay) compared to the biological activity that the composition had prior to storage (eg, storage at 2-8° C. for up to 3, 6, 9, 12, or 24 months (or longer)).

Antioxidants and Stabilizers

[0071] The GCB/IFM and GCB/IFMT compositions described herein may additionally contain an antioxidant. One suitable antioxidant is cysteine. Cysteine may be present in an amount of 0.030% to 0.100%, 0.050% to 0.080%, 0.040% to 0.070%, 0.030% to 0.060%, 0.060% to 0.090%, 0.070% to 0.100%, 0.065% up to 0.080%, 0.060% to 0.075%, 0.055% to 0.070%, from 0.050% to 0.065%, from 0.070% to 0.085%, from 0.075% to 0.090%, about 0.065%, about 0.080%, 0.065%, 0.070%, 0.075% or 0.080%. Without wishing to be bound by theory, cysteine may further stabilize GCB.

[0072] The GCB/IFM and GCB/IFMT compositions described herein may additionally contain a carbohydrate such as sucrose or trehalose. A carbohydrate, such as sucrose or trehalose, may be present in an amount of 12% to 19%, 13% to 18%, 14% to 17%, 12% to 15%, 13% to 16%, 15% to 17%, about 16%, or 16%. Without wishing to be bound by theory, sucrose or trehalose may further stabilize GCB by reducing the availability of thiol (-SH) groups.

[0073] The GSB/IFM and GSB/IFMT compositions herein may additionally contain a detergent. The detergent may be polysorbate 20 (which is particularly suitable for the practice of this invention), or any amount of a poloxamer compound.

[0074] In some embodiments, the stability of GS is at least 5-80% greater (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% more), under preselected conditions, than the stability of a GCB in a composition that characterized by the absence of a carbohydrate (sucrose or trehalose), an antioxidant, or both a carbohydrate and an antioxidant.

[0075] The GCB/IFM and GCB/IFMT compositions can be deoxygenated prior to storage in the container. In addition, the container is ideally gas-tight, so as to prevent the ingress of oxygen. GCB in the compositions described herein, for example, liquid compositions containing GCB, may have prolonged stability. For example, under preselected conditions, such as storage in a gas-tight container, at 2-8°C for up to 3, 6, 9, 12, or 24 months (or longer in some embodiments), the GS in the composition will retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the stability it had prior to storage.

[0076] A suitable protein concentration can be verified by preparing a composition containing 0.075% cysteine, 16% sucrose, adjusting the pH to 5.7, adjusting the GCB to the intended concentration, and purging the composition with O ₂ . The stability of the GB in the GB/IFM composition, eg the GB/IFMT composition at the appropriate concentration, measured eg as percentage aggregation or degradation, at a predetermined time, is compared to one or more standards. The stability of GCB at each concentration is compared. Suitability can be demonstrated by a suitable concentration having a comparable or better effect on stability than the concentration described herein.

[0077] The stability of GCB can be measured using any of the methods described in this application, for example, by measuring protein aggregation or protein degradation. Protein aggregation can be determined, for example, by size exclusion chromatography, non-denaturing EPAG or other sizing methods, etc. Protein degradation can be determined, for example, by reverse phase HPLC, non-denaturing EPAG, ion exchange chromatography, SEC, SEC HPLC, peptide mapping, or the like.

Surfactants

[0078] The GSB/IFM and GSB/IFMT compositions described herein may additionally contain one or more surfactants. Without wishing to be bound by any theory, surfactants can enhance the stability of a protein, for example by providing an air/liquid interface that can reduce degradation of the protein during agitation or during transport. A surfactant may be selected that enhances the stability of the protein, for example without causing degradation of the protein, in a particular liquid composition. A typical surfactant is poloxamer 188 or Pluronic F68. The surfactant may be present in an amount of about 0.005% to about 5%, such as about 0.01% to about 1%, such as about 0.025% and about 0.5%, such as about 0.03% and about 0.25%, for example, from about 0.04% to about 0.1%, for example, from about 0.05% to about 0.075%, for example, 0.05%. The ideal surfactant is one that is not modified or degraded by GCB.

[0079] For example, a suitable surfactant can be tested by preparing a formulation containing 2 mg/mL GCB, some IFM, 0.075% cysteine, 16% sucrose, then adjusting the pH to 5.7, then adding the appropriate surfactant substance and blowing the composition O ₂ . The stability of a GCB/IFM composition containing a suitable surfactant is measured, for example, as percentage aggregation or degradation at a given time compared to one or more standards. For example, a suitable standard may be a composition similar to the test conditions, except that no surfactant is added to the composition. The stability of the treated (surfactant-containing) and untreated (surfactant-free) formulations can be compared under conditions that simulate "real world" scenarios, such as storage and transportation. The standard may be a composition similar to the test composition, except that a different surfactant is used instead of poloxamer 188. Poloxamer 188 will then be the standard for comparison. Suitability can be shown by a suitable surfactant having a comparable or better effect on stability than the surfactant described herein. If the appropriate surfactant is determined to be appropriate (eg, it increases the stability of the composition over one of the standards), the concentration of the appropriate surfactant may be adjusted. For example, the concentration can be increased or decreased over a range of values and compared to the standard and other test concentrations to determine which concentration causes the greatest increase in stability.

[0080] Alternatively, a combination of two or more surfactants is used in the compositions described herein. The suitability of the combination can be tested as described above by comparing the stability of the GCB/IFM formulation with the test combination of surfactants with the stability of the GCB/IFM formulation with poloxamer 188.

[0081] The stability of proteins can be measured, for example, by measuring protein aggregation or protein degradation. Protein aggregation can be determined, for example, by size exclusion chromatography, non-denaturing EPAG or other sizing methods, etc. Protein degradation can be determined, for example, by reverse phase HPLC, non-denaturing EPAG, ion exchange chromatography, peptide mapping, or the like.

Pharmaceutically acceptable salt

[0082] The pharmaceutical composition may further comprise a salt or a pharmaceutically acceptable salt.

[0083] Suitable pharmaceutically acceptable acid addition salts can be derived from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acids. Suitable organic acids may be selected from the aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic , maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenyl acetate, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilamino , stearic, alginic, β-hydroxybutyric, salicylic, galactaric, oxalic, malonic and galacturonic acids. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates. All of these acid addition salts can be prepared from isophagomine or GCB by reacting, for example, the corresponding acid with the compound.

[0084] Suitable pharmaceutically acceptable isophagomine base addition salts include, for example, metal salts, including alkali metals, alkaline earth metals, and transition metal salts, such as, for example, calcium, magnesium, potassium, sodium, and a zinc salt. Pharmaceutically acceptable base addition salts also include organic salts derived from basic amines such as, for example, N,N'-dibenzylethylenediamine, chlorprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples of non-pharmaceutically acceptable base addition salts include lithium salts and cyanate salts. All of these base addition salts can be prepared from isophagomine by reacting, for example, a suitable base with the compound.

Pharmaceutical carriers

[0085] Pharmaceutical compositions containing GCB may include one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any solvents, excipients, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and adsorption-retarding agents, and the like that are compatible with pharmaceutical administration. The pharmaceutical composition is well known technology and is further described in, for example, 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). Except where any conventional carrier or agent is incompatible with the active compound, such carriers may be used in the compositions of the invention. Additional active compounds may also be included in the compositions.

[0086] The pharmaceutical compositions described herein may further include carriers that protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. The compositions can be prepared in accordance with methods known to those skilled in the art, for example as described in US Pat. No. 4,522,811.

[0087] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it can be easily injected through a syringe. The composition must also be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating agent such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. Prevention of the activity of microorganisms can be achieved with various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it will be preferable to include isotonic agents, eg sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Long-term stability of injectable compositions can be achieved by the inclusion of an adsorption-retarding agent, such as aluminum monostearate, human serum albumin, and gelatin.

[0088] Sterile injectable solutions can be prepared by adding HCB/IFM to a suitable solvent with one or a combination of the ingredients listed above, as needed, followed by filtered sterilization. Typically, dispersions are prepared by adding the active compound to a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the formulation of sterile injectable solutions, the preferred formulation methods are vacuum drying and lyophilization, for example lyophilization, which yields a powder of the active ingredient plus any additional desired ingredient from their pre-sterile filtered solution.

[0089] Active compounds (eg, the GCB compositions described herein) can be formulated with carriers that will protect the compound from rapid excretion from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. Materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. The compositions can be prepared in accordance with methods known to those skilled in the art, for example as described in US Pat. No. 4,522,811.

Packing and shipping

[0090] The GSB/IFM and GSB/IFMT compositions described herein can be administered using a variety of medical devices. For example, the composition described herein can be administered using a needle-free hypodermic injection device such as those described in US Pat. Examples of well-known implants and modules used in the invention include: US Patent No. 4,487,603, which describes an implantable microinfusion pump for dosing a drug at a controlled rate; US patent No. 4,486,194, which describes a therapeutic device for the introduction of drugs through the skin; US Pat. No. 4,447,233, which describes a drug infusion pump for drug delivery at a precise infusion rate; US patent No. 4,447,224, which describes an implantable infusion device with variable flow for continuous drug delivery; US Pat. No. 4,439,196, which describes an osmotic drug delivery system having multi-chamber compartments; and US Pat. No. 4,475,196, which describes an osmotic drug delivery system. Of course, many other such implants, delivery systems and modules are also known.

[0091] The GCB/IFM and GCB/IFMT compositions described herein can be placed in two syringe chambers. For example, GCB/IFM and GCB/IFMT formulations in lyophilized form may be placed in the first chamber of the syringe and liquid may be present in the second chamber of the syringe (see, for example, US Published Application No. 2004-0249339).

[0092] The GSB/IFM and GSB/IFMT compositions described herein can be placed in a needleless syringe (see, for example, US Pat. Nos. 6,406,455 and 6,939,324). Briefly, as one example, the injection device includes: a gas chamber containing a gas or gas source; a port that can allow the release of gas from the gas chamber; a piston, which, when the gas is released from the gas chamber, can cause movement of at least the first piston; first piston; second piston; a first chamber, such as a medicine storage and mixing chamber; a piston housing in which the first piston, the second piston and the first chamber are located; a biasing element that can, regardless of the driving force of the gas from the gas chamber, cause the movement of one or both of the first and second pistons (the biasing element may be a piston or a separate element); an opening suitable for needleless injection in communication with the first chamber; wherein the first and second pistons are slidably arranged in the piston housing, and the biasing element, the gas source and the piston are located so that: in the first position of the pistons - the second chamber, for example, the liquid reservoir in the piston housing is determined by the first piston, the piston housing and the second piston, the biasing member may move one or both of the pistons to a second position wherein the first piston is in such a position that the second chamber, which may be a fluid reservoir, communicates with the first chamber, which may be a drug storage and mixing chamber, and the second piston moves towards the first piston, thereby reducing the volume of the second chamber and ensuring the transfer of liquid from the second chamber to the first chamber, the plunger, after releasing gas from the gas chamber, causes the first piston to move so as to reduce the volume of the first chamber, allowing the substance to be pushed through the hole and from the camera and, for example, the subject.

[0093] The needleless syringe may include separate modules for a first component, such as a dry or liquid component, and a second component, such as a liquid component. The modules may be provided as two separate components and assembled, for example, by a subject who will administer the component to themselves, or by another person, such as a person who is providing or providing medical care. Together, these modules may form all or part of the piston housing of the devices described herein. The devices can be used to provide any first and second component where it is desirable to store or provide the components separately and combine them prior to administration to a subject.

Methods of treatment

[0094] Any of the GCB/IFM and GCB/IFMT compositions described herein may be administered to a patient. The GCB/IFM and GCB/IFMT compositions described herein are for use in methods of treating a disorder associated with GCase pathway dysfunction, in particular Gaucher's disease. The compositions are also used in the manufacture of a medicament for the treatment of such diseases using the methods of treatment described herein.

[0095] The dose may be about 60 units/kg or 60 units/kg administered every other week. The dose may be about 30 units/kg or 30 units/kg given every week. Alternatively, the dose may be 30 to 80 units/kg administered every other week, 40 to 70 units/kg administered every other week, 50 to 80 units/kg administered every other week, 45 to 65 units/kg kg administered every other week, every other week: 40 to 60 units/kg administered every other week, 35 to 55 units/kg administered every other week, 30 to 50 units/kg administered every other week, from 45 to 65 units/kg given every other week, 50 to 70 units/kg given every other week, 55 to 75 units/kg given every other week, 60 to 80 units/kg given every other week , 55 to 65 units/kg administered every other week, 45 to 55 units/kg administered every other week, 35 to 45 units/kg administered every other week, or 65 to 75 units/kg administered every other week. Alternatively, the dose may vary from 15 to 40 units/kg given every week, 20 to 35 units/kg given every week, 25 to 40 units/kg given every week, 22.5 to 32.5 units /kg administered every week, 20 to 30 units/kg administered every week, 17.5 to 22.5 units/kg administered every week, 15 to 25 units/kg administered every week, from 22, 5 to 32.5 units/kg administered weekly, 25 to 35 units/kg administered weekly, 22.5 to 37.5 units/kg administered weekly, 30 to 40 units/kg administered weekly, 27.5 to 32.5 units/kg administered weekly, 22.5 to 27.5 units/kg administered weekly, 17.5 to 22.5 units/kg administered weekly , or 32.5 to 37.5 units/kg administered every week. Typically, the dose is 15-60 units/kg administered every other week, in particular 60 units/kg administered every other week. Dose adjustments may be made on an individual basis to achieve and maintain therapeutic goals.

[0096] The dose may be about 1.5 mg/kg or 1.5 mg/kg administered every other week. The dose may be about 0.75 mg/kg or 0.75 mg/kg given every week. Alternatively, the dose may be 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, from 0.75 to 1.25 mg/kg given every other week, 1.215 to 1.625 mg/kg given every other week, 1.25 to 1.75 mg/kg given every other week, 1.375 to 1.875 mg/kg given every other week, 1.5 to 2.0 mg/kg given every other week, 1.375 to 1.625 mg/kg given every other week, 1.125 to 1.375 mg/kg given every other week 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 dose may be 0.375 to 1.0 mg/kg given every week, 0.5 to 0.875 mg/kg given every week, 0.625 to 1.0 mg/kg given every week, 0, 5625 to 0.8125 mg/kg given every week, 0.5 to 0.75 mg/kg given every week, 0.4375 to 0.5625 mg/kg given every week, 0.375 to 0.625 mg /kg administered every week, 0.5625 to 0.8125 mg/kg administered every week, 0.625 to 0.875 mg/kg administered every week, 0.5625 to 0.9375 mg/kg administered every week, 0.75 to 1.0 mg/kg given every week, 0.6875 to 0.8125 mg/kg given every week, 0.5625 to 0.6875 mg/kg given every week, 0.4375 to 0.5625 mg/kg given every week or 0.8125 to 0.9375 mg/kg given every week. Typically, the dose is 15 mg/kg administered every second week, in particular by subcutaneous administration. Dose adjustments may be made on an individual basis to achieve and maintain therapeutic goals.

[0097] Any of the GCB/IFM and GCB/IFMT compositions described herein may be administered to a patient. The dose may be from about 90 to 180 units/kg administered every other week. The dose may be about 90 units/kg or 90 units/kg given every week. Alternatively, the dose may be 90 to 150 units/kg administered every other week, 110 to 160 units/kg administered every other week, 120 to 180 units/kg administered every other week, 120 to 150 units/kg kg administered every other week 90 to 120 units/kg administered every other week 100 to 130 units/kg administered every other week 110 to 140 units/kg administered every other week 120 to 150 units /kg administered every other week, 130 to 160 units/kg administered every other week, 140 to 170 units/kg administered every other week, or 150 to 180 units/kg administered every other week. Alternatively, the dose may be 90 to 110 units/kg administered every other week, 100 to 120 units/kg administered every other week, 110 to 130 units/kg administered every other week, 120 to 140 units/kg kg administered every other week, 130 to 150 units/kg administered every other week, 140 to 160 units/kg administered every other week, 150 to 170 units/kg administered every other week, or 160 to 180 units/kg administered every other week.

[0098] The dose may be about 2.25 mg/kg or 4.5 mg/kg administered every other week. Alternatively, the dose may be 2.25 to 3.75 mg/kg administered every other week, 2.75 to 4.0 mg/kg administered every other week, 3.0 to 4.5 mg/kg administered every other week, 3.0 to 3.75 mg/kg administered every other week, 2.25 to 3.0 mg/kg administered every other week, 2.5 to 3.25 mg/kg kg given every other week, 2.75 to 3.5 mg/kg given every other week, 3.0 to 3.75 mg/kg given every other week, 3.25 to 4.0 mg /kg administered every other week, 3.5 to 4.25 mg/kg administered every other week, or 3.75 to 4.5 mg/kg administered every other week. Alternatively, the dose may be 2.25 to 2.75 mg/kg administered every other week, 2.5 to 3.0 mg/kg administered every other week, 2.75 to 3.25 mg/kg administered every other week, 3.0 to 3.5 mg/kg administered every other week, 3.25 to 3.75 mg/kg administered every other week, 3.5 to 4.0 mg/kg kg administered every other week, 3.75 to 4.25 mg/kg administered every other week, or 4.0 to 4.5 mg/kg administered every other week.

[0099] Administration of the GCB/IFM and GCB/IFMT compositions may be undertaken to treat a disorder associated with dysfunction of the GCase pathway, such as lysosomal storage diseases. Typical lysosomal storage diseases include Gaucher's disease, Fabry's disease, Pompe's disease, mucopolysaccharidosis, and multiple system atrophy. The compositions described herein are particularly suitable for the treatment of Gaucher's disease. The disorder may be a neurodegenerative disorder, such as Parkinson's disease, Alzheimer's disease, or dementia with Lewy bodies. Alternatively, the disorder may include dysregulation of alpha-synuclein.

[00100] In the treatment of the disorder, the GCB/IFM and GCB/IFMT compositions may be administered intravenously or subcutaneously. Subcutaneous administration includes subcutaneous injection, which is particularly suitable for carrying out the invention. Various dosing regimens may be used to administer the compositions. For example, the composition may be administered once a week, once every two weeks, or once a month. The composition can be administered, for example, once every three days, every four days, every five days, every six days, every eight days, every nine days, every 10 days, every 11 days, every 12 days, every 13 days, every 15 days, or every 16 days. The frequency of administration can be changed throughout the course of treatment due to various factors. Typically, the compositions described herein are administered by subcutaneous injection either once or twice a week, or once every other week.

[00101] If the compositions described herein are for subcutaneous administration, care should be taken to minimize patient discomfort during administration. Therefore, as a rule, the total volume administered to the patient per injection does not exceed 5 ml. More typically, the subcutaneously injected volume will be less than 2.5 ml per injection. If multiple subcutaneous injections are required to achieve a therapeutically effective dose, they may be given at different sites. Alternatively, the treatment interval may be reduced. Dose adjustments may be made on an individual basis to achieve and maintain therapeutic goals.

EXAMPLES

[00102] The present invention is also described and demonstrated using the following examples. However, the use of these and other examples anywhere in the description is illustrative only and in no way limits the scope and meaning of the invention or any exemplary term. In addition, the present invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this description, and such variations may be made without departing from the spirit of the invention, in spirit or scope. Therefore, the invention is to be limited only by the terms of the appended claims together with the full scope of equivalents to which those claims are entitled.

Example 1: GS concentration

[00103] HCB (5 ml 10 mg/ml) was thawed after storage at -80°C. The GCB was then concentrated by centrifugal filtration at 3800 rpm, 4° C. for 30 minutes. Then GCB was diluted 50 times and the concentration was measured at A280. Received the concentration of GCB 100 mg/ml. Then 1% polysorbate 20 was added to a final concentration of 0.1%. To some solution was added 20 mg of pH-adjusted isophagomine.

[00104] Filtration was carried out through a membrane with a pore size of 0.22 μm. The specific process is shown in FIG. 1.

Example 2: Addition of IFM, without adjusted pH, destabilizing GCB

[00105] SDS-EPAG was used to analyze various GB samples as shown below. The samples were denatured at 37°C for 15 minutes. SDS-EPAG was run on an 8-16% gel pre-treated with Novex™ trixglycine. Dithiothretiol (50 mmol) was used as a reducing agent. Isophagomine was added to some samples, the pH of which was not adjusted. The results obtained on the first day are shown in FIG. 2A:

Lane 1: Molecular weight markers

Lane 2: 0.5% assay control (60 ng HCB)

Lane 3: 1% assay control (120 ng HCB)

Strip 4: 12 µg reference GB, reconstituted

Strip 5: 12 mcg GCB (4°C) Day 1, not reconstituted

Strip 6: 12 µg GCB (4°C) Day 1, reconstituted

Strip 7: 12 mcg HCB (4°C) Day 1 with 12 mcg isophagomine, not reconstituted

Strip 8: 12 mcg HCB (4°C) Day 1 with 12 mcg isophagomine, reconstituted

Strip 9: 12µg GCB (-80°C) Day 1, not reconstituted

Strip 10: 12µg GCB (-80°C) Day 1, reconstituted

Strip 11: 12 mcg HCB (-80°C) Day 1 with 12 mcg isophagomine, not reconstituted

Strip 12: 12 mcg GCB (-80°C) Day 1 with 12 mcg isophagomine, reconstituted

[00106] The results after two weeks are shown in FIG. 2B:

Lane 1: Molecular weight markers

Lane 2: 0.5% assay control (60 ng HCB)

Lane 3: 1% assay control (120 ng HCB)

Strip 4: 12 µg reference GB, reconstituted

Strip 5: 12µg GCB (4°C) Week 2, not reconstituted

Strip 6: 12µg GCB (4°C) Week 2, reconstituted

Strip 7: 12 mcg HCB (4°C) Week 2 with 12 mcg isophagomine, not reconstituted

Strip 8: 12 mcg GCB (4°C) Week 2 with 12 mcg isophagomine, reconstituted

Strip 9: 12µg GCB (-80°C) Week 2, not reconstituted

Strip 10: 12µg GCB (-80°C) Week 2, reconstituted

Strip 11: 12µg HCB (-80°C) Week 2 with 12µg isophagomine, not reconstituted

Strip 12: 12µg HCB (-80°C) Week 2 with 12µg Isophagomine, reconstituted

[00107] The concentration procedure itself can cause cysteine-related oligomerization of GCB, as indicated by weak bands from 150 kDa to 200 kDa in lanes 4-12, which may include about 0.5% of the total protein. When adding isophagomine to GCB at 4°C, there were noticeably more fragments of GCB than when adding isophagomine to GCB at -80°C, as shown by several faint bands at sizes less than 50 kDa in lanes 7 and 8 in FIG. 2A The appearance of weak bands may be associated with the destabilization of GCB by acidic isophagomine.

[00108] The addition of isophagomine gave GCB fragments at 4°C but not at -80°C. See lanes 5-8 in FIG. 2A and 2B.

Example 3 pH Adjustment of IPMT and Subsequent Lyophilization

[00109] When IPMT is dissolved in water, an acidic solution is formed. In particular, when 103 mg of isophagomine tartrate is dissolved in 5 ml of water, the pH of the solution is 3.25. The pH was adjusted to 6.0 by adding 15 μl of 10 M sodium hydroxide to the solution.

[00110] Aliquots of 500 μl of pH-adjusted IPMT solution were added to 2 ml Eppendorf tubes. Eppendorf tubes containing solutions were frozen on dry ice for one hour, covered with Parafilm, which was pierced with a needle and lyophilized overnight. Eppendorf tubes with lyophilisates are shown in FIG. 3.

Example 4: Addition of HCB to pH Adjusted IFM

[00111] Before adding HCB to the IPMT adjusted to pH 6.0, the pH of the HCB solution is also adjusted to 6.0 with sodium citrate. In particular, 100 mg/ml HCB in 50 mmol sodium citrate gives a solution with a pH of 6.0. When 100 mmol/ml IPMT (at pH 6.0) was added to 100 mg/ml HCB in 50 mmol sodium citrate, the pH was 6.0.

Example 5 Addition of pH Adjusted IFM Non-Destabilizing GCB

[00112] SDS-EPAG was used to analyze multiple samples of GCB obtained on the same day (Day 0) and after three days of storage (Day 3), as shown below, which contain GCB added to pH adjusted isophagomine. The samples were denatured at 37°C for 15 minutes. SDS-EPAG was run on an 8-16% gel pre-treated with Novex™ trixglycine. Dithiothretiol (50 mmol) was used as a reducing agent. Isophagomine was added to some samples, the pH of which was not adjusted.

Lane 1: Molecular weight markers

Lane 2: 0.5% assay control (60 ng HCB)

Lane 3: 1% assay control (120 ng BMK)

Strip 4: 12 µg of reference GS, not reconstituted

Strip 5: 12µg GCB at 100mg/mL, not reconstituted

Lane 6: 12 µg GCB from 100 mg/ml, reconstituted

Lane 7: 12µg GCB from 100mg/mL with 12µg pH-adjusted isophagomine, not reconstituted

Lane 8: 12 µg GCB from 100 mg/mL with 12 µg pH-adjusted isophagomine, reconstituted

[00113] SEC HPLC was performed on day 3 samples to confirm stability. Parameters included Gibco FSBD with 400 mmol sodium chloride as mobile phase, flow rate 0.8 ml/min, Sepax Zenix-C SEC-150. 3 µm, 150 A, 7.8 x 300 mmol as SEC column and column temperature 25°C.

[00114] The results are shown in FIG. 4A for Day 0 and FIG. 4B for Day 3. The GS band is visible on bands 4-8. No smaller bands below 50 kD were observed on either Day 0 or Day 3. Adjusting the pH of isophagomine to 6.0, which is similar to that of GCB, can minimize destabilization of GCB.

Example 6: Analysis of the effect of pH-adjusted IFMT on the stability of GCB

[00115] GCB was concentrated to 100 mg/ml. The pH of 100 mg/ml isophagomine tartrate (IPMT) was adjusted to 6.0. IPMT was then mixed with GCB. When no IPMT pH adjustment was made, GCB/IPMT was not stable and protein cut-off was observed with SDS-EPAG.

[00116] When the pH was adjusted, the HCB in solution was stable for at least three days as measured by SEC. Mobile phase was Gibco PBS supplemented with 400 mmol sodium chloride, flow rate was 0.8 ml/min, ECR column was Sepax Zenic-C SEC-150, 3 μm, 150 A, 7.8 x 300 mmol. The column temperature was 25°C. Four samples were analyzed by SEC as shown in FIG. 5. GS in DS buffer, reference GS 98.8% and GS 98.7% were used as standards. Isophagomine-neutralized HCB (pH adjusted to 6.0) that had been stored for three days was also analyzed by SEC and appeared to be stable.

Example 7 Size Exclusion Chromatography Analysis of the Effect of pH Adjusted IFM on GCB Stability.

[00117] ECR separation analysis was performed on at least the following samples listed in Table 1 below:

[00118] The following parameters were used for ECR: Gibco PBS supplemented with 400 mmol sodium chloride was used as the mobile phase. The flow rate was 0.8 ml/min. The ECR column was a Sepax Zenix-C SEC-150. 3 µm, 150 A, 7.8 x 300 mmol. The column temperature was 25°C.

[00119] The results are shown in Figures 6A and 6B. Peptide fragments observed with SDS-EPAG appear in a peak eluting after about 10 minutes and 30 seconds to 10 minutes and 45 seconds. The sample at -80°C with isophagomine and GCB has a smaller peak associated with peptide fragments than the sample at 4°C or even the GCB sample at -80°C.

Example 8: Effect of GCB and IFM Concentration on Viscosity

[00120] Different compositions of (a) GCB and (b) GCB mixed with isophagomine (GCB/IFM) were prepared. GCB formulations included 10 mg/ml GCB, 25 mg/ml GCB, 50 mg/ml GCB, 75 mg/ml GCB, and 100 mg/ml GCB. GCB/IFM compositions included 10 mg/ml GCB with 5 mg/ml IFM tartrate, 25 mg/ml GCB with 12.5 mg/ml IFM tartrate, 50 mg/ml GCB with 25 mg/ml IFM tartrate, 75 mg/ml GCB with 37.5 mg/ml IFM tartrate and 100 mg/ml GCB with 50 mg/ml IFM tartrate. The viscosity and shear rate of each composition was measured in a viscometer (m-VROC from RheoSense, San Ramon, CA, USA). Approximately 200 µl of samples are required for each measurement. Viscosity results are shown with an R-square slope of >0.98. The results are shown in the following tables:

[00121] Viscosity correlates with the concentration of HCB. The composition with 100 mg/ml GCB and 50 mg/ml IFM tartrate has a viscosity of about 5 cps, which is amenable to subcutaneous injection.

Example 9 Binding of IFM to GCB at pH 7.4 and pH 5.0 measured with Biacore

[00122] Experiments were performed to characterize the affinity and binding kinetics of GCB and isophagomine at pH 7.4 and 5.0 using surface plasmon resonance (SPR). These pH values can illustrate how GCB and isophagomine bind to each other in different environments such as plasma, cytoplasm and lysosome compartments that have different pH values.

[00123] All SPR experiments were performed on Biacore S-200 by the single cycle kinetics method. For experiments at pH 7.4, 2 mg/mL GCB was diluted in GE acetate buffer pH 5.0 to a final concentration of 100 µg/mL. Immobilization working buffer was 10 mM HEPES, 5 mM EDTA, 0.01% P-20, pH 7.4. This buffer was subsequently used directly in the binding assay.

[00124] For experiments at pH 5.0, 2 mg/ml GCB was diluted in GE acetate buffer pH 5.0 to a final concentration of 100 μg/ml. The immobilization working buffer was 20 mmol sodium phosphate, 2.7 mmol potassium chloride, 137 mmol sodium chloride, 5 mmol tartrate, 0.01% P-20, pH 5.0. Tartrate was added to running buffer to eliminate the solute effect introduced by isophagomine tartrate. GCB was immobilized on the CM5 chip using the usual imine addition procedure. The target level of immobilization is 4000 RU.

[00125] For Biacore experiments at pH 7.4, the concentration range was 0.39-100 nmol. In total there were 9 points with 2-fold serial dilution. For Biacore experiments at pH 5.0, the concentration range was 0.39-100 nmol. In total there were 9 points with 2-fold serial dilution.

[00126] The conditions for the binding assay were as follows: flow rate 30 μl/min, association time 120 s, dissociation time 600 s, and 3 M magnesium chloride as regeneration reagent. Isophagomine concentrations ranging from 0.3 to 100 μmol were passed through immobilized velaglucerase alfa in a single cycle mode without surface regeneration.

[00127] For each of the pH 5.0 and pH 7.4 studies, two replicates were performed. The data obtained are presented in the following tables and in FIG. 7A-7D. The black lines represent the actual data and the red lines represent the fit of the model.

[00128] The K _D of GCB/IPM binding at pH 5.0 is 198-251 nmol. K _D binding GCB/IFM at pH 7.4 is 6.4-9.4 nmol.

Example 10 Elevation of the Melting Point of Velaglucerase Alfa by Isophagomine

[00129] The thermal stability of velaglucerase alone or in combination with various ratios of isophagomine was assessed using nano-differential scanning fluorometry (nano-DSF) (FIG. 8). Samples were initially prepared at the indicated molar ratio of isophagomine at a velaglucerase alfa concentration of 40 mg/mL. Samples were diluted to velaglucerase alpha concentrations of 2 mg/mL prior to loading into the nano-DSF apparatus. The sample conditions listed in FIG. 8, the following:

Control: isophagomine D-tartrate (IFMT) absent

Sample 1: 100x IFMT molar ratio

Sample 2: 30x IFMT molar ratio

Sample 3: 10x IFMT molar ratio

Sample 4: 3x molar ratio of IPMT

Sample 5: IFMT is absent

Sample 7: 100x molar ratio of isophagomine hydrochloride

Sample 8: 100x molar ratio of isophagomine acetate

[00130] Binding of isophagomine to velaglucerase alfa was also determined using GCB enzyme activity assay. Enzymatic reactions were carried out for 1 hour at 37°C. Isophagomine tartrate was pre-incubated with velaglucerase alfa for about 10 minutes.

[00131] Assay concentrations for isophagomine tartrate are as shown in the graphs in FIG. 9A-9C. Final concentrations for velaglucerase alfa assay were ~1 nmol at pH 5.0 and ~10 nmol at pH 7.4. FIG. 9A shows the activity inhibition curve with the pNP-GPS synthetic colorimetric substrate. FIG. 9B shows the activity inhibition curve with the 4MU-GPS synthetic fluorometric substrate. FIG. 9C shows inhibition of activity by natural glycosphingolipid substrate C12-GluCer. C12-GluCer cleavage response was assessed by measuring glucose production using a glucose oxidase assay kit.

Example 11 Stability Study after Three Weeks

[00132] Four different mixtures of GCB and isophagomine D-tartrate were prepared as shown in Table 8 below.

[00133] At the initial time point and after storage for three weeks at 40°C, the specific activity and purity measured by each of the SEC, RP-HPLC and SDS-EPAG were analyzed. SEC can detect soluble macromolecular species, while RP-HPLC provides information on the chemical stability of GCB, such as oxidation stability. SDS-EPAG can detect clipping and protein aggregation. For specific activity, the activities of the reference standard were 16 µmol/min/mg (day 0) and 18 µmol/min/mg (week 3). Significant daily variability was observed in the analysis of activity based on fluorescence. All stability samples had slightly higher activity than the reference standard.

[00134] A visual inspection was also performed. Sample images are shown in FIG. 10A. The data are presented in the tables below. In the SDS-EPAG results shown in FIG. 10B, the following strips correspond to the above samples:

Lane 1: Molecular weight markers

Strip 2: 12 µg of reference GS, not reconstituted

Strip 3: 12 µg Group 1 GS, not reconstituted

Strip 4: 12 µg Group 2 GS, not reconstituted

Strip 5: 12 µg Group 3 GS, not reconstituted

Strip 6: 12 µg Group 4 GS, not reconstituted

[00135] The 1:1 molar ratio of GCB to IFM was too low to be stable for three weeks at 40°C. In SDS-EPAG analysis, aggregates were visible and the solution appeared cloudy after three weeks. However, at a molar ratio of GCB to IFM of 1:3 or higher, after three weeks the purity was at least 98% in SDS-EPAG and the solutions appeared clear.

Example 12 Pharmacokinetic study of intravenous GCB and subcutaneous GCB with IFM in cynomolgus monkeys

[00136] Two groups of cynomolgus monkeys were tested for the pharmacokinetics of GCB. In Group 1, GCB was administered once by intravenous injection. In Group 2, the composition of GCB with IFM was administered once by subcutaneous injection. Three samples from the liver and spleen were taken every 1 hour, 2 hours, 8 hours and 24 hours after dosing. Additional details of the study design are provided in Table 13 below:

[00137] Liver and spleen sampling was performed at 1, 2, 8, and 24 hours (n=3) post-dose.

[00138] Histological analysis of all samples was then performed. Liver and spleen fixed with 10% NBF were treated for paraffin blockade. 5 micron sections were prepared for HCB IHC (primary antibody TK36 mouse anti-hGCB at a ratio of 1:10,000) and stained with hematoxylin and eosin.

[00139] FIG. 11 shows negative and positive controls for HCB IHC staining on monkey tissues in liver and spleen. Slight staining was seen in the absence of GCB IHC antibody (upper panels). In the presence of the GCB IHC antibody, faint background staining was observed in untreated liver (lower left panel). Dark staining was observed in treated liver and spleen in the presence of GCB IHC antibody (lower middle and lower right panels). In particular, the liver showed positive staining in Kupffer cells, endothelium and hepatocyte for GCB, and the spleen showed positive staining of endothelium and macrophages.

[00140] The biodistribution of GCB in the liver was studied after delivery of GCB by intravenous injection and GCB with IFM by subcutaneous injection. In monkey liver treated by subcutaneous injection, strong GCB was seen at 8 hours. Strong staining of GCB was observed at time points of one and two hours in the liver of a monkey receiving intravenous injection. The results are shown in FIG. 12 (2x magnification) and FIG. 13 (20x magnification).

[00141] Similar results were observed in the spleen, as shown in FIG. 14 (2x magnification) and FIG. 15 (20x magnification). These data indicate that subcutaneous administration of GCB with IFM can provide a comparable effect of GCB on tissues as intravenous administration of GCB.

Example 13: Correlation of velaglucerase alfa activity with liver and spleen protein levels

[00142] Protein and velaglucerase alpha activity levels were assessed in liver and spleen homogenates following intravenous dosing in cynomolgus monkeys. Tissues were collected at predetermined time points post-dose (0.5-24 hours). The results are shown in FIG. 16A and 16B. In particular, in FIG. 16A shows the results of intravenous administration of velaglucerase alfa only in the range of 2-10 mg/kg. FIG. 16B shows the results of SC administration of velaglucerase alfa in the range of 1.5-10 mg/kg combined with an appropriate amount of isophagomine (0.0075-5 mg/kg) such that the molar ratio of velaglucerase alfa to isophagomine is 1:3.

Example 14 Serum GCB Activity Levels in Cynomolgus Macaques Following Subcutaneous Administration of Isophagomine Tartrate

[00143] Serum GCB activity levels were analyzed in cynomolgus monkeys following subcutaneous (SC) administration of velaglucerase alfa with isophagomine tartrate. The data is shown in FIG. 16C. Endogenous serum GCB activity was determined in carrier and pre-dose animals (n=39) treated with GCB and ranged from 4-14 ng/mL or 0.07-0.25 nmol 4 IU/min/mL. Isophagomine tartrate may increase serum HCB activity above the upper limit of normal at a SC dose of 2.5 mg/kg. This increase in serum activity is probably due to the prevention of the degradation processes of native GCB that constantly occur in the serum. The dose of isophagomine tartrate included in Vela-3xIFMT (0.0225 mg/kg) will not increase endogenous GCB serum activity based on the higher dose data of 0.025 mg/kg.

Example 15: IFM provides >25-fold increase in subcutaneous exposure to serum velaglucerase alfa

[00144] Velaglucerase alfa and IFM at a 1:3 molar ratio administered subcutaneously to cynomolgus monkeys at a dose of 4 mg/kg were able to provide more than a 25-fold improvement in serum exposure compared to 4 mg/kg intravenous velaglucerase alfa . IFM ratios from 3-fold to 100-fold molar excess compared to velaglucerase alfa resulted in a similar increase in serum exposure. The increase in serum bioavailability determined from the ECL ELISA assay was confirmed by the GCB activity assay (4MU-GPS substrate). The results are shown in FIG. 17A and 17B. The addition of 0.07 mg/kg IPMT to 4 mg/kg GCB significantly increased the amount of GCB in serum (16A) as well as the total enzymatic activity of GCB (16B). Thus, the data demonstrates that when GCB is combined with an IFM, e.g., IFMT, especially in a molar ratio of at least 1:3 (GCB:IFM, e.g., GCB:IFMT), it can provide serum bioavailability that allows SC administration.

Example 16: Excellent tissue biodistribution with subcutaneous VPRIV with IFM in a 1:100 molar ratio compared to intravenous VPRIV alone

[00145] Velaglucerase alfa and IFM at a molar ratio of 1:100 administered subcutaneously to cynomolgus monkeys at a dose of 4 mg/kg were able to provide tissue uptake of velaglucerase alfa that exceeded the intravenous dose of velaglucerase alfa of 10 mg/kg. The standard of care for the IV infusion dosage of VPRIV is 1.5 mg/kg. In some embodiments, a target subcutaneous dose of about 1.5 mg/kg may be used. About 250 mg of tissue was homogenized in 1 ml of HEPES/Triton X-100 lysis buffer. Tissue velaglucerase alfa content was measured by ECL ELISA assay and normalized to total protein content as determined by BHA assay. The results are shown in FIG. 18A and 18B. The exposure profile of GCB present in the liver (18A) and spleen (18B) after subcutaneous administration of a molar ratio of GCB:IFM of 1:100 was higher than with intravenous administration of GCB for 5 days (120 hours).

Example 17: Comparison of Tissue Biodistribution of Subcutaneous VPRIV with IFM in a 1:3 Molar Ratio with IV VPRIV Only

[00146] Velaglucerase alfa and IFM at a 1:3 molar ratio administered subcutaneously to cynomolgus monkeys at a clinical target dose of 1.5 mg/kg were able to provide tissue uptake of velaglucerase alfa comparable to that of 2 mg/kg intravenously. inject velaglucerase alfa. The standard of care for the IV infusion dosage of VPRIV is 1.5 mg/kg. About 250 mg of tissue was homogenized in 1 ml of HEPES/Triton X-100 lysis buffer. Tissue velaglucerase alfa content was measured by ECL ELISA assay and normalized to total protein content as determined by BHA assay. The results are shown in FIG. 19A and 19B. The amount of HCB present in the liver after subcutaneous administration of a molar ratio of HCB:IFM of 1:3 is comparable to that of intravenous HCB at time points of 8 and 24 hours. Similarly, the effect of GCB on tissues in the spleen after subcutaneous administration of a molar ratio of GCB:IFM of 1:3 is comparable to that with intravenous administration of GCB at time points of 8 and 24 hours.

[00147] Similarly, the effect of GCB on tissues in the spleen after subcutaneous injection of a molar ratio of GCB:IFM of 1:3 is comparable to that of intravenous administration of GCB at time points of 8 and 24 hours. Thus, the addition of an IFM, eg, IFMT, to a GCB composition allows subcutaneous administration of the compositions containing the GCB.

Example 18: Ratio of Isophagomine as low as 1:1, provides the same effect on serum as a higher molar ratio of Isophagomine

[00148] Velaglucerase alpha and IFM in a 1:1 molar ratio administered subcutaneously to cynomolgus monkeys at 1.5 mg/kg are able to provide the same serum GCB exposure as higher ratios of isophagomine. For molar ratios between 1:1 and 1:100, there was no apparent difference in the effect of HCB on serum. The increase in serum bioavailability determined from the ECL ELISA assay was confirmed by the GCB activity assay (4MU-GPS substrate). The results are shown in FIG. 20A and 20B.

[00149] Test specimens for administration were prepared as frozen compositions in vivarium prior to administration. The test samples were thawed from about 1 to 3 hours before administration. Thus, the data show that while the obligation to store at room temperature can be circumvented by storage at low temperatures (e.g., frozen), then when GSB is mixed with an IPM, e.g., IPMT, especially in a molar ratio of at least 1: 1 (GCB:IFM, eg GCB:IFMT), it can provide sufficient serum bioavailability to allow subcutaneous administration.

Example 19 Isophagomine Protects VPRIV from Thermal Denaturation at 37° C. in Human Serum

[00150] Serum that contained 10 nmol VPRIV (a form of GCB) was tested to determine if IFM could stabilize GCB. IFM was added to VPRIV such that IFM had the following serum IFM concentrations: 1 nmol, 3 nmol, 10 nmol, 30 nmol, 100 nmol, 300 nmol and 1000 nmol. A negative control without the addition of IFM was used.

[00151] Enzyme activity was measured by cleaving the substrate 4-methylumbelliferone b-D-glucopyranoside. Activity decreased from 100% to about 40% within 60 minutes with the negative control, 1 nmol IFM and 10 nmol IFM. See FIG. 21. However, the addition of concentrations of 30 nmol (3 times molar ratio) and above prevented most of the loss of activity. IFM may be effective in protecting HCB from heat denaturation in serum. IFM- and IPMT-provided protection of GCB from thermal degradation can increase the bioavailability of GCB, increase the stability of GCB in serum and provide a longer duration of GCB uptake by cells and tissues.

***

[00152] The present invention should not be limited in scope to the specific embodiments described in this application. Indeed, various modifications of the present invention, in addition to those described in this application, will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be within the scope of the appended claims. It is further to be understood that all values are approximate and are intended to be descriptive.

[00153] All publications, patent applications, patents and other references mentioned in this document are incorporated by reference in their entirety. In the event of any conflict, this application, including definitions, shall prevail. In addition, materials, methods, and examples are provided for illustration only and are not intended to be limiting.

Claims (73)

1. A composition for the treatment of GCase pathway dysfunction comprising glucocerebrosidase (GCB) and isophagomine (IPM) in a molar ratio of at least about 1:2.5, wherein the composition is intended to be administered subcutaneously. 2. Composition according to claim 1, characterized in that GCB is alpha-velaglucerase. 3. A composition according to claim 1 or 2, characterized in that the pH of the composition is about 6.0, about 6.5, or about 7.0. 4. The composition according to any one of paragraphs. 1-3, characterized in that the molar ratio of GCB to IFM is from about 1:2.5 to about 1:30. 5. The composition according to any one of paragraphs. 1-3, characterized in that the molar ratio of GCB to IFM is from about 1:2.5 to about 1:10. 6. Composition according to any one of paragraphs. 1-3, characterized in that the molar ratio of GCB to IFM is from about 1:10 to about 1:30. 7. The composition according to any one of paragraphs. 1-3, characterized in that the molar ratio of GCB to IFM is from about 1:2.5 to about 1:3.5. 8. The composition according to any one of paragraphs. 1-3, characterized in that the molar ratio of GCB to IFM is about 1:3.0. 9. The composition according to any one of paragraphs. 1-3, characterized in that the molar ratio of GCB to IFM is 1:3.0. 10. The composition according to any one of paragraphs. 1-9, characterized in that the temperature of the composition is at least 20°C. 11. Composition according to any one of paragraphs. 1-9, characterized in that the temperature of the composition is from 0°C to 20°C. 12. The composition according to any one of paragraphs. 1-9, characterized in that the temperature of the composition is less than 0°C. 13. The composition according to any one of paragraphs. 8-12, characterized in that the composition is an aqueous solution. 14. The composition according to any one of paragraphs. 1-9 and 12, characterized in that the composition is a lyophilisate. 15. The composition according to any one of paragraphs. 1-14 further comprising a pharmaceutically acceptable excipient, a pharmaceutically acceptable salt, or both a pharmaceutically acceptable excipient and a pharmaceutically acceptable salt. 16. The composition according to any one of paragraphs. 1-15, characterized in that the IFM is isophagomine tartrate. 17. Composition according to claim 16, characterized in that the IFM is isophagomine D-tartrate. 18. Composition according to any one of paragraphs. 1-13 and 15-17, characterized in that the composition is a liquid. 19. The composition according to any one of paragraphs. 1-18, additionally containing an antioxidant. 20. The composition according to any one of paragraphs. 1-19, additionally containing carbohydrate. 21. The composition according to any one of paragraphs. 1-20, additionally containing a surfactant. 22. The composition according to any one of paragraphs. 1-21, characterized in that the composition contains 45-120 mg/ml GCB and from 0.2 to 1.8 mg/ml isophagomine. 23. Composition according to claim 22, characterized in that the composition contains 60 mg/ml GCB and 0.9 mg/ml isophagomine. 24. Composition according to claim 22 or 23, characterized in that the composition additionally contains 50 mmol of sodium citrate or sodium phosphate and 0.01% of polysorbate-20. 25. Composition according to claim 24, characterized in that the composition contains 5-20 mmol sodium citrate and 0.01% polysorbate-20. 26. Composition according to claim 25, characterized in that the composition contains 10 mmol sodium citrate and 0.01% polysorbate-20. 27. Composition according to claim 24, characterized in that the composition contains 5-20 mmol of sodium phosphate and 0.01% of polysorbate-20. 28. Composition according to claim 27, characterized in that the composition contains 10 mmol sodium phosphate and 0.01% polysorbate-20. 29. The composition according to any one of paragraphs. 22-28, characterized in that the pH of the composition is about 6.0. 30. The composition according to any one of paragraphs. 22-28, characterized in that the pH of the composition is 6.0. 31. A container containing a composition according to any one of paragraphs. 1-30. 32. The container according to claim 31, characterized in that the container is selected from the group consisting of a pre-filled syringe, vial or ampoule. 33. The method of obtaining a composition according to any one of paragraphs. 1-30, including dissolving IFM in water, bringing the pH to about 6.0 and adding GCB to obtain a composition. 34. The method according to p. 33, further comprising lyophilization of IFM before adding HCB. 35. The method of claim 33 or 34, further comprising adding 0.01% polysorbate 20. 36. The method according to any one of paragraphs. 33-35, further comprising filtering the composition through a membrane with a pore size of 0.22 microns. 37. The method according to any one of paragraphs. 33-36, characterized in that the IFM is present in an amount sufficient to maintain the stability of the GCB in the composition. 38. The method according to any one of paragraphs. 33-36, characterized in that the IFM is present in an amount sufficient to maintain the stability of the GCB in the composition for at least three days at 0-50°C. 39. The method according to any one of paragraphs. 33-36, characterized in that the IFM is present in an amount sufficient to maintain the stability of the GCB in the composition for at least 6 months at 0-40°C. 40. A method for treating dysfunction of the GCase pathway, comprising administering a composition according to any one of paragraphs. 1-11, 13 and 15-30 to the patient in need of it. 41. The method of claim 40, wherein the method is effective for treating the dysfunction. 42. The method according to p. 40, characterized in that the subcutaneous administration of the composition is carried out by subcutaneous injection. 43. The method according to any one of paragraphs. 40-42, characterized in that the composition is administered twice a week. 44. The method according to any one of paragraphs. 40-42, characterized in that the composition is administered once a week. 45. The method according to any one of paragraphs. 40-42, characterized in that the composition is administered less than once a week. 46. The method according to any one of paragraphs. 40-42, characterized in that the composition is administered once every two weeks. 47. The method according to any one of paragraphs. 40-46, characterized in that said dysfunction includes a defect in GCase activity. 48. The method of claim 47 wherein said defect in GCase activity includes reduced enzymatic activity. 49. The method according to any one of paragraphs. 40-46, characterized in that said dysfunction includes dysregulation of alpha-synuclein. 50. The method according to any one of paragraphs. 40-49, characterized in that said dysfunction is a lysosomal storage disease. 51. The method of claim 50 wherein said lysosomal storage disease is selected from Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidosis, and multiple system atrophy. 52. The method according to any one of paragraphs. 40-51, characterized in that said dysfunction is a neurodegenerative disorder. 53. The method of claim 52, wherein the neurodegenerative disorder is selected from Parkinson's disease, Alzheimer's disease, and dementia with Lewy bodies. 54. The method according to any one of paragraphs. 40-53, characterized in that the subject is a human. 55. A method for treating dysfunction of the GCase pathway, comprising administering to a subject a composition containing a dose of 0.5 to 5.0 mg/kg of GCB, and IFM in at least about 3-fold molar excess relative to the GCB, in which the composition is administered subcutaneously . 56. The method according to p. 55, characterized in that the dose contains from 0.8 to 4.0 mg/kg GCB. 57. The method according to p. 56, characterized in that the dose contains from 1 to 3.0 mg/kg GCB. 58. The method according to p. 56, characterized in that the dose contains from 1.2 to 2.0 mg/kg GCB. 59. The method according to p. 56, characterized in that the dose contains about 1.5 mg/kg GCB. 60. The method according to p. 56, characterized in that the dose contains 1.5 mg/kg GCB. 61. The method according to p. 55, characterized in that the dose contains from 2.0 to 5.0 mg/kg GCB. 62. The method according to p. 55, characterized in that the dose contains from 2.25 to 4.5 mg/kg GCB. 63. The method according to p. 55, characterized in that the dose contains from 2.25 to 3.75 mg/kg GCB. 64. The method according to p. 55, characterized in that the dose contains from 3.5 to 5.0 mg/kg GCB. 65. The method according to any one of paragraphs. 56-64, characterized in that the IFM is in a 3 to 10-fold molar ratio to GCB. 66. The method according to any one of paragraphs. 56-64, characterized in that the IFM is in a 10 to 30-fold molar ratio to GCB. 67. The method according to any one of paragraphs. 56-64, characterized in that the IFM is in a 30 to 100-fold molar ratio to GCB. 68. The method according to any one of paragraphs. 56-64, characterized in that the IFM is in a 3-fold molar ratio to GCB. 69. The method according to any one of paragraphs. 40-68, characterized in that the impact, activity or bioavailability of GCB in the spleen is increased. 70. The method according to any one of paragraphs. 40-69, characterized in that the effect, activity or bioavailability of GCB in the liver increases. 71. The method according to any one of paragraphs. 40-70, characterized in that the effect, activity or bioavailability of GCB in serum increases. 72. The composition according to any one of paragraphs. 1-11, 13 and 15-30 for use in the method according to any one of paragraphs. 40-71. 73. The use of a composition according to any one of paragraphs. 1-11, 13 and 15-30 upon receipt of a medicinal product for the method according to any one of paragraphs. 40-72.

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