Denosumab preparations

Abstract

The aqueous pharmaceutical formulation with improved stability comprises denosumab and a poloxamer, and preferably a histidine buffer and / or a sugar or sugar alcohol. The formulation is for use in the treatment or prevention of skeletal-related events associated with osteoporosis, reduced bone mass, multiple myeloma, solid tumor bone metastasis, giant cell tumor of bone, or hypercalcemia.

Description

[Technical Field]

[0001] The present disclosure relates to an aqueous pharmaceutical formulation of denosumab. [Background technology]

[0002] Any discussion of related art throughout this specification should not be taken in any way as an admission that such art is widely known or forms part of the common general knowledge in the art.

[0003] Denosumab is a fully human monoclonal IgG antibody that binds with high affinity and specificity to receptor activator of NF-κB ligand (RANKL). Two commercially available denosumab products exist: Prolia® and Xgeva® (Amgen Corporation), which contain 60 mg / mL and 70 mg / mL denosumab, respectively. Both Prolia® and Xgeva® are parenteral formulations suitable for administration by subcutaneous injection. A lower-concentration denosumab product, Prolia®, is indicated for the treatment of osteoporosis on a dosing schedule of one dose every six months; each 1 mL dose contains 60 mg denosumab, 47 mg sorbitol, 1 mg acetate, 0.1 mg polysorbate 20, and sodium hydroxide to adjust to pH 5.2 in water for injection. Xgeva® is indicated for the treatment of skeletal events associated with cancer or cancerous tumors on a dosing schedule of one dose every four weeks, with each 1.7 mL dose containing 120 mg of denosumab, 78.1 mg of sorbitol, 1.8 mg of acetate, 0.17 mg of polysorbate 20, and sodium hydroxide to adjust the pH to 5.2 in water for injection.

[0004] Product stability is an essential quality attribute for pharmaceutical formulations that is important for both efficacy and safety, and is a regulatory requirement for approval by health authorities. It refers to the retention of chemical, physical, and biological properties and characteristics after manufacture, formulation, transportation, storage, and administration. Stability can be particularly challenging for biologics, which are prone to aggregation, degradation, oxidation, isomerization, adsorption, and denaturation, which can be accelerated by agitation and environmental stresses such as changes in pH and temperature.

[0005] Surfactants play a vital role in the stabilization of biopharmaceuticals, which may include protection from interfacial stresses, such as interface-induced aggregation, precipitation, and adsorption, resulting from exposure to liquid-air interfaces and contact with purification and other processing, storage, and delivery equipment. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO 2018 / 200918 Summary of the Invention [Problem to be solved by the invention]

[0007] One object of the present invention is to provide a stable formulation of denosumab. [Means for solving the problem]

[0008] Polyoxyethylene (PEO) surfactants, including polysorbates, have been identified as promising surfactants for use in biologics. However, the present inventors surprisingly found that polysorbates can be unstable and apparently caused particulates and opacity during the identification and evaluation of denosumab formulations, particularly when co-formulated with histidine buffers. Applicants have discovered that stable denosumab formulations can be produced using poloxamer surfactants, particularly when used in combination with histidine buffers.

[0009] According to a first aspect, the present disclosure relates to an aqueous pharmaceutical formulation comprising 50-80 mg / mL denosumab, 200-300 mM sugar, 1-10 mM histidine buffer, 0.005-0.1% (w / v) poloxamer, and water, and having a pH of 5.5-6.5. In one embodiment, the aqueous pharmaceutical formulation comprises 55-65 or 65-75 mg / mL denosumab, 230-250 mM sugar, 4-6 mM histidine buffer, 0.01-0.05% (w / v) poloxamer, and water, and has a pH of 6.0-6.4. In a further embodiment, the aqueous pharmaceutical formulation comprises 60 or 70 mg / mL denosumab, 240 mM sugar, 5 mM histidine buffer, 0.03% (w / v) poloxamer, and water, and has a pH of 6.2.

[0010] According to a second aspect, the present disclosure relates to an aqueous pharmaceutical formulation comprising denosumab and a poloxamer, the formulation optionally comprising a histidine buffer.

[0011] According to a third aspect, the present disclosure relates to a denosumab formulation for parenteral injection, comprising a sugar or sugar alcohol, a buffer, and a surfactant, wherein the surfactant is a poloxamer. In embodiments, the buffer is a histidine buffer.

[0012] According to a fourth aspect, the present disclosure relates to an aqueous pharmaceutical formulation comprising denosumab and a surfactant, wherein the formulation exhibits a decrease in surfactant concentration of 40% or less when stored at 25° C. for 1 month and / or a decrease in surfactant concentration of 50% or less when stored at 40° C. for 1 month.

[0013] According to a fifth aspect, the present disclosure relates to a formulation as described herein for use in the treatment or prevention of osteoporosis or reduced bone mass.

[0014] According to a sixth aspect, the present disclosure relates to a formulation as described herein for use in the treatment or prevention of skeletal-related events associated with multiple myeloma, solid tumor bone metastases, or giant cell tumor of bone.

[0015] According to a seventh aspect, the present disclosure relates to a formulation as described herein for use in the treatment or prevention of hypercalcemia.

[0016] According to an eighth aspect, the present disclosure relates to a method for treating or preventing osteoporosis or reduced bone mass, comprising administering to a subject in need of such treatment a formulation described herein.

[0017] According to a ninth aspect, the present disclosure relates to a method for treating or preventing skeletal-related events associated with multiple myeloma, solid tumor bone metastasis, or giant cell tumor of bone, comprising administering to a subject in need of such treatment a formulation as described herein.

[0018] According to a ninth aspect, the present disclosure relates to a method for treating or preventing hypercalcemia, comprising administering to a subject in need of such treatment a formulation described herein.

[0019] According to a tenth aspect, the present disclosure relates to the use of a formulation as described herein in the manufacture of a medicament for treating or preventing osteoporosis or reduced bone mass.

[0020] According to an eleventh aspect, the present disclosure relates to the use of a formulation as described herein in the manufacture of a medicament for treating or preventing skeletal-related events associated with multiple myeloma, solid tumor bone metastasis, or giant cell tumor of bone.

[0021] According to a twelfth aspect, the present disclosure relates to the use of a formulation described herein in the manufacture of a medicament for treating or preventing hypercalcemia.

[0022] The foregoing paragraphs have been provided by way of general introduction and are not intended to limit the scope of the claims that follow. The described embodiments, together with further advantages, will be best understood by reference to the following detailed description taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a graph showing surfactant concentration over time for several inventive and comparative denosumab formulations stored at 5° C. [Figure 2] 1 is a graph showing surfactant concentration over time for several inventive and comparative denosumab formulations stored at 25° C. [Figure 3] 1 is a graph showing surfactant concentration over time for several inventive and comparative denosumab formulations stored at 40° C. DETAILED DESCRIPTION OF THE INVENTION

[0024] Embodiments of the present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the disclosure are shown.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] As used herein, the words "a," "an," and the like, retain the meaning of "one or more." Furthermore, within the description of this disclosure, when numerical limits or ranges are set forth, the endpoints are included unless otherwise stated. Also, all values ​​and subranges within the numerical limits or ranges, including subranges excluding the endpoints, are specifically included as if expressly and fully recited.

[0027] As used herein, the terms "optionally" or "optionally" mean that the subsequently described event may or may not occur, or that the subsequently described ingredient may or may not be present (e.g., 0 wt %).

[0028] The term "comprising" is an open-ended term synonymous with terms such as "including," "containing," or "having," and is used herein to claim aspects of the invention that may include additional steps, components, functionality, and / or structures. The term "consisting essentially of" is used herein to claim additional, unrecited steps or components that would not materially affect aspects of the invention and the basic and novel properties of the formulations or methods according to the present disclosure. The basic and novel properties of the present disclosure include improved surfactant stability compared to known denosumab formulations. For formulations described herein, "consisting essentially of" serves to exclude the addition of unrecited active substances, sugars, buffers, surfactants, etc., but would allow for the addition of minor agents, such as pH adjusters, antioxidants, or preservatives. The term "consisting of" is used to claim aspects of the invention that include only those features explicitly recited in the claim, thus excluding other steps or components not explicitly or essentially included in the claim. For formulations described herein, "consisting of" excludes all unrecited active substances, sugars, buffers, surfactants, etc. For clarity, impurities at levels sufficiently low to be pharmaceutically acceptable, such as those incidental to pH adjustment, are not excluded.

[0029] It is expressly intended that the features of the various features of the formulations and methods described herein be used interchangeably and in combination. That is, to the extent that ingredients, ingredient amounts, or other parameters are mentioned with respect to one embodiment, it is expressly intended that such parameters be substituted as appropriate for use in other embodiments. The inventors contemplate such combinations and intend them to be encompassed by this disclosure, even if not fully described.

[0030] Hereinafter, unless otherwise indicated, "%" means "% (w / v)" calculated as [100 x mx / vtot] (mx is the mass of component x in g and vtot is the total volume of all components in mL).

[0031] As used herein, the term "substantially" shall mean, where appropriate, including more than 50% by weight, unless otherwise indicated.

[0032] The recitation of numerical ranges using endpoints includes those endpoints and all numbers subsumed within the range (e.g., 1 to 10 includes 1, 1.5, 2, 5, 5.75, 7.1, 8.5, 9, 10, etc.).

[0033] As used herein, the terms "about" and "approximately" are used interchangeably. Both terms are intended to cover any normal fluctuations or variations understood by one of ordinary skill in the art. In some embodiments, "about" and "approximately" refer to ±10% of a reference value, or ±5%, or ±2%, or ±1%, or ±0.1% of a reference value. The present disclosure is intended to encompass alternative embodiments in which numbers not modified by the term "about" or "approximately" are so modified.

[0034] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of denosumab that elicits a biological or medical response in a subject, e.g., inhibits a disease and its progression (e.g., inhibits a disease, condition, or disorder, e.g., prevents further development of the disease symptoms and / or symptomology in an individual experiencing or exhibiting the disease, condition, or disorder's symptoms or symptomology; and / or ameliorates a disease (e.g., reduces the symptoms, reverses the disease symptoms and / or symptomology in an individual experiencing or exhibiting the disease, condition, or disorder's symptoms or symptomology).

[0035] As used herein, the phrases "treating," "treat," and "treatment," etc., include inhibiting a disease and / or its progression (e.g., inhibiting a disease, condition, or disorder in an individual experiencing or exhibiting the symptoms or symptomology of the disease, condition, or disorder, e.g., arresting further onset of the symptoms and / or symptomology of the disease; and / or ameliorating a disease (e.g., reducing the symptoms, reversing the symptoms and / or symptomology of the disease) in an individual experiencing or exhibiting the symptoms or symptomology of the disease, condition, or disorder.

[0036] As used herein, the terms "preventing," "prevent," and "prevention," etc., include the prevention of the recurrence and / or onset of one or more symptoms of a disorder or disease, particularly in a subject analyzed as being susceptible to or likely to develop the disease.

[0037] As used herein, the phrase "subject in need thereof" means that the subject has been identified as in need of a treatment described herein.

[0038] As used herein, the terms "subject" and "patient" are interchangeable and can be taken to mean any living organism that can be treated with the compounds of the invention. Thus, the terms "patient" and "subject" can include human adults or children.

[0039] All references cited herein, including patents, patent applications, publications, and databases, whether or not previously specifically incorporated, are hereby incorporated by reference in their entirety.

[0040] The inventors have surprisingly discovered that certain formulations under development exhibit remarkable performance in surfactant concentration stability, i.e., prevention of surfactant degradation, during storage compared to Prolia® and other alternative denosumab formulations. The inventors believe that surfactant stability in denosumab formulations is positively affected by the use of a poloxamer surfactant, optionally in conjunction with a histidine buffer.

[0041] Described herein is an aqueous pharmaceutical formulation comprising 50-80 mg / mL denosumab, 200-300 mM sugar, 1-10 mM histidine buffer, 0.005-0.1% (w / v) poloxamer, and water. The formulation has a pH of 5.5-6.5. In one embodiment, the formulation comprises 55-65 or 65-75 mg / mL denosumab, 230-250 mM sugar, 4-6 mM histidine buffer, 0.01-0.05% (w / v) poloxamer, and water, and has a pH of 6.0-6.4. In a further embodiment, the aqueous pharmaceutical formulation comprises 60 or 70 mg / mL denosumab, 240 mM sugar, 5 mM histidine buffer, 0.03% (w / v) poloxamer, and water, and has a pH of 6.2.

[0042] The denosumab formulations described herein are suitable for pharmaceutical administration, e.g., parenteral administration, particularly subcutaneous injection, e.g., by syringe, pre-filled disposable syringe, or auto-injector.

[0043] water The primary component (or carrier) of the denosumab formulation is pharmaceutical-grade water. "Pharmaceutical-grade water" refers to water that meets at least the purity and quality requirements suitable for making parenteral denosumab formulations as set forth in the USP for "Water for Injection" (or "WFI"). As will be understood by those skilled in the art, WFI is potable water that has been purified to remove chemicals and microorganisms by distillation or a purification method equivalent to or superior to distillation. WFI contains no additives. Pharmaceutical-grade water can be sterilized before and / or after being combined with the other components of the denosumab formulation, but sterilization is not required.

[0044] The denosumab formulations herein contain several components dissolved and / or dispersed, typically completely dissolved, in pharmaceutical-grade water. These include denosumab, a sugar, a histidine buffer, and a poloxamer. In some embodiments, the denosumab formulations herein also contain a histidine buffer as the only buffering agent, excluding the antibody (denosumab).

[0045] Denosumab The formulations described herein include the antibody denosumab, the encoded amino acid sequence of which comprises 1326 amino acids, including, for example, the denosumab sequence disclosed in WO 2018 / 200918, the contents of which are incorporated by reference, including any acceptable variations or biosimilars thereof.

[0046] In embodiments, denosumab has the variable light chain sequence of denosumab:

[0047] [ka]

[0048] The amino acid sequence is generated from one or more vectors encoding the amino acid sequence comprising:

[0049] In a further embodiment, denosumab has the light chain sequence of denosumab:

[0050] [ka]

[0051] The amino acid sequence is generated from one or more vectors encoding the amino acid sequence comprising:

[0052] In a further embodiment, denosumab is provided with the variable heavy chain sequence of denosumab on the same or a separate vector as the variable light chain:

[0053] [ka]

[0054] The vectors are generated from one or more vectors encoding the

[0055] In a further embodiment, denosumab is provided with the heavy chain sequence of denosumab on the same or a separate vector as the light chain:

[0056] [ka]

[0057] The vectors are generated from one or more vectors encoding the

[0058] Because denosumab is produced by a biological process, certain minor variations may occur from batch to batch, including, but not limited to, "lysine clipping," in which the C-terminal lysine of the constant heavy chain is clipped. This variation is considered denosumab and has no clinically meaningful impact. As understood by those skilled in the art, a biosimilar refers to a biological product that is highly similar to a reference product (in this case, denosumab produced by Amgen Inc.) and has no clinically meaningful differences in efficacy and safety. The high degree of similarity between a biosimilar and a reference product generally includes physical, chemical, and biological properties, and minor differences that are tolerated are clinically insignificant. This understanding is used by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), as well as by pharmaceutical companies seeking marketing authorization for biosimilars of reference products. In the United States, biosimilars may be designated by an extension following the name of the reference product. For the avoidance of doubt, in the disclosure herein, all such extensions of denosumab should be considered within the scope of the term "denosumab" as used herein. The concentration of denosumab in the denosumab formulations described herein may be 50 mg / mL to 80 mg / mL, for example, 55 mg / mL to 65 mg / mL or 65 mg / mL to 75 mg / mL, and in particular, 60 mg / mL or 70 mg / mL.

[0059] sugar The denosumab formulations described herein preferably contain a sugar in an amount of 200-300 mM. The primary purpose of the sugar is to stabilize denosumab and to change the osmolality of the formulation to an isotonic level. The sugar can be any suitable sugar. In some embodiments, the sugar is selected from sucrose, trehalose, maltose, and lactose, individually or in any suitable combination of two or more. In one embodiment, the sugar is sucrose. Alternatively, or in addition, a sugar alcohol such as sorbitol may be used.

[0060] The sugar may be present in the denosumab formulations described herein at any suitable concentration between 200 and 300 mM. In some embodiments, the sugar is present at a concentration of 200 mM to 225 mM, or 225 mM to 250 mM, or 210 mM to 240 mM, or 235 mM to 245 mM, or 235 mM to 265 mM, or 250 mM to 275 mM, or 265 mM to 285 mM, or 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, or 300 mM. In some embodiments, sugars are present in the denosumab formulations described herein at a concentration of 200-250 mM. In one embodiment, the denosumab formulations herein contain about 240 mM sucrose.

[0061] Poloxamer The denosumab formulations described herein contain a poloxamer as a surfactant. Poloxamers are nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers can protect protein formulations from agitation-induced stress and / or exposure of proteins to surfaces and / or air-liquid interfaces. The term poloxamer as used herein is not intended to be particularly limiting. In certain embodiments, the poloxamer may be poloxamer 188, also known as P188, where the first two digits (18) are multiplied by 100 to obtain the approximate molecular mass of the polyoxypropylene core (i.e., 1800) and the last digit (8) is multiplied by 10 to obtain the percentage of polyoxyethylene content (i.e., 80%).

[0062] The denosumab formulations described herein include poloxamer in an amount of 0.005-0.1% (w / v) based on the total formulation. In some embodiments, the poloxamer is present in an amount of 0.005%-0.01%, 0.01%-0.05%, 0.05%-0.1%, 0.0075%-0.0125%, 0.01%-0.075%, 0.01%-0.04%, or 0.03%-0.08%, or in an amount of 0.005%, 0.0075%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% (w / v).

[0063] Osmotic pressure The denosumab formulations described herein have an osmolality within the parenterally acceptable range, generally between 200 and 600 mOsm / kg. Typically, the osmolality of the denosumab formulations described herein is within the range of 240 to 400 mOsm / kg. In some embodiments, the denosumab formulations have an osmolality within the range of 260 to 380 mOsm / kg, typically between 270 and 360 mOsm / kg. In other embodiments, the osmolality is within the range of 285 to 345 mOsm / kg. Typically, values ​​within these ranges can be achieved by balancing the type and amount of sugar, the amount of histidine buffer, and, to a lesser extent, the type and amount of poloxamer, taking into account the amount of denosumab in pharmaceutical-grade water. Osmolality can also be adjusted by adding additional agents, such as salts (e.g., NaCl).

[0064] pH The denosumab formulations described herein have a pH of 5.5 to 6.5. In embodiments, the pH is 5.6 to 5.8, 6.1 to 6.3, about 5.7 to about 6.2, or any subrange therein. In further embodiments, the pH is 5.7, about 5.7, 6.2, or about 6.2. The pH can be adjusted, if necessary, by adding a pH adjuster. In some embodiments, the pH adjuster can be an acid or a base. In some embodiments, the acid is HCl or acetic acid. In some embodiments, the base is NaOH.

[0065] Histidine Buffer The denosumab formulation herein may contain a buffer, preferably a histidine buffer, preferably at a concentration of 1 to 10 mM. The concentration of the histidine buffer does not necessarily refer to the concentration of the starting components used to make the buffer. Instead, it refers to the concentration of the histidine species in the buffer after the starting components are added and undergo ionic dissociation in the equilibration buffer. For example, a denosumab formulation may contain L-histidine and L-histidine monohydrochloride monohydrate as starting components. Upon dissociation in the buffer, these concentrations of the components result in a histidine buffer concentration of 1 to 10 mM.

[0066] In some embodiments, the denosumab formulations herein contain 1-10 mM histidine buffer. In other embodiments, the formulation contains 3-7 mM histidine buffer. In other embodiments, the formulation contains 4-6 mM histidine buffer. In some embodiments, the formulation contains 4 mM, 5 mM, about 5 mM, or 6 mM histidine buffer. It is understood that antibodies, such as denosumab, can contribute to the buffering capacity of the formulation.

[0067] stability The denosumab formulations described herein are relatively stable. Typically, the formulations have satisfactory bulk stability for at least 36 months, or at least 24 months, or at least 12 months, or at least 6 months. Generally, such stability includes the chemical and physical stability of denosumab such that only minor fragmentation and aggregation of the antibody is observed.

[0068] The denosumab formulations described herein exhibit remarkable surfactant concentration stability, or, alternatively, resistance to surfactant degradation. In embodiments, the denosumab formulations exhibit minimal decrease in surfactant concentration during storage. The decrease in surfactant concentration can be expressed as a percentage of the original surfactant concentration, determined by comparing the surfactant concentration after storage under specified conditions with the concentration of the as-prepared denosumab formulation. The surfactant concentration can be measured after storage, for example, by ultra-performance liquid chromatography coupled with evaporative light scattering detection (UPLC / ELSD).

[0069] By way of non-limiting illustrative example, the surfactant stability of the denosumab formulations described herein may result in part from increased resistance to oxidation and hydrolysis, both of which are enzymatic from residual host cell proteins (e.g., lipases) and / or histidine (e.g., along with trace metals) that are not removed during clarification and purification.

[0070] In embodiments, the denosumab formulation exhibits a decrease in surfactant concentration of 15% or less after storage at 5° C. for 1 month. In embodiments, the denosumab formulation exhibits a decrease in surfactant concentration of 40% or less, 35% or less, 25% or less, 20% or less, 15% or less, or 10% or less after storage at 25° C. for 1 month. In embodiments, the denosumab formulation exhibits a decrease in surfactant concentration of 50% or less, 55% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 15% or less after storage at 40° C. The decrease in surfactant concentration can be measured, for example, as described in the Examples below.

[0071] formulation In one embodiment, the denosumab formulation comprises 50-80 mg / mL denosumab; 200-300 mM sugar; 1-10 mM histidine buffer; 0.005-0.1% (w / v) poloxamer; and water, and has a pH of 5.5-6.5.

[0072] In one embodiment, the denosumab formulation comprises about 60 mg / mL denosumab; about 240 mM sucrose; about 5 mM histidine buffer; about 0.03% (w / v) poloxamer 188; and water; and has a pH of about 6.2.

[0073] In one embodiment, the denosumab formulation consists essentially of about 60 mg / mL denosumab; about 240 mM sucrose; about 5 mM histidine buffer; about 0.03% (w / v) poloxamer 188; and water; and has a pH of about 6.2.

[0074] In one embodiment, the denosumab formulation consists of about 60 mg / mL denosumab; about 240 mM sucrose; about 5 mM histidine buffer; about 0.03% (w / v) poloxamer 188; and water; and has a pH of about 6.2.

[0075] In one embodiment, the denosumab formulation comprises 60 mg / mL denosumab; 240 mM sucrose; 5 mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; and has a pH of 6.2.

[0076] In one embodiment, the denosumab formulation consists essentially of 60 mg / mL denosumab; 240 mM sucrose; 5 mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; and has a pH of 6.2.

[0077] In one embodiment, the denosumab formulation consists of 60 mg / mL denosumab; 240 mM sucrose; 5 mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; and has a pH of 6.2.

[0078] In one embodiment, the denosumab formulation comprises about 70 mg / mL denosumab; about 240 mM sucrose; about 5 mM histidine buffer; about 0.03% (w / v) poloxamer 188; and water; and has a pH of about 6.2.

[0079] In one embodiment, the denosumab formulation consists essentially of about 70 mg / mL denosumab; about 240 mM sucrose; about 5 mM histidine buffer; about 0.03% (w / v) poloxamer 188; and water; and has a pH of about 6.2.

[0080] In one embodiment, the denosumab formulation consists of about 70 mg / mL denosumab; about 240 mM sucrose; about 5 mM histidine buffer; about 0.03% (w / v) poloxamer 188; and water; and has a pH of about 6.2.

[0081] In one embodiment, the denosumab formulation comprises 70 mg / mL denosumab; 240 mM sucrose; 5 mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; and has a pH of 6.2.

[0082] In one embodiment, the denosumab formulation consists essentially of 70 mg / mL denosumab; 240 mM sucrose; 5 mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; and has a pH of 6.2.

[0083] In one embodiment, the denosumab formulation consists of 70 mg / mL denosumab; 240 mM sucrose; 5 mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; and has a pH of 6.2.

[0084] In addition to the specific formulation variants identified above, the present disclosure is more broadly directed to denosumab formulations that achieve the desired effects described herein. For example, denosumab formulations that include a poloxamer surfactant, optionally with a histidine buffer, can provide improved surfactant stability.

[0085] In embodiments, the aqueous pharmaceutical formulation comprises denosumab and a poloxamer. Optionally, the formulation may contain 50 to 80 mg / mL of denosumab. Optionally, the poloxamer may be poloxamer 188. Optionally, the formulation may contain 0.005 to 0.1% (w / v) poloxamer. Optionally, the formulation may contain a histidine buffer. Optionally, the formulation may contain 1 to 10 mM histidine buffer. Optionally, the formulation may contain a sugar. Optionally, the formulation may contain sucrose. Optionally, the formulation may contain 200 to 300 mM sugar. Optionally, the formulation may have a pH of 5.5 to 6.5. In addition to these options, the formulation may contain any combination of the ingredients, amounts of ingredients, and properties described herein, including, in particular, the specific formulation variant aspects listed above.

[0086] In embodiments, a denosumab formulation for parenteral injection comprises a sugar or sugar alcohol, a buffer, and a surfactant, wherein the surfactant is a poloxamer. Optionally, the formulation may contain 50-80 mg / mL of denosumab. Optionally, the poloxamer may be poloxamer 188. Optionally, the formulation may contain 0.005-0.1% (w / v) poloxamer. Optionally, the formulation may contain a histidine buffer. Optionally, the formulation may contain 1-10 mM histidine buffer. Optionally, the formulation may contain a sugar. Optionally, the formulation may contain sucrose. Optionally, the formulation may contain 200-300 mM sugar. Optionally, the formulation may have a pH of 5.5-6.5. In addition to these options, the formulation may contain any combination of the ingredients, amounts of ingredients, and properties described herein, including, in particular, the specific formulation variant aspects listed above.

[0087] In embodiments, the aqueous pharmaceutical formulation comprises denosumab and a surfactant, and the formulation exhibits a surfactant concentration loss of 40% or less when stored at 25°C for 1 month. Optionally, the formulation exhibits a surfactant concentration loss of 15% or less. Optionally, the formulation comprises a poloxamer. Optionally, the formulation comprises a histidine buffer. In addition to these options, the formulation may include any combination of the ingredients, ingredient amounts, and properties described herein, including, in particular, the aspects of the specific formulation variants listed above.

[0088] In embodiments, the aqueous pharmaceutical formulation comprises denosumab and a surfactant, and the formulation exhibits a decrease in surfactant concentration of 50% or less when stored at 40°C for 1 month. Optionally, the formulation exhibits a decrease in surfactant concentration of 15% or less. Optionally, the formulation comprises a poloxamer. Optionally, the formulation comprises a histidine buffer. In addition to these options, the formulation may comprise any combination of the ingredients, ingredient amounts, and properties described herein, including, in particular, the aspects of the specific formulation variants listed above.

[0089] Manufacturing of pharmaceutical preparations The denosumab formulations described herein can be made by combining denosumab, sugar, histidine buffer, and poloxamer in pharmaceutical-grade water to form a solution and / or suspension. The order of addition and method of combination are generally not critical. For convenience and commercial practicality, denosumab can be formulated into the formulations described herein using well-known ultrafiltration / diafiltration (UF / DF) techniques. Generally, expressed denosumab is captured and purified by various methods and then subjected to UF / DF for further purification, concentration, and modification of the liquid medium. The denosumab solution obtained by the previous purification process step can be exchanged by diafiltration, as is well known in the art. In making the formulations described herein, poloxamer can be added during or after the UF / DF process is completed, and denosumab can be diafiltered with a solution of sugar and histidine buffer in WFI. The poloxamer can be added as a solution in WFI or the diafiltration solution. If necessary, the pH can be adjusted before, during, or after the UF / DF procedure, such as by the introduction of HCl or other pH adjusting agents suitable for parenteral formulations. However, denosumab formulations are not limited to the use of UF / DF, and any suitable method or means for combining the listed components at the desired concentrations can be used to make the denosumab formulations described herein.

[0090] Use of the formulation The denosumab formulations described herein are useful for preventing or treating a variety of diseases and disorders or their symptoms that respond to denosumab or anti-RANKL antibody treatment, including osteoporosis, low bone mass, and skeletal problems in patients with multiple myeloma or solid tumor bone metastases, giant cell tumor of bone, and hypercalcemia; particularly, treatment of postmenopausal women with osteoporosis who are at high risk of fracture, treatment to increase bone mass in men at high risk of fracture who are receiving androgen deprivation therapy for non-metastatic prostate cancer, treatment to increase bone mass in women at high risk of fracture who are receiving adjuvant aromatase inhibitor therapy for breast cancer, prevention of skeletal-related events in patients with bone metastases from solid tumors, and treatment of adults and skeletally mature adolescents with giant cell tumor of bone that is unresectable or where surgical resection would result in significant morbidity.

[0091] In embodiments, the present disclosure relates to methods for treating or preventing the aforementioned diseases or conditions, wherein an effective amount of a formulation described herein is administered to a subject, e.g., a human patient, in need of such treatment or prevention. In embodiments, such administration can be a subcutaneous injection of a predetermined amount of a formulation described herein. Such subcutaneous injection can be administered by the subject (patient) themselves or by a caregiver using a syringe filled from a vial, a prefilled syringe, or an autoinjector. The subcutaneous injection can be into the subject's upper arm, upper thigh, or abdomen. The vial, prefilled syringe, or autoinjector can contain, for example, 1.0 or 1.7 mL of a formulation described herein. Administration of a formulation described herein can also include co-administration with calcium (e.g., 1,000 mg daily) and / or vitamin D (e.g., 400 IU daily).

[0092] Dosages and regimens are well known, for example, from the administration of Prolia® and Xgeva®, as approved by the US FDA or EMA. As mentioned above, for example, the denosumab formulation herein can be loaded into a pre-filled syringe to provide 1.0 mL of formulation, which corresponds to a 60 mg dose of denosumab, or can be provided in an injection vial in an amount of 1.7 mL of formulation, which corresponds to a 120 mg dose of denosumab. Exemplary dosing regimens include: administering 120 mg every 4 weeks as a subcutaneous injection into the upper arm, upper thigh, or abdomen (e.g., to treat bone metastases from solid tumors); administering 120 mg every 4 weeks as a subcutaneous injection into the upper arm, upper thigh, or abdomen, with additional 120 mg doses on days 8 and 15 of the first month of therapy (e.g., to treat giant cell tumor of bone); and administering 60 mg every 6 months as a subcutaneous injection into the upper arm, upper thigh, or abdomen.

[0093] In various exemplary embodiments, the present disclosure relates to the following and all combinations thereof: 1. Denosumab 50–80 mg / mL; 200-300mM sugar; 1–10 mM histidine buffer; 0.005 to 0.1% (w / v) poloxamer; and water; Including, An aqueous pharmaceutical formulation having a pH of 5.5 to 6.5. 2. Denosumab at approximately 60 mg / mL; approximately 240 mM sucrose; approximately 5 mM histidine buffer; Approximately 0.03% (w / v) poloxamer 188; and water; Including, The formulation of item 1 or any item subordinate thereto, having a pH of about 6.2. 3. Denosumab at approximately 60 mg / mL; approximately 240 mM sucrose; approximately 5 mM histidine buffer; Approximately 0.03% (w / v) poloxamer 188; and water 2. A formulation of item 2 or any item subordinate thereto, consisting essentially of: 4. Denosumab at approximately 60 mg / mL; approximately 240 mM sucrose; approximately 5 mM histidine buffer; Approximately 0.03% (w / v) poloxamer 188; and water A formulation according to item 2 or any item subordinate thereto, comprising: 5. Denosumab 60 mg / mL; 240 mM sucrose; 5mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; Including, 6. A formulation of item 1 or any item subordinate thereto having a pH of 6.2. 6. Denosumab 60 mg / mL; 240 mM sucrose; 5mM histidine buffer; 0.03% (w / v) poloxamer 188; and water 5. A formulation of item 5 or any item subordinate thereto, consisting essentially of 7. Denosumab 60 mg / mL; 240 mM sucrose; 5mM histidine buffer; 0.03% (w / v) poloxamer 188; and water A formulation according to item 5 or any item subordinate thereto, comprising: 8. Denosumab at approximately 70 mg / mL; approximately 240 mM sucrose; approximately 5 mM histidine buffer; Approximately 0.03% (w / v) poloxamer 188; and water; Including, The formulation of item 1 or any item subordinate thereto, having a pH of about 6.2. 9. Denosumab at approximately 70 mg / mL; approximately 240 mM sucrose; approximately 5 mM histidine buffer; Approximately 0.03% (w / v) poloxamer 188; and water 10. The formulation of item 8 or any item subordinate thereto, consisting essentially of 10. Denosumab at approximately 70 mg / mL; approximately 240 mM sucrose; approximately 5 mM histidine buffer; Approximately 0.03% (w / v) poloxamer 188; and water A formulation according to item 8 or any item subordinate thereto, comprising: 11. Denosumab 70 mg / mL; 240 mM sucrose; 5mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; Including, 6. A formulation of item 1 or any item subordinate thereto having a pH of 6.2. 12. Denosumab 70 mg / mL; 240 mM sucrose; 5mM histidine buffer; 0.03% (w / v) poloxamer 188; and water 12. A formulation according to item 11 or any item subordinate thereto, consisting essentially of 13. Denosumab 70 mg / mL; 240 mM sucrose; 5mM histidine buffer; 0.03% (w / v) poloxamer 188; and water 10. A formulation according to item 11 or any item subordinate thereto, comprising: 14. A formulation of item 1 or any item subordinate thereto containing 60 to 70 mg / mL denosumab. 15. A formulation of item 1 or any subordinate item comprising 220 to 260 mM sugar. 16. A formulation of item 1 or any item subordinate thereto, comprising 0.1 to 0.5% (w / v) poloxamer. 17. The formulation of item 1 or any item subordinate thereto, wherein the sugar is at least one selected from the group consisting of sucrose, trehalose, maltose, and lactose. 18. A formulation of item 1 or any item subordinate thereto, wherein the sugar is sucrose. 19. The formulation of item 1 or any item subordinate thereto, wherein the poloxamer is poloxamer 188. 20. The formulation of item 1 or any item subordinate thereto, having a pH of about 5.7 to about 6.2. 21. The formulation of item 1 or any item subordinate thereto, having a pH of about 5.7. 22. The formulation of item 1 or any item subordinate thereto, having a pH of about 6.2. 23. An aqueous pharmaceutical formulation comprising denosumab and poloxamer. 24. A formulation of item 23 or any item subordinate thereto containing 50 to 80 mg / mL of denosumab. 25. The formulation of item 23 or any item subordinate thereto, wherein the poloxamer is poloxamer 188. 26. A formulation of item 23 or any item subordinate thereto, containing 0.005 to 0.1% (w / v) poloxamer. 27. The formulation of item 23 or any item dependent thereon, further comprising a histidine buffer. 28. The formulation of item 27 or any item dependent thereon, comprising 1 to 10 mM histidine buffer. 29. The formulation of item 23 or any item subordinate thereto, further comprising a sugar. 30. A formulation of item 29 or any item subordinate thereto, wherein the sugar is sucrose. 31. A formulation of item 29 or any of its dependent items, comprising 200 to 300 mM sugar. 32. A formulation of item 23 or any item subordinate thereto having a pH of 5.5 to 6.5. 33. A denosumab formulation for parenteral injection, comprising a sugar or sugar alcohol, a buffer, and a surfactant, wherein the surfactant is a poloxamer. 34. A formulation of item 33 or any item subordinate thereto containing 50 to 80 mg / mL of denosumab. 35. The formulation of item 33 or any item subordinate thereto, wherein the poloxamer is poloxamer 188. 36. A formulation of item 33 or any item subordinate thereto, containing 0.005 to 0.1% (w / v) poloxamer. 37. The formulation of item 33 or any item dependent thereon, wherein the buffer is a histidine buffer. 38. The formulation of item 37 or any item dependent thereon, comprising 1 to 10 mM histidine buffer. 39. A formulation of item 33 or any item subordinate thereto, wherein the sugar or sugar alcohol is sucrose. 40. A formulation of item 33 or any of the items subordinate thereto, comprising 200 to 300 mM sugar or sugar alcohol. 41. A formulation of item 33 or any item subordinate thereto having a pH of 5.5 to 6.5. 42. An aqueous pharmaceutical formulation containing denosumab and a surfactant, which shows a decrease in surfactant concentration of 40% or less when stored at 25°C for 1 month. 43. The formulation of item 42 or any item subordinate thereto, wherein the reduction in surfactant concentration is 15% or less. 44. The formulation of item 42 or any item dependent thereon, wherein the surfactant comprises a poloxamer. 45. The formulation of item 44 or any item dependent thereon, further comprising a histidine buffer. 46. ​​Denosumab 50–80 mg / mL; 200-300mM sugar; 1–10 mM histidine buffer; 0.005 to 0.1% (w / v) poloxamer; and water; Including, 45. The formulation of claim 45 or any of the items subordinate thereto, having a pH of 5.5 to 6.5. 47. Denosumab 60 mg / mL; 240 mM sucrose; 5mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; consists essentially of 46. ​​The formulation of claim 46 or any of the items subordinate thereto, having a pH of 5.7 to 6.2. 48. An aqueous pharmaceutical formulation containing denosumab and a surfactant, which shows a decrease in surfactant concentration of 50% or less when stored at 40°C for 1 month. 49. The formulation of item 48 or any item subordinate thereto, wherein the reduction in surfactant concentration is 15% or less. 50. The formulation of item 48 or any item subordinate thereto, wherein the surfactant comprises a poloxamer. 51. The formulation of item 50 or any item dependent thereon, further comprising a histidine buffer. 52. Denosumab 50–80 mg / mL; 200-300mM sugar; 1–10 mM histidine buffer; 0.005 to 0.1% (w / v) poloxamer; and water; Including, 5. The formulation of claim 51 or any of the items subordinate thereto, having a pH of 5.5 to 6.5. 53. Denosumab 60 mg / mL; 240 mM sucrose; 5mM histidine buffer; 0.03% (w / v) poloxamer 188; and water; consists essentially of 52. The formulation of claim 52 or any of the items subordinate thereto, having a pH of 5.7 to 6.2. 54. A formulation of any one of items 1 to 53 or any item subordinate thereto for use in the treatment or prevention of osteoporosis or reduced bone mass. 55. A formulation of any one of items 1 to 53 or any item subordinate thereto for use in the treatment or prevention of skeletal-related events associated with multiple myeloma, solid tumor bone metastases or giant cell tumor of bone. 56. A formulation of any one of items 1 to 53 or any item subordinate thereto for use in the treatment or prevention of hypercalcemia. 57. A method for treating osteoporosis or reduced bone mass, comprising administering to a subject in need of such treatment a formulation of any one of items 1 to 53 or any item subordinate thereto. 58. A method for treating skeletal-related events associated with multiple myeloma, solid tumor bone metastasis, or giant cell tumor of bone, comprising administering to a subject in need of such treatment a formulation of any one of items 1 to 53 or any item subordinate thereto. 59. A method for treating hypercalcemia, comprising administering a formulation of any one of items 1 to 53 or any item subordinate thereto to a subject in need of such treatment. 60. A method for preventing osteoporosis or loss of bone mass, comprising administering a formulation of any one of items 1 to 53 or any item subordinate thereto to a subject in need of such treatment. 61. A method for preventing skeletal-related events associated with multiple myeloma, solid tumor bone metastasis, or giant cell tumor of bone, comprising administering a formulation of any one of items 1 to 53 or any item subordinate thereto to a subject in need of such treatment. 62. A method for preventing hypercalcemia, comprising administering a formulation of any one of items 1 to 53 or any item subordinate thereto to a subject in need of such treatment. 63. Use of a formulation of any one of items 1 to 53 or any item subordinate thereto in the manufacture of a medicament for treating or preventing osteoporosis or reduced bone mass. 64. Use of a formulation of any one of items 1 to 53 or any item subordinate thereto in the manufacture of a medicament for treating or preventing skeletal-related events associated with multiple myeloma, solid tumor bone metastases, or giant cell tumor of bone. 65. Use of a formulation of any one of items 1 to 53 or any item subordinate thereto in the manufacture of a medicament for treating or preventing hypercalcemia. [Example]

[0094] The present invention will now be described with reference to the following examples, which should be considered in all respects as illustrative and non-limiting. It will be understood by those skilled in the art that the embodiments of the present disclosure can be embodied in many other forms; in particular, the features of any one of the various described examples can be provided in any combination in any of the other described examples.

[0095] Aqueous denosumab formulations, Examples 1 and 2 (E1 and E2) and Comparative Examples 1-6 (CE1-CE6), were prepared using the compositions specified in Table 1A and Table 1B below. E1 and E2 are inventive denosumab formulations containing a poloxamer surfactant. CE1 is similar to Prolia®. CE2-CE6 are alternative denosumab formulations containing a polysorbate surfactant. CE5 and CE6 differ from CE2 and CE3 in that a post-harvest filtration step was omitted when preparing the denosumab used in CE5 and CE6.

[0096] [Table 1]

[0097] [Table 2]

[0098] Surfactant concentrations were monitored for denosumab formulations E1, E2, and CE1-CE6 at various storage temperatures over various time periods. For each formulation, vials with approximately 0.5 mL fill volume were prepared, stored under specified conditions, analyzed at designated pull points, and returned to the specified storage conditions after analysis. Surfactant concentrations were detected at the indicated times by ultra-performance liquid chromatography coupled with evaporative light scattering detection (UPLC / ELSD).

[0099] Denosumab formulations E1, E2, and CE1-CE4 were stored at 5°C, and surfactant concentrations were measured for all formulations after 1 month and for E1 and E2 after 3 months. Figure 1 is a graph showing the measured surfactant concentrations, and TABLE 2A shows the calculated percent remaining surfactant at each measurement.

[0100] [Table 3]

[0101] Denosumab formulations E1, E2, and CE1-CE6 were stored at 25°C, and surfactant concentrations were measured for all formulations after 0.5 and 1 month. Figure 2 is a graph showing the measured surfactant concentrations, and TABLE 2B shows the calculated percent remaining surfactant at each measurement.

[0102] [Table 4]

[0103] Denosumab formulations E1, E2, and CE1-CE4 were stored at 40°C, and surfactant concentrations were measured for all formulations after 0.25, 0.5, and 1 month, and for E1 after 2 months. Figure 3 is a graph showing the measured surfactant concentrations, and TABLE 2C shows the calculated percent remaining surfactant at each measurement.

[0104] [Table 5]

[0105] As is evident from the above results, denosumab formulations E1 and E2, which used poloxamer 188 as the surfactant, demonstrated remarkable performance in surfactant concentration stability, i.e., prevention of surfactant degradation, during storage compared to the Prolia® formulation containing polysorbate 20 as the surfactant and an alternative denosumab formulation containing polysorbate 80 as the surfactant. Superior performance was observed under intended (5°C), accelerated (25°C), and stressed (40°C) storage conditions.

[0106] In some cases, surfactant concentrations showed slight increases over time, suggesting some variability in the evaluation. Comparison of CE2 and CE3 with CE5 and CE6 indicates that the filtration step omitted in CE5 and CE6 had no lasting effect on surfactant concentrations. The results for CE4 indicate that the addition of EDTA had no beneficial effect on surfactant concentrations.

[0107] The foregoing discussion merely discloses and describes exemplary embodiments of the present disclosure. As will be understood by those skilled in the art, the present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the disclosure of the present invention is intended to be illustrative, but not limiting, of the scope of the present disclosure and other claims. The present disclosure, including any readily discernible variants of the teachings herein, defines in part the scope of the terms in the preceding claims so as not to subject the subject matter to the public.

Claims

1. An aqueous pharmaceutical formulation comprising denosumab and poloxamer.

2. 10. The aqueous formulation of claim 1, comprising about 50 to about 80 mg / mL of denosumab, preferably about 60 to about 70 mg / mL of denosumab.

3. 3. The aqueous formulation of claim 1, wherein the poloxamer is poloxamer 188.

4. 4. The aqueous formulation of claim 1, comprising about 0.005 to about 0.1% (w / v) of poloxamer.

5. 5. The aqueous formulation according to any one of claims 1 to 4, further comprising a histidine buffer, preferably from about 1 to about 10 mM of histidine buffer.

6. 6. The aqueous formulation according to any one of claims 1 to 5, further comprising a sugar or sugar alcohol, preferably wherein the sugar is at least one selected from the group comprising sucrose, trehalose, maltose and lactose, more preferably sucrose, in an amount of about 200 to about 300 mM, preferably about 220 to about 260 mM.

7. 7. The aqueous formulation according to any one of claims 1 to 6, having a pH of about 5.5 to about 6.5, preferably a pH of about 5.5 to about 6.5, more preferably a pH of about 5.7 to about 6.

2.

8. about 60 or about 70 mg / mL denosumab; approximately 240 mM sucrose; approximately 5 mM histidine buffer; approximately 0.03% (w / v) poloxamer 188; water; Including, 10. The aqueous formulation of claim 1, having a pH of about 6.

2.

9. 9. Aqueous formulation according to any one of claims 1 to 8 for parenteral injection.

10. 10. The aqueous formulation according to claim 1, which exhibits a decrease in surfactant concentration of 40% or less when stored at 25°C for 1 month, preferably a decrease in surfactant concentration of 15% or less.

11. 11. The aqueous formulation according to claim 1, which exhibits a decrease in surfactant concentration of 50% or less when stored at 40°C for 1 month, preferably a decrease in surfactant concentration of 15% or less.

12. 12. An aqueous formulation according to any one of claims 1 to 11 for use in the treatment or prevention of skeletal-related events associated with osteoporosis, low bone mass, multiple myeloma, solid tumor bone metastases, giant cell tumor of bone or hypercalcemia.

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