Formulations for improving the stability of recombinant human parathyroid hormone

Abstract

To provide improved PTH formulations.SOLUTION: The invention relates to pharmaceutical compositions and formulations comprising full-length recombinant human parathyroid hormone (rhPTH(l-84)). The invention further relates to new and / or improved PTH compositions having improved in-use stability that are resistant to protein degradation in response to physical and chemical stresses.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to new and improved pharmaceutical compositions and dosage forms containing recombinant human parathyroid hormone (rhPTH(1-84)) that have improved in-use stability. [Background technology]

[0002] Parathyroid hormone (PTH) is an 84-amino acid product secreted by the mammalian parathyroid gland that regulates serum calcium levels through its actions on various tissues, including bone. Human studies with certain forms of PTH have demonstrated anabolic effects on bone, greatly stimulating interest in its use for the treatment of osteoporosis and related bone disorders.

[0003] Unlike other successfully formulated proteins, PTH is particularly susceptible to various forms of degradation. Furthermore, its N-terminal sequence must remain intact to maintain bioactivity. For example, oxidation can occur at methionine residues at positions 8 and 18, generating the oxidized PTH species ox-M(8)-PTH and ox-M(18)-PTH, while deamidation can occur at asparagine at position 16, generating d16-PTH. The polypeptide chain is truncated by peptide bond cleavage at both the N- and C-termini. Furthermore, PTH can also adsorb to surfaces, form nonspecific aggregates, and / or precipitate, reducing the available concentration of the drug. All of these degradative reactions, and their combinations, can lead to partial or complete loss of PTH bioactivity.

[0004] Commercial use of parathyroid hormone requires an acceptable formulation for storage and in-use stability, as well as ease of preparation and reconstitution. Because parathyroid hormone is a protein and is much more unstable than traditional low-molecular-weight drugs, formulation of parathyroid hormone presents challenges not typically encountered in the pharmaceutical industry.

[0005] Full-length rhPTH(1-84) was recently approved as a safe and effective treatment for hypoparathyroidism (marketed by Shire Pharmaceuticals under the brand name NATPARA® / NATPAR®). It is the first specific hormone replacement therapy for hypoparathyroidism and is a once-daily subcutaneous injection formulation taken as an adjunct to calcium and vitamin D treatment. NATPARA® is currently supplied as a multi-dose, dual-chamber glass cartridge containing a sterile, lyophilized powder and diluent in various dosage strengths. The sterile, lyophilized powder contains 0.40 mg, 0.80 mg, 1.21 mg, or 1.61 mg of parathyroid hormone, as well as 4.5 mg of sodium chloride, 30 mg of mannitol, and 1.26 mg of citric acid monohydrate, depending on the dosage strength. The sterile diluent weighs 1.13 g and contains a 3.2 mg / mL aqueous solution of m-cresol. Upon reconstitution, each dose consists of a solution of rhPTH(1-84) at pH 5-6.

[0006] Disposable NATPARA® drug cartridges are designed for use with a reusable mixer for product reconstitution and a reusable Q-Cliq pen for drug delivery. The Q-Cliq pen delivers a fixed-volume dose of 71.4 μL. Using the Q-Cliq pen, each NATPARA® dual-chamber cartridge delivers 14 doses of NATPARA®.

[0007] It has been observed that under certain circumstances, reconstituted NATPARA® solutions may form protein particles during the in-use period. Therefore, greater robustness of NATPARA® formulations to normal processing conditions, physical and chemical stresses encountered during the product's shelf life and in-use life is desirable.

[0008] Thus, there is a need for improved PTH formulations, including full-length rhPTH(1-84), particularly formulations that prevent physical and chemical degradation of PTH, have improved in-use stability, and are easy to prepare, reconstitute, and use. Summary of the Invention [Means for solving the problem]

[0009] Various non-limiting aspects and embodiments of the present invention are described below.

[0010] In one aspect, a stable liquid pharmaceutical formulation comprising recombinant human parathyroid hormone (rhPTH(1-84)) is provided. The formulation is designed for direct use as an injectable solution without a powder reconstitution step. In one embodiment, the pharmaceutical formulation comprises: (a) a therapeutically effective amount of recombinant human parathyroid hormone (rhPTH(1-84)); (b) surfactants; (c) tonicity agent; (d) antioxidants; (e) preservatives; (f) a pharmaceutically acceptable buffer; and (g) water wherein the pharmaceutical preparation is formulated as an injectable solution, and the preparation is physically and chemically stable, remains clear, colorless, and free of visible particles for at least 48 hours.

[0011] In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 72 hours. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 96 hours. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 7 days. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 14 days. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 21 days.

[0012] In one embodiment, the surfactant is a poloxamer. In one embodiment, the surfactant is poloxamer 188. In one embodiment, the surfactant is poloxamer 188 present at about 0.03 to about 3 w / v% of the formulation.

[0013] In one embodiment, the tonicity agent is selected from sodium chloride, sucrose, and glycerol, or a combination thereof. In one embodiment, the tonicity agent is sodium chloride, present at about 0.2 to about 20 w / v% of the formulation. In one embodiment, the tonicity agent is sucrose, present at about 0.2 to about 20 w / v% of the formulation. In one embodiment, the tonicity agent is glycerol, present at about 0.2 to about 20 w / v% of the formulation.

[0014] In one embodiment, the preservative is m-cresol present at about 0.03 to about 3 w / v% of the formulation. In one embodiment, the preservative is m-cresol present at about 0.3 w / v% of the formulation.

[0015] In one embodiment, the pharmaceutically acceptable buffer is acetate buffer, phosphate buffer, L-histidine buffer, or succinate buffer. In one embodiment, the pharmaceutically acceptable buffer is present at a concentration of about 5 mM to about 50 mM, or about 20 mM.

[0016] In one embodiment, the antioxidant is methionine, which is present at a concentration of about 0.015 to about 1.50% w / v of the formulation. In one embodiment, the antioxidant is methionine, which is present at about 0.15% w / v or 10 mM.

[0017] In one embodiment, the pharmaceutical formulation has a pH of about 3.8 to about 6.2, or about 5.5.

[0018] In one embodiment, the pharmaceutical formulation is in a single dose vial, a multi-dose vial, a cartridge, a pre-filled syringe, an auto-injector, or an injection pen.

[0019] In one embodiment, the pharmaceutical formulation comprises: (a) about 0.2 to about 2.0 mg / mL recombinant human parathyroid hormone (rhPTH(1-84)); (b) about 0.03 to about 3.0 w / v % surfactant; (c) about 0.2 to about 20 w / v % of an isotonic agent; (d) about 0.015 to about 1.50 w / v % antioxidant; (e) about 0.03 to about 3% preservative; (f) about 5 mM to about 50 mM of a pharmaceutically acceptable buffer, and (g) water; wherein the pharmaceutical preparation is formulated as an injectable solution, and the preparation is physically and chemically stable, remains clear, colorless, and free of visible particles for at least 48 hours.

[0020] In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 72 hours. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 96 hours. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 7 days. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 14 days. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 21 days.

[0021] In another aspect, a pharmaceutical formulation comprising recombinant human parathyroid hormone (rhPTH(1-84)) is provided as a lyophilized powder that is reconstituted prior to injection. In one embodiment, the pharmaceutical formulation comprises: (a) a therapeutically effective amount of recombinant human parathyroid hormone (rhPTH(1-84)); (b) bulking agents; (c) a cryoprotectant; and (d) a pharmaceutically acceptable buffer wherein the pharmaceutical preparation is formulated as a lyophilized powder that is reconstituted prior to injection, and the preparation is physically and chemically stable and remains clear, colorless, and free of visible particles for at least 48 hours after reconstitution.

[0022] In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 72 hours. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 96 hours. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 7 days. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 14 days. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 21 days.

[0023] In one embodiment, the bulking agent is mannitol. In one embodiment, the bulking agent is mannitol, which is present at about 0.3 to about 30 w / v % of the formulation.

[0024] In one embodiment, the cryoprotectant is sucrose. In one embodiment, the cryoprotectant is sucrose present at about 0.2 to about 20 w / v% of the formulation.

[0025] In one embodiment, the pharmaceutically acceptable buffer is a phosphate buffer, an L-histidine buffer, or a succinate buffer. In one embodiment, the pharmaceutically acceptable buffer is present at a concentration of about 5 mM to about 50 mM, or about 20 mM. In one embodiment, the pharmaceutically acceptable buffer is an L-histidine buffer. In one embodiment, the pharmaceutically acceptable buffer is a succinate buffer.

[0026] In one embodiment, the pharmaceutical formulation further comprises an antioxidant. In one embodiment, the antioxidant is methionine. In one embodiment, the antioxidant is methionine, which is present at a concentration of about 0.015 to about 1.50 w / v% of the formulation. In one embodiment, the antioxidant is methionine present at about 0.15 w / v% or 10 mM.

[0027] In one embodiment, the pharmaceutical formulation further comprises a surfactant. In one embodiment, the surfactant is a poloxamer. In one embodiment, the surfactant is poloxamer 188. In one embodiment, the surfactant is poloxamer 188 present at about 0.03 to about 3 w / v% of the formulation.

[0028] In one embodiment, the pharmaceutical formulation has a pH of about 3.8 to about 6.2, or about 4.3, or about 5.5.

[0029] In one embodiment, the pharmaceutical formulation comprises: (a) about 0.02 to about 2.0 mg / mL recombinant human parathyroid hormone (rhPTH(1-84)); (b) about 0.3 to about 30 w / v % bulking agent; (c) about 0.2 to about 20 w / v % cryoprotectant; and (d) about 5 mM to about 50 mM of a pharmaceutically acceptable buffer wherein the pharmaceutical preparation is formulated as a lyophilized powder that is reconstituted prior to injection, and the preparation is physically and chemically stable and remains clear, colorless, and free of visible particles for at least 48 hours after reconstitution.

[0030] In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 72 hours. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 96 hours. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 7 days. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 14 days. In one embodiment, the pharmaceutical formulation remains clear and free of visible particles for at least 21 days.

[0031] These and other aspects of the present invention will become apparent to those skilled in the art after reading the following detailed description of the invention, including the appended claims.

[0032] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0033] [Figure 1] A comparison of the opalescence of the reference suspension (RS) is shown. [Figure 2] Figure 1 shows the appearance of rhPTH formulated in various buffers when agitated (220 revolutions per minute (rpm), orbital shaking) in 2R glass vials at ambient conditions. [Figure 3A] RP-HPLC data of the main peak of rhPTH for pH screening samples stored at 40° C. for up to 6 months are shown. [Figure 3B] RP-HPLC data of the main peak of rhPTH for pH screening samples stored at 25° C. for up to 6 months are shown. [Figure 3C] RP-HPLC data of the main peak of rhPTH for pH screening samples stored at 5° C. for up to 6 months are shown. [Figure 4A]RP-HPLC data for oxidized Met8 rhPTH impurity for pH screening samples stored at 40° C. for up to 6 months are shown. [Figure 4B] RP-HPLC data for oxidized Met8 rhPTH impurity for pH screening samples stored at 25° C. for up to 6 months are shown. [Figure 4C] RP-HPLC data for oxidized Met8 rhPTH impurity for pH screening samples stored at 5° C. for up to 6 months are shown. [Figure 5A] RP-HPLC data for oxidized Met18 rhPTH impurity for pH screening samples stored at 40° C. for up to 6 months are shown. [Figure 5B] RP-HPLC data for oxidized Met18 rhPTH impurity for pH screening samples stored at 25° C. for up to 6 months are shown. [Figure 5C] RP-HPLC data for oxidized Met18 rhPTH impurity for pH screening samples stored at 5° C. for up to 6 months are shown. [Figure 6A] RP-HPLC data for IsoAsp33 rhPTH from pH screening samples stored at 40° C. for up to 6 months are shown. [Figure 6B] RP-HPLC data for IsoAsp33 rhPTH from pH screening samples stored at 25° C. for up to 6 months are shown. [Figure 6C] RP-HPLC data for IsoAsp33 rhPTH from pH screening samples stored at 5° C. for up to 6 months are shown. [Figure 7A] Figure 1 shows RP-HPLC data for rhPTH ((1-30) + (1-33)) impurity for pH screening samples stored at 40°C for up to 6 months. [Figure 7B] Figure 1 shows RP-HPLC data for rhPTH ((1-30) + (1-33)) impurity for pH screening samples stored at 25°C for up to 6 months. [Figure 7C]Figure 1 shows RP-HPLC data for rhPTH((1-30)+(1-33)) impurity for pH screening samples stored at 5°C for up to 6 months. [Figure 8A] RP-HPLC data for rhPTH(1-45) fragment impurities for pH screening samples stored at 40° C. for up to 6 months are shown. [Figure 8B] RP-HPLC data for rhPTH(1-45) fragment impurities for pH screening samples stored at 25° C. for up to 6 months are shown. [Figure 8C] RP-HPLC data for rhPTH(1-45) fragment impurities for pH screening samples stored at 5° C. for up to 6 months are shown. [Figure 9A] RP-HPLC data of the main peak of rhPTH when samples were formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients and stored at 40° C. are shown. [Figure 9B] RP-HPLC data of the main peak of rhPTH are shown for samples stored at 25° C. formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 9C] RP-HPLC data of the main peak of rhPTH are shown for samples formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients and stored at 5° C. [Figure 10A] RP-HPLC data for the oxidized Met8 rhPTH impurity when samples were stored at 40° C. formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 10B] RP-HPLC data for the oxidized Met8 rhPTH impurity are shown for samples stored at 25° C. formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 10C]RP-HPLC data for the oxidized Met8 rhPTH impurity are shown for samples stored at 5° C. formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 11A] RP-HPLC data for the oxidized Met18 rhPTH impurity when samples were stored at 40° C. formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 11B] RP-HPLC data for the oxidized Met18 rhPTH impurity are shown for samples stored at 25° C. formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 11C] RP-HPLC data for the oxidized Met18 rhPTH impurity are shown for samples stored at 5° C. formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 12A] Figure 1 shows RP-HPLC data for IsoAsp33 rhPTH impurity when samples were stored at 40°C formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 12B] Figure 1 shows RP-HPLC data for IsoAsp33 rhPTH impurity when samples were stored at 25°C formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 12C] Figure 1 shows RP-HPLC data for IsoAsp33 rhPTH impurity when samples were stored at 5°C formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients. [Figure 13] 1 shows the appearance of a lyophilized cake of an rhPTH formulation according to various embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Furthermore, each of the examples given in connection with various embodiments of the present invention is intended to be illustrative and not limiting. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a representative basis for teaching those skilled in the art how to use the present invention in various ways.

[0035] 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 belongs.

[0036] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0037] As used in this application, the terms "about" and "approximately" are used interchangeably. Any numbers used in this application, whether or not they are about / approximate, are intended to cover normal variations recognized by those of ordinary skill in the relevant art. As used herein, the term "approximately" or "about," when applied to one or more target values, refers to a value similar to the stated reference value. In one embodiment, the term "approximately" or "about" refers to a numerical range that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less of the stated reference value in any direction (greater or less), unless otherwise specified or apparent from the context (except where such numerical value exceeds 100% of possible values).

[0038] As used herein, the terms "carrier" and "diluent" refer to pharmaceutically acceptable (e.g., safe and non-toxic for human administration) carriers or diluents useful in preparing pharmaceutical formulations. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0039] The terms "treat" or "treatment" of a condition, disorder, or state include (1) preventing, delaying, or reducing the likelihood of the onset and / or appearance of at least one clinical or asymptomatic symptom of a developing condition, disorder, or state in a subject who may be afflicted with or susceptible to the condition, disorder, or state, but who has not yet experienced or manifested a clinical or asymptomatic symptom of the condition, disorder, or state; (2) inhibiting the condition, disorder, or state, i.e., arresting, reducing, or delaying the progression of the disease or its recurrence, or at least one clinical or asymptomatic symptom thereof; and (3) palliating the disease, i.e., causing regression of the condition, disorder, or state, or at least one clinical or asymptomatic symptom thereof. The benefit to a treated subject is either statistically significant or at least perceptible to the subject or the clinician.

[0040] As used herein, "subject" or "patient" or "individual" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0041] As used herein, the term "effective" as applied to a dose or amount refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that is effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug used, the method of administration, etc.

[0042] The phrase "pharmaceutically acceptable" as used in connection with the compositions of the present invention refers to molecular entities and other components of such compositions that are physiologically tolerable and do not normally produce adverse reactions when administered to mammals (e.g., humans). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a U.S. federal or state government regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in mammals, particularly humans.

[0043] The compositions of the present invention provide improved in-use stability of rhPTH(1-84) compared to commercially available rhPTH(1-84) formulations. As used herein, the term "in-use" refers to the period of time during which a multidose formulation can be used while maintaining quality within acceptable specifications after the multidose container has been opened. Thus, "in-use stability" refers to the stability of a multidose formulation during the in-use period. In some embodiments of the present invention, the in-use period is 7 days. In some embodiments of the present invention, the in-use period is 14 days. In some embodiments of the present invention, the in-use period is 21 days. In some embodiments of the present invention, the in-use period is 1 month.

[0044] rhPTH(1-84) The compositions disclosed herein incorporate as an active ingredient the full-length 84 amino acid form of human parathyroid hormone, obtained recombinantly, by peptide synthesis, or by extraction from human body fluids. Recombinant human PTH, abbreviated herein as rhPTH(1-84), has the amino acid sequence reported by Kimura et al., Biochem Biophys Res Comm, 114(2):493.

[0045] As an alternative to full-length human PTH, the compositions of the present invention can incorporate a homolog, fragment, or variant of human PTH that has human PTH activity as determined in the ovariectomized rat model of osteoporosis reported by Kimmel et al., Endocrinology, 1993, 32(4):1577 (incorporated herein by reference).

[0046] In one embodiment, the parathyroid hormone compositions of the present invention are provided in single or multiple unit liquid dosage forms (liquids) as injectable aqueous hormone solutions that do not require reconstitution, dilution, or mixing.

[0047] In one embodiment, the parathyroid hormone compositions of the present invention are provided in a lyophilized powder form containing 3% or less water by weight, which is obtained by lyophilization of a sterile aqueous hormone solution prepared by mixing the selected parathyroid hormone, a non-volatile buffer, and excipients.

[0048] The PTH compositions of the present invention incorporate PTH in a therapeutically effective amount, the term "therapeutically effective amount" being used in reference to an amount useful therapeutically or in medical diagnosis. The specific amount of parathyroid hormone incorporated into the preparation can be predetermined based on the type of PTH selected and the intended end use of the preparation. In one embodiment, the composition is utilized for therapeutic purposes, particularly for the treatment of osteoporosis and related bone disorders, and hypoparathyroidism. In one embodiment, such treatment involves the administration of a liquid and / or reconstituted lyophilized composition by injection (e.g., subcutaneous injection) in a unit dose reflecting the designated treatment regimen. In one embodiment, a treatment regimen may include administering recombinant human PTH(1-84) in the range of about 0.01 mg PTH / mL to 5 mg PTH / mL of injectable solution per patient, for example, in an injection volume of about 0.3 mL to about 2.3 mL, or about 0.5 mL to about 2 mL, or about 1 mL to about 1.75 mL, or about 1.2 mL, or about 1.3 mL, or about 1.4 mL, or about 1.5 mL, or about 1.6 mL, or about 1.7 mL. Thus, in one embodiment, purified and sterile-filtered PTH is incorporated with a buffer and excipients to form an aqueous solution containing PTH at a concentration range of 0.01 mg / mL to 5 mg / mL, or about 0.02 mg / mL to about 2.5 mg / mL, or about 0.025 mg / mL to about 1 mg / mL, or about 0.025 mg / mL to about 0.5 mg / mL, or about 0.025 mg / mL to about 0.25 mg / mL. In one embodiment, PTH is incorporated with a buffer and excipients to form an aqueous solution containing PTH at a concentration range, or about 0.025 mg / mL, or about 0.05 mg / mL, or about 0.075 mg / mL, or about 0.1 mg / mL.

[0049] If desired, molar equivalents of substantially equipotent forms of PTH, such as variants and fragments of PTH(1-84), can likewise be incorporated in place of human PTH(1-84).

[0050] In some embodiments, the composition of the present invention further comprises pharmaceutically acceptable excipients and / or carriers.Examples of suitable excipients are provided in Pramanick, S. et al., Excipient Selection in Parenteral Formulation Development, Pharma Times, 2013, 45, 3, 65-77, the contents of which are incorporated herein by reference in their entirety.Non-limiting examples of suitable excipients are shown below.

[0051] surfactants In some embodiments, the formulations disclosed herein further comprise a surfactant. In some embodiments, the surfactant may be selected from poloxamer (e.g., poloxamer 188), polyethylene glycol, cetyl hydroxyethyl cellulose, hydrophobically modified hydroxyethyl cellulose, polyoxyethylene glycol alkyl ether, polyoxypropylene glycol alkyl ether, glucoside alkyl ether, polyoxyethylene glycol alkylphenol ether, glycerol alkyl ester, polysorbate (e.g., polysorbate 20 and polysorbate 80), cocamide monoethanolamine (MEA), cocamide diethanolamine (DEA), dodecyl dimethylamine oxide, or any combination thereof. In one embodiment, the surfactant is selected from poloxamer 188, polysorbate 20, polysorbate 80, and polyethylene glycol, and combinations thereof.

[0052] In one embodiment, the surfactant is a poloxamer. In one embodiment, the surfactant is poloxamer 188.

[0053] The surfactant may be present at a concentration of about 0.01% to about 20% by weight, about 0.01% to about 15%, about 0.01% to about 10%, about 0.01% to about 5%, about 0.02% to about 4%, about 0.03% to about 3%, about 0.03% to about 1%, about 0.05% to about 0.5%, about 0.1% to about 0.5%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 2.5%, 0.1% to about 1%, or about 0.1% to about 0.7%, or about 0.1%, or about 0.2%, or about 0.3%, or about 0.4%, or about 0.5%. In one embodiment, the surfactant is poloxamer 188, which is present at about 0.3% w / v of the composition.

[0054] Isotonic agent In some embodiments, the composition of the present disclosure further comprises a tonicity agent. Tonicity is a measure of the effective osmotic gradient (defined by the water potential of the two solutions) between two solutions separated by a semipermeable membrane. Tonicity is commonly used to describe the response of cells immersed in an external solution. In other words, tonicity is the relative concentration of a solution, determining the direction and extent of diffusion. Body fluids typically have an osmotic pressure equivalent to that of a 0.9% sodium chloride solution. A composition (e.g., a solution or gel) is considered isotonic when its tonicity is approximately equal to that of a 0.9% sodium chloride solution (i.e., 290 mOsm / kg). A composition is isotonic with a body fluid solution when the magnitude of the salt is equal between the composition and the physiological solution. In physiological solutions, tonicity equilibrium is reached by water moving across the membrane while the salt remains in the original solution. A solution is isotonic with living cells when cells are in contact with the solution and there is no net gain or loss of water or other changes within the cell.

[0055] In some embodiments, the tonicity adjusting agent used in the compositions disclosed herein is an electrolyte, a monosaccharide or disaccharide, an inorganic salt (e.g., sodium chloride, calcium chloride, sodium sulfate, magnesium chloride), a polyol, or a combination thereof. In some embodiments, the tonicity adjusting agent is glucose, sucrose, sodium chloride, potassium chloride, calcium chloride, sodium sulfate, magnesium chloride, dextrose, mannitol, glycerol, or any combination thereof. In one embodiment, the tonicity adjusting agent is selected from sodium chloride, sucrose, and glycerol, or a combination thereof. In one embodiment, the tonicity adjusting agent is sucrose. In one embodiment, the tonicity adjusting agent is sodium chloride. In one embodiment, the tonicity adjusting agent is glycerol.

[0056] The tonicity agent may be present at any concentration necessary to achieve isotonic conditions. In some embodiments, the tonicity agent may be present at a concentration of about 0.01% to about 50%, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.02% to about 20%, about 0.03% to about 20%, about 0.05% to about 15%, about 0.1% to about 10%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 9%, about 0.2% to about 10%, 0.5% to about 10%, or about 1% to about 10%, or about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9% (w / v) of the composition. In one embodiment, the tonicity agent is sucrose, which is present at about 0.2% to about 20% of the composition, or about 8.5% (w / v) of the composition. In one embodiment, the tonicity agent is glycerol, which is present at about 0.2% to about 20% of the composition, or about 2.3% (w / v) of the composition. In one embodiment, the tonicity agent is sodium chloride, which is present at about 0.2% to about 20% of the composition, or about 0.8% (w / v) of the composition.

[0057] preservatives In some embodiments, the compositions of the present disclosure are sterile and preservative-free. In other embodiments, the compositions of the present disclosure optionally contain a preservative. In certain embodiments, the preservative is a paraben-free preservative. Parabens are a series of parahydroxybenzoates or esters of parahydroxybenzoic acid, which are known to cause cytokine release and stimulation and are associated with several types of cancer. Examples of parabens include methylparaben, ethylparaben, propylparaben, butylparaben, heptylparaben, isobutylparaben, isopropylparaben, benzylparaben, and their sodium salts.

[0058] Exemplary paraben-free preservatives include methylphenols (cresols), such as 3-methylphenol (metacresol or m-cresol), phenol, phenethyl alcohol, caprylyl glycol, phenoxyethanol, sorbate, potassium sorbate, sodium sorbate, sorbic acid, sodium benzoate, benzoic acid, acemannan, oleuropein, carvacrol, cranberry extract, gluconolactone, green tea extract, sunflower seed oil (Helianthus annuus seed oil), Lactobacillus ferment, Usnea barbata extract, polyaminopropyl biguanide, polyglyceryl-3 palmitate, polyglyceryl-6 caprylate, pomegranate extract, Populus tremuloides bark extract, resveratrol, rosemary leaf extract (Rosmarinus officinalis leaf extract), and the like. extract), benzyl alcohol, or any combination thereof.

[0059] In one embodiment, the preservative is selected from m-cresol, phenol, benzyl alcohol, sodium benzoate, and propylparaben, and combinations thereof. In one embodiment, the preservative comprises m-cresol.

[0060] In some embodiments, the compositions of the present disclosure comprise from about 0.005% to about 10% by weight of the composition, from about 0.005% to about 5%, from about 0.01% to about 5%, from about 0.02% to about 4%, from about 0.03% to about 3%, from about 0.05% to about 2%, from about 0.1% to about 1%, from about 0.2% to about 0.5%, from about 0.01% to about 10%, from about 0.01% to about 5%, from about 0.01% to about 1%. The composition may contain a preservative at a concentration of about 0.03% to about 3% of the composition. In one embodiment, the preservative is m-cresol, which is present at about 0.03% to about 3% of the composition. In one embodiment, m-cresol is present at 0.3% of the composition.

[0061] Pharmaceutically acceptable buffers In some embodiments, the compositions of the present invention may contain a pharmaceutically acceptable buffer by incorporating a buffering agent. In one embodiment, the buffering agent incorporated into the composition is selected from those capable of buffering the preparation to a pH within a physiologically acceptable range. A physiologically acceptable pH is one that causes minimal or no discomfort to the patient when the formulation is administered and, therefore, may vary depending on the method of administration. For preparations that are diluted prior to administration, such as by dissolution in a stock infusion solution, the pH of the preparation itself may vary widely, for example, from about pH 3 to about pH 9. For preparations to be administered immediately after reconstitution, PTH preparations are buffered within a pH range of 3.5 to 7.5. Therefore, suitable buffers are pharmaceutically acceptable agents capable of buffering the pH of the preparation within the desired pH range, including acetate buffer, phosphate buffer, L-histidine buffer, and succinate buffer.

[0062] While any pharmaceutically acceptable buffer may be suitable for formulations according to the present invention, it has surprisingly been found that the nature of the buffering agent has a significant impact on the stability of the rhPTH solution.

[0063] For example, the citrate buffer currently used in NATPARA® results in the formation of rhPTH protein particles after only 24 hours of stirring at ambient conditions. However, rhPTH solutions prepared in acetate, phosphate, and L-histidine buffers remain clear and colorless and free of visible particles even after 24 hours of stirring.

[0064] To provide a parathyroid hormone formulation stable during use according to the present invention, a selected buffering agent is incorporated to provide a final pH within the range of 3.5 to 6.5, with the buffer being present at a concentration of about 5 mM to about 50 mM. In some embodiments of the present invention, the pH provided by the buffering agent is in the range of 3.8 to 6.2, with the buffer concentration being about 10 mM to about 30 mM. In one embodiment, the pH of the formulation is 5.5. In one embodiment, the pH of the formulation is 4.3. In one embodiment, the buffer is an acetate buffer present at a concentration of about 20 mM. In one embodiment, the buffer is an L-histidine buffer present at a concentration of about 20 mM. In one embodiment, the buffer is a succinate buffer present at a concentration of about 20 mM.

[0065] antioxidants In some embodiments, the formulations of the present invention may further include one or more antioxidants to provide oxidative stability to the rhPTH protein during periods of use. Suitable antioxidants include, but are not limited to, acetone sodium bisulfite, argon, ascorbyl palmitate, ascorbic acid (salt / acid), sodium bisulfite, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), cysteine / cysteinate HCl, sodium dithionite (sodium hydrosulfite, sodium sulfoxylate), gentisic acid, gentisic acid ethanolamine, monosodium glutamate, glutathione, sodium formaldehyde sulfoxylate, potassium metabisulfite (potassium pyrosulfite), methionine, monothioglycerol (thioglycerol), nitrogen, propyl gallate, sodium sulfite, alpha (α)-tocopherol, alpha tocopherol hydrogen succinate, sodium thioglycolate, or a combination of two or more thereof. In one embodiment, the antioxidant can be methionine.

[0066] The antioxidant may be present at any concentration necessary to achieve oxidative stability of the formulation. In some embodiments, the antioxidant may be present at a concentration of about 0.0001% to about 20% by weight, about 0.001% to about 10%, about 0.01% to about 5%, about 0.01% to about 2%, about 0.02% to about 2%, about 0.03% to about 2%, about 0.05% to about 1.5%, or about 0.1% to about 1% (w / v). In one embodiment, the antioxidant is methionine present in an amount of about 0.015% to about 1.5% of the composition. In one embodiment, the antioxidant is methionine present in an amount of about 0.15% w / v of the composition.

[0067] Novel freeze-dried formulation In one embodiment, the parathyroid hormone compositions of the present invention are provided in lyophilized powder form containing 3% or less water by weight, which is obtained by lyophilization of a sterile aqueous hormone solution prepared by mixing the selected parathyroid hormone, a non-volatile buffer, and excipients.

[0068] In one embodiment of the present invention, the lyophilized composition is provided in a form that, upon reconstitution in about 1 to 1.5 mL (0.7 to 1.8 mL) of reconstitution vehicle, results in a unit dose of about 0.05 mg / mL to about 0.15 mg / mL of recombinant human PTH(1-84); therefore, the vial is filled with about 1 to 1.5 mL of aqueous PTH preparation for subsequent lyophilization.

[0069] In one embodiment of the present invention, the PTH preparation to be lyophilized contains 25-250 μg / mL human PTH(1-84), about 0.3 to about 30% w / v bulking agent, about 0.2 to about 20% w / v isotonicity agent, and a physiologically acceptable buffer in an amount sufficient to buffer the preparation to a pH within the range of 3.5 to 6.5 upon reconstitution in sterile water. In certain embodiments of the invention, the buffer is incorporated in an amount sufficient to buffer the pH to 5.5±0.3, or 4.3±0.3.

[0070] bulking agent In some embodiments, the novel freeze-dried formulations may further include one or more bulking agents for optimal cake structure and appearance. Suitable bulking agents include compatible carbohydrates, polypeptides, amino acids, or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, and sorbose; disaccharides such as lactose and trehalose; cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin; polysaccharides such as raffinose, maltodextrin, and dextran; and alditols such as mannitol and xylitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine. In one embodiment, the novel freeze-dried formulations may include one or more bulking agents selected from mannitol, glycine, poly(ethylene glycol), ammonium sulfate, sucrose, trehalose, and combinations thereof. In one embodiment, the novel freeze-dried formulations may include mannitol.

[0071] The bulking agent may be present at any concentration necessary to achieve optimal structure and appearance of the lyophilized powder. In some embodiments, the bulking agent may be present at a concentration of about 0.01% to about 50% by weight of the composition, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.02% to about 20%, about 0.03% to about 20%, about 0.05% to about 15%, about 0.1% to about 10%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 9%, about 0.2% to about 10%, 0.5% to about 10%, or about 1% to about 10%, or about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9% (w / v). In one embodiment, the bulking agent is mannitol, which is present in about 0.2% to about 20% of the composition, or about 2% to about 8% of the composition, or about 3% of the composition, or about 4% of the composition (w / v).

[0072] cryoprotectants In some embodiments, the novel lyophilized formulation may further comprise one or more cryoprotectants to provide stability to the rhPTH protein during the lyophilization process and product storage. Suitable cryoprotectants include compatible carbohydrates such as sugars and polyols. Suitable carbohydrates may include glucose, sucrose, trehalose, ethylene glycol, propylene glycol, 2-methyl-2,4-pentaglycol, and glycerol. In one embodiment, the novel lyophilized formulation may comprise one or more cryoprotectants selected from sucrose, glycine, mannitol, disaccharides, poly(ethylene glycol), and combinations thereof. In one embodiment, the novel lyophilized formulation may comprise sucrose.

[0073] The cryoprotectant may be present at any concentration necessary to achieve stability of the lyophilized powder. In some embodiments, the cryoprotectant may be present at a concentration of about 0.01% to about 50% by weight of the composition, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.02% to about 20%, about 0.03% to about 20%, about 0.05% to about 15%, about 0.1% to about 10%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 9%, about 0.2% to about 10%, 0.5% to about 10%, or about 1% to about 10%, or about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9% (w / v). In one embodiment, the cryoprotectant is sucrose, which is present at about 0.2% to about 20% of the composition, or about 1% to about 8% of the composition, or about 2% of the composition, or about 3% (w / v) of the composition.

[0074] Dosage form The compositions may be provided in single-dose or multi-dose injectable form, for example in the form of a pen. The compositions may be prepared by any suitable pharmaceutical method, as already mentioned, which includes the step of bringing into contact the active ingredient with the carrier (which may consist of one or more additional ingredients).

[0075] In certain embodiments, the pharmaceutical composition may be provided with a device for application (e.g., with a syringe, injection pen, or auto-injector, such as a Q-cliq pen). Such a device may be provided separately from the pharmaceutical composition or may be pre-filled with the pharmaceutical composition. [Example]

[0076] The following examples illustrate specific aspects of this description and should not be construed as limiting, as they merely provide specific understandings and implementations of the embodiments and their various aspects.

[0077] The formulations were prepared as follows: rhPTH(1-84) drug substance (active pharmaceutical ingredient) was exchanged into the respective base formulation buffer using dialysis methods commonly known to those skilled in the art. Further pH adjustment of the solution was performed using acidic or basic stock solutions, as needed. Stock solutions of excipients were prepared separately in the base buffer and mixed with the dialyzed peptide solution to create the final formulation with the desired peptide and excipient concentrations. The formulations were sterile filtered and filled into either glass vials or glass cartridges. Liquid formulations were stoppered and crimped before storage. The formulations for lyophilization were subjected to a preprogrammed lyophilization cycle consisting of freezing, annealing, primary drying, and secondary drying steps, followed by stoppering and crimping.

[0078] Example 1: Composition of a novel liquid formulation of rhPTH Table 1 below summarizes exemplary embodiments of novel liquid formulations of rhPTH according to the present invention. As shown in Table 1, liquid formulations #1-3 have the following compositions:

[0079] Liquid Formulation #1 underwater, 0.35–1.40 mg / mL rhPTH; 20mM acetate buffer; 10mM methionine; 130mM sodium chloride; 0.3 w / v% Poloxamer 188, and 0.3 w / v% m-cresol.

[0080] Liquid Formulation #2 underwater, 0.35–1.40 mg / mL rhPTH; 20mM acetate buffer; 10mM methionine; 8.5w / v% sucrose; 0.3 w / v% Poloxamer 188, and 0.3 w / v% m-cresol.

[0081] Liquid Formulation #3 underwater, 0.35–1.40 mg / mL rhPTH; 20mM acetate buffer; 10mM methionine; 2.3v / v% glycerol; 0.3 w / v% Poloxamer 188, and 0.3 w / v% m-cresol.

[0082] The pH of formulations #1 to #3 is 5.5.

[0083] [Table 1]

[0084] Example 2: Composition of a novel lyophilized powder formulation of rhPTH Table 2 below summarizes exemplary embodiments of novel lyophilized powder formulations of rhPTH according to the present invention. As shown in Table 2, lyophilized formulations #1 to #3 have the following compositions:

[0085] Lyophilized Formulation #1 underwater, 0.35–1.40 mg / mL rhPTH; 20mM L-histidine buffer; 4 w / v% mannitol, and 2% sucrose.

[0086] Lyophilized formulation #2 underwater, 0.35–1.40 mg / mL rhPTH; 20mM L-histidine buffer; 10mM methionine; 4w / v% mannitol; 2% w / v sucrose, and 0.3 w / v% Poloxamer 188.

[0087] Lyophilized formulation #3 underwater, 0.35–1.40 mg / mL rhPTH; 20mM succinate buffer; 10mM methionine; 3 w / v% mannitol, and 3 w / v% sucrose.

[0088] The pH of lyophilized formulations #1 and #2 is 5.5. The pH of lyophilized formulation #3 is 4.3.

[0089] [Table 2]

[0090] Example 3: Agitation studies of rhPTH formulated in various buffers Agitation (shaking) of actual drug product storage containers / closures or small-scale representative primary containers is often applied in protein drug development to serve as a test of stability under physical stress conditions encountered in real-world processes. The overall purpose of these "stress tests" is to accelerate protein degradation / aggregation, which would otherwise occur at a much slower rate, thereby increasing experimental throughput and speeding up the determination of stability-critical process parameters. The results are useful for determining key parameters in formulation development.

[0091] Table 3 below and Figure 2 show appearance data from agitation studies of rhPTH formulated with various buffers. rhPTH was formulated in various buffers (10 mM) containing sodium chloride (140 mM) in 2R glass vials. Agitation studies were conducted at ambient conditions for at least 48 hours, using an orbital shaker at 220 rpm with the vials in a horizontal position. At regular agitation intervals, the appearance of the agitated samples was compared to reference suspensions (RS I-IV) and a standard of opalescence (SOP) according to standard procedures, such as those outlined in the European Pharmacopoeia (European Pharmacopoeia 5.0, 2.2.1 "Clarity and Degree of Opalescence in Liquids"). Figure 1 shows the opalescence of reference suspensions RS I-IV and SOP. Water was used for comparison. Figure 2 shows the appearance of suspensions of rhPTH formulated in various buffers.

[0092] [Table 3]

[0093] As shown in Table 3 and Figure 2, acetate buffer exhibits the best stability against agitation-induced particle formation of rhPTH, followed by phosphate buffer and then L-histidine buffer, and all three buffers exhibit better stability than the citrate buffer currently used in the NATPARA® formulation.

[0094] Example 4: Agitation Test of rhPTH in a Novel Liquid Formulation Table 4 below shows appearance data from agitation testing of various liquid formulations of rhPTH according to the present invention. Liquid formulations #1-#3 were compounded in dual-chamber cartridges and agitation testing (220 rpm, orbital shaking) was performed at ambient conditions. At regular agitation intervals, the appearance of the agitated samples was compared to reference suspensions (RS I-IV) and opalescence standards (SOP) according to standard procedures. Data from the commercially available NATPARA® formulation are also provided for comparison. The opalescence at various time points is summarized in Table 4.

[0095] [Table 4]

[0096] As shown in Table 4, all three novel liquid formulations #1-#3 remained clear and free of visible particles for at least 72 hours. In contrast, the current commercial formulation showed significant particle presence and higher opalescence as early as just a 5-hour stirring period.

[0097] Example 5: Stirring test of rhPTH in a novel freeze-dried formulation Table 5 below shows appearance data from agitation testing of various reconstituted lyophilized formulations of rhPTH according to the present invention. Lyophilized formulations #1-#3 were compounded in dual-chamber cartridges and subjected to agitation testing (220 rpm, orbital shaking) at ambient conditions. At regular agitation intervals, the appearance of the agitated samples was then compared to reference suspensions (RS I-IV) and opalescence standards (SOP) according to standard procedures. Data from the commercially available NATPARA® formulation are also provided for comparison. The opalescence at various time points is summarized in Table 5.

[0098] [Table 5]

[0099] As the above examples demonstrate, the novel lyophilized formulations can be observed to significantly improve the physical stability of rhPTH. As shown above, the novel formulations of rhPTH remain clear, colorless, and free of visible particles for at least 24 hours, and / or at least 48 hours, and / or at least 72 hours, and / or at least 90 hours.

[0100] Example 6: In-use stability testing of liquid and lyophilized formulations Table 6 below shows appearance data from in-use testing of a liquid formulation according to an embodiment of the invention, exemplified by Liquid Formulation #2, and a reconstituted lyophilized formulation of rhPTH according to an embodiment of the invention, exemplified by Lyophilized Formulation #2.

[0101] [Table 6]

[0102] As shown in Table 6 above, the novel liquid and lyophilized formulations of rhPTH, exemplified by Liquid Formulation #2 and Lyophilized Formulation #2, remain clear and free of visible particles for an in-use period of at least 1 day, or at least 7 days, or at least 14 days, or at least 21 days.

[0103] Example 7: Solution pH Screening of rhPTH for Optimal Physicochemical Stability Recombinant human parathyroid hormone was formulated in 10 mM citrate buffer containing 140 mM sodium chloride at a solution pH range of 3.5 to 7.5 in 0.5 pH unit intervals. Samples were dispensed into 2 mL type I borosilicate glass vials and maintained at temperatures of 5 ± 3°C (5°C), 25 ± 2°C (25°C), and 40 ± 2°C (40°C). At predetermined intervals, samples were withdrawn, observed visually, and analyzed for rhPTH stability using chromatographic assays (size exclusion chromatography (SEC) and reverse-phase chromatography (RP-HPLC)) with some modifications, as appropriate.

[0104] The supplied drug substance was thawed and dialyzed against each pH buffer solution in a 2 kDa molecular weight cut-off (MWCO) dialysis cassette. Dialysis was performed at 5 ± 3°C and included at least three buffer exchange cycles over approximately 24 hours. After dialysis, the pH of the sample was checked and adjusted with 0.2 N sodium hydroxide, if necessary. A280 measurements were performed and found to be 0.584 (mL / mg). -1 cm -1 The rhPTH concentration was calculated using the extinction coefficient of . The final solutions were prepared aseptically in a laminar flow hood. For each solution pH, rhPTH was prepared at a concentration of 1.0 mg / mL using the respective buffer as the dilution medium. The prepared samples were filtered through a 0.22 μm PVDF filter and filled into 2 mL Type I borosilicate glass vials at a volume of 1.5 mL, followed by stoppering and crimping.

[0105] Each vial was observed for solution appearance on a light box. A baseline sample was separated, aliquoted into polypropylene tubes, and stored at -80°C. The remaining vials were incubated at 5°C, 25°C, and 40°C. At predetermined intervals, sample vials were withdrawn from each incubation condition, observed for appearance, aliquoted into polypropylene tubes, and stored at -80°C until analysis. Samples were tested for physical and chemical changes using Natpara® validated assays, including SEC and RP-HPLC, with several modifications to injection volume and injection order.

[0106] To demonstrate physical stability, Tables 7 and 8 show the appearance results of rhPTH stability samples stored for 6 months at 40°C and 25°C, respectively. When measured, opalescence is recorded compared to the reference suspension. White, particulate flocculants were observed in the pH 7.0 and 7.5 samples within 2 weeks of storage at 40°C. This particle formation appeared to progress over time from the basic to the acidic end of the solution pH. By 3 months, most samples stored at 40°C contained particles. Samples stored at 25°C showed a similar trend toward particle formation as observed at 40°C, but the reaction rate was slower. Particle size also varied depending on the solution pH. Samples formulated in the pH range of 6.5 to 7.5 contained aggregates, while samples at lower pHs contained fine particles. Samples stored at 5°C maintained their initial appearance of clear, colorless fluid with no visible particles for 6 months.

[0107] [Table 7]

[0108] [Table 8]

[0109] To demonstrate chemical stability, Table 9 provides protein concentration data for stability samples stored at 40°C and 25°C, respectively. Samples were thoroughly centrifuged (17,000g for 5 minutes) and the supernatant was used for A280 measurements. Appropriate light scattering correction (A320 subtraction) was performed. The decrease in protein concentration at 40°C roughly correlated with the tendency of the samples to form particles during storage. No change in protein concentration over time was observed for samples stored at 25°C (Table 9) and 5°C.

[0110] [Table 9]

[0111] To further demonstrate chemical stability, Figures 3-8 show RP-HPLC data for rhPTH and associated impurities for pH screening samples stored for up to 6 months at 40 °C, 25 °C, and 5 °C. At the 40 °C storage condition, only data up to 1 month are shown because the samples subsequently deteriorated too much to perform peak integration.

[0112] Main Peak: As shown in Figures 3A-3C, a bell-shaped trend for the main peak was observed at all storage temperatures, with maximum peak recovery occurring around pH 5.0-6.0.

[0113] Oxidized Met8: The oxidation of Met8 was also observed to follow a bell-shaped trend (similar to the main peak). When stored at 40°C and 25°C, maximum Met8 oxidation was observed in the pH range of approximately 4.0–5.5. At 5°C, no trend was observed up to 6 months of storage, as shown in Figures 4A–4C.

[0114] Oxidized Met18: The oxidation rate of Met18 was found to be highest toward the basic solution pH range and gradually decrease as the solution pH became more acidic. As shown in Figures 5A-5C, this trend was primarily observed at both 40 °C and 25 °C storage conditions.

[0115] IsoAsp33: The formation of isoaspartic acid from asparagine 33 was observed to be minimized as the formulation pH became more acidic and to increase significantly once the solution pH exceeded approximately 5.5. This trend was evident at all storage temperatures, as shown in Figures 6A-6C.

[0116] rhPTH((1-30)+(1-33)): These rhPTH impurities increased significantly with storage in samples formulated at pHs below 5.0 and above 6.0. However, the increase in impurities at pHs above 6.0 occurred to a much lesser extent than that observed at lower pH values. As shown in Figures 7A-7C, this increase in impurities was observed to be minimal in the pH range of 5.0-6.0.

[0117] rhPTH(1-45): This fragment-related impurity was observed to increase significantly in samples formulated at pHs below 5.0 and did not change significantly between pH 5.0 and 7.5. This trend was observed at all storage temperatures, as shown in Figures 8A-8C.

[0118] This example demonstrates the effect of solution pH on the physicochemical stability of rhPTH when formulated in the pH range of 3.5 to 7.5 and exposed to thermal stress. Physical stability characteristics observed using appearance (visible particle formation) and SEC (aggregate and fragment formation), and chemical stability characteristics observed using RP-HPLC (oxidation, deamidation, and fragmentation) suggest that a solution pH range of 5.0 to 6.0 is optimal for the physical and chemical stability of rhPTH.

[0119] Example 8: Excipient screening of rhPTH for optimal physicochemical stability Recombinant human parathyroid hormone (rhPTH) was formulated in a 20 mM sodium acetate buffer solution at pH 5.5 with 50 mM sodium chloride (NaCl). Excipient stocks were added to this base formulation to achieve the desired target levels of a given excipient. Samples were stabilized at temperatures of 5 ± 3°C (5°C), 25 ± 2°C (25°C), and 40 ± 2°C (40°C). At predetermined intervals, samples were removed, observed visually, and analyzed for rhPTH stability using reverse-phase high-performance liquid chromatography (RP-HPLC). A baseline (time 0 (t0)) sample was also subjected to multiple freeze-thaw cycles and orbital agitation, and the solution visually observed.

[0120] Visual data from static storage heat stress, freeze-thaw stress, and agitation stress indicated that the presence of arginine and high levels (≥150 mM) of NaCl resulted in significant levels of visible particle formation compared to other excipients. RP-HPLC stability data showed significantly higher levels of oxidized Met8 and Met18 in samples containing glycine, lysine, or arginine at all incubation temperatures. Meanwhile, samples containing methionine exhibited significantly reduced rates of rhPTH oxidation. Agitation testing results indicated that the presence of the surfactant poloxamer 188 prevented the formation of visible particles upon shaking.

[0121] The supplied drug substance was thawed and dialyzed against base buffer solution in a 2 kDa MWCO dialysis cassette. Dialysis was performed at 5 ± 3°C and included at least three buffer exchange cycles over approximately 30 hours. After dialysis, the pH of the sample was checked and adjusted with 0.2 N sodium hydroxide, if necessary. An A280 measurement was performed and found to be 0.584 (mL / mg). -1 cm -1The concentration of rhPTH was calculated based on the extinction coefficient of . Preparation of the final solution was performed aseptically in a laminar flow hood. For each excipient, rhPTH was prepared at a concentration of 1.0 mg / mL by using base buffer as the dilution medium and adding excipient stock to achieve the desired excipient concentration. Additionally, m-cresol was added to each formulation at a level of 0.3% (v / v).

[0122] Table 10 provides a description of the various formulations used in the excipient screening study. Prepared samples were filtered through 0.22 μm PVDF filters and filled into 2R Type I glass vials at 1.5 mL volumes, followed by stoppering / crimping. Each vial was observed for solution appearance under a light box. A baseline sample was aliquoted into polypropylene tubes and stored at -80°C. The remaining vials were incubated at 5°C, 25°C, and 40°C. At predetermined intervals, sample vials were withdrawn from each incubation condition, observed for appearance, aliquoted into polypropylene tubes, and stored at -80°C until further analysis. Samples were tested for physical and chemical changes using Natpara-validated assays, including SEC and RP-HPLC, with some modifications to injection volume and injection order.

[0123] Baseline samples were subjected to repeated freeze / thaw cycles (freezing at -80°C for 5-12 hours and thawing at room temperature) and the appearance of the solutions was observed in a light box. A different set of baseline samples in vials was agitated horizontally under ambient temperature conditions using an orbital shaker at 220 rpm and the appearance of the solutions was observed at regular intervals in a light box. Preliminary results from the agitation studies were used to select additional formulations for orbital agitation in 2R vials and dual-chamber cartridges.

[0124] [Table 10]

[0125] Tables 11-13 below show the appearance results for rhPTH stability samples stored at 40°C, 25°C, and 5°C, respectively, for up to 6 months. Opalescence, when measured, is recorded relative to the reference suspension. When stored at 40°C, samples containing arginine (150 mM) showed a significant presence of proteinaceous particles that appeared at 2 weeks and increased over time. Samples containing other excipients showed an appearance comparable to baseline up to 3 months when stored at 40°C. By the end of 6 months of storage at 40°C, most samples had visible particles and opalescence of various colors. Similarly, for samples stored at 25°C, the solution containing arginine (150 mM) was the first sample to show particle formation, which did not appear until 6 months of storage; all other samples maintained their baseline appearance. Samples stored at 5°C exhibited an appearance similar to baseline at the end of 3 months of storage, but unlike the results at 25°C, by the end of 6 months many samples (particularly NaCl, glycerol, glycine, lysine, and arginine) had visible particles.

[0126] [Table 11]

[0127] [Table 12]

[0128] [Table 13]

[0129] To demonstrate chemical stability, Figures 9-12 show RP-HPLC data for rhPTH and related impurities for samples formulated in acetate buffer, pH 5.5, containing 50 mM NaCl with various excipients and stored at 40°C, 25°C, and 5°C. Results are shown for samples where reasonable peak integration was possible without shifts in the reported relative retention times.

[0130] Main Peak: Samples containing glycine, lysine, and arginine showed a significantly faster decrease in the main peak compared to the other excipients upon storage at both 40°C and 25°C. A similar trend was observed upon storage at 5°C. See Figures 9A-9C.

[0131] Oxidized Met8 and Met18: Compared to the other excipients, glycine, lysine, and arginine samples exhibited significantly higher levels of oxidized Met8 and Met18 at all storage temperatures. Samples containing methionine showed the least change in Met8 and Met18 oxidation over time at all storage temperatures. See Figures 10A-10C (Met8) and Figures 11A-11C (Met18).

[0132] IsoAsp33: When stored at 25°C and 40°C, samples containing 150 mM and 300 mM NaCl exhibited slightly lower rates of IsoAsp33 formation, but no significant differences were observed between excipients. (The significantly lower and inconsistent IsoAsp33 levels observed in the glycine, lysine, and arginine samples may be due to issues integrating missing / slightly shifted peaks in the chromatograms of these samples.) See Figures 12A-12C.

[0133] Freeze-thaw (F / T) test Table 14 shows the appearance of solutions of various formulations upon repeated freeze-thaw cycles in 2R vials. Visible particles, if observed, are reported along with opalescence. Samples with 150 mM NaCl or higher were significantly affected by repeated freeze-thaw cycles and were found to contain white, fibrous, proteinaceous particles. Samples containing 0.02% PS20 initially exhibited a granular appearance due to sand-like (non-proteinaceous) particles. Solutions containing 8% glycerol showed a worsening of opalescence after each freeze-thaw cycle without the formation of visible particles.

[0134] [Table 14]

[0135] Stirring test Table 15 shows the appearance results from agitation tests conducted on three 2R vials in a horizontal position at 220 rpm under ambient conditions. All samples were clear and colorless at baseline, with no visible particles present, except for PS20, which had sand-like particles present. Samples containing NaCl showed the earliest signs of particle formation, the rate of which increased with increasing NaCl content. By 24 hours, samples containing 150 mM NaCl and PS20 had developed a cloudy appearance. All samples, except those containing poloxamer 188 (P-188), showed a cloudy appearance by the end of 48 hours. The P-188 sample maintained its baseline appearance until the end of the test (72 hours).

[0136] [Table 15]

[0137] Based on the preliminary results from the storage stability, freeze-thaw, and agitation studies described above, the formulation was narrowed down and NaCl, mannitol, sucrose, and glycerol were identified as excipients due to their stabilizing / isotonicity capabilities, along with methionine and m-cresol to reduce oxidation and support multi-dose formulations, respectively.

[0138] Table 16 shows the results of horizontal agitation tests conducted in 2R vials at 220 rpm under ambient conditions with NaCl removed from the base formulation. The presence of m-cresol resulted in opalescence formation significantly earlier than in formulations without m-cresol. Despite the removal of 50 mM NaCl from the base formulation, all solutions, except those containing poloxamer 188, still developed a cloudy appearance by the end of 48 hours of agitation. These formulations were also subjected to agitation in the container / closure currently used for the commercially available Natpara® (1 mL siliconized cartridge with siliconized middle and end rubber stoppers and aluminum seals, using a 1.1 mL formulation fill volume). Appearance results similar to those obtained with agitation in 2R vials (Table 15) were observed, with poloxamer 188 significantly preventing / delaying particle formation.

[0139] [Table 16]

[0140] All tested formulations in Table 16 maintained their baseline (clear) appearance at the end of 72 hours (2R vials) or 48 hours (1 mL cartridges) when shaken at 2-8°C.

[0141] As the examples demonstrate, sodium chloride, sucrose, mannitol, and glycerol are suitable excipients for providing stability to rhPTH.Methionine shows a high potential for significantly inhibiting peptide oxidation.Poloxamer 188 has been found to be important for preventing the formation of visible particles when stirred.

[0142] Example 9: Formulation optimization study of rhPTH aimed at liquid dosage forms Recombinant human parathyroid hormone was formulated in a pH 5.5 solution containing 20 mM acetate buffer and 0.3% (w / v) m-cresol. This base formulation was prepared with varying levels of methionine (antioxidant) and poloxamer 188 (surfactant), excipients identified as important for rhPTH stability during initial formulation screening (see Example 7). Sodium chloride, sucrose, glycerol, and mannitol were evaluated to render the formulation isotonic and further improve formulation stability. To screen excipients and optimize their concentrations, samples were subjected to heat and agitation stress. For heat stress, samples were stabilized in 2R type I glass vials at 5 ± 3°C (5°C), 25 ± 2°C (25°C), and 40 ± 2°C (40°C). At predetermined intervals, samples were removed, observed visually, and analyzed for rhPTH stability using reverse-phase high-performance liquid chromatography (RP-HPLC). For agitation stress, baseline samples in 2R Type I glass vials and 1 mL siliconized dual-chamber cartridges were separately subjected to orbital agitation, and the appearance of the solutions was observed over time.

[0143] Upon heat stress, no differences in the oxidation profile of rhPTH were observed between formulations containing 50 mM, 25 mM, and 10 mM methionine. The formation of visible particles in rhPTH solutions upon agitation was not observed to be dependent on the concentration of poloxamer 188. Stabilizers / tonics in the form of NaCl, sucrose, and glycerol were selected from a combination of chemical and physical changes in the molecules observed upon heat and agitation stress performed during preliminary excipient screening (see Example 7). The concentrations of the stabilizers / tonics were selected to render the solution isotonic. Overall, three formulation matrices aimed at liquid dosage forms were identified: a) pH 5.5, 20 mM acetate buffer, 10 mM methionine, 0.3 w / v% poloxamer 188, 130 mM sodium chloride, 0.3 w / v% m-cresol b) pH 5.5, 20 mM acetate buffer, 10 mM methionine, 0.3 w / v% poloxamer 188, 8.5 w / v% sucrose, 0.3 w / v% m-cresol c) pH 5.5, 20 mM acetate buffer, 10 mM methionine, 0.3 w / v% poloxamer 188, 2.3 v / v% glycerol, 0.3 w / v% m-cresol

[0144] The supplied drug substance was thawed and dialyzed against buffer in a 2 kDa MWCO dialysis cassette. Dialysis was performed at 5 ± 3°C and included at least three buffer exchange cycles over approximately 24-48 hours. After dialysis, the pH of the sample was checked and adjusted with 0.2 N sodium hydroxide, if necessary. A280 measurements were performed and found to be 0.584 (mL / mg). -1 cm -1 The concentration of rhPTH was calculated based on the extinction coefficient of . The final solution was prepared aseptically in a laminar flow hood. rhPTH was prepared at a concentration of 1.0 mg / mL by using base buffer as the dilution medium and adding excipient stocks to achieve the desired excipient concentration. Additionally, m-cresol was added to each formulation at a level of 0.3 w / v%.

[0145] Optimization of Methionine and Poloxamer 188 Concentrations: Table 17 provides a description of the various formulations used in the methionine and P-188 concentration optimization studies.

[0146] [Table 17]

[0147] Stabilizer / Tonicity Agent Optimization: Table 18 provides a description of the various formulations used to evaluate the effect of stabilizers / tonicity agents on the stability of rhPTH.

[0148] [Table 18]

[0149] The samples were filtered through a 0.22 μm PVDF filter and filled into 2R Type I glass vials (for agitation) at 1.5 mL volume or into 2R Type I glass vials (for storage stability) at 1 mL volume, followed by stoppering / crimping. Each vial was observed for solution appearance on a light box. All baseline samples in Tables 17 and 18 were subjected to horizontal agitation at ambient temperature using an orbital shaker at 220 rpm, and the solution appearance was observed at regular intervals on the light box.

[0150] Samples from Table 17 containing 0.3% poloxamer 188 with 0 mM, 10 mM, 25 mM, and 50 mM methionine, as well as samples from Table 18, were also placed on shelf-stable storage. The baseline sample was separated, aliquoted into polypropylene tubes, and stored at -80°C. The remaining vials were incubated at 5°C, 25°C, and 40°C. At predetermined intervals, sample vials were removed from each incubation condition, observed visually, aliquoted into polypropylene tubes, and stored at -80°C until analysis. Samples were tested for physical and chemical changes using Natpara-validated assays, including SEC and RP-HPLC, with several modifications to injection volume and injection order.

[0151] Appearance: Table 19 shows the appearance results of rhPTH stability samples with various levels of methionine concentration when stored for up to 6 months at 40° C., 25° C., and 5° C. All samples remained clear and colorless and free of visible particles throughout the study period.

[0152] [Table 19]

[0153] RP-HPLC data over the storage stability period of rhPTH formulated at various methionine concentrations shows a significant reduction in peptide oxidation when methionine is included as part of the formulation, although within assay variability, no significant differences were observed in the percentage of Met8 and Met18 oxidation peaks or the main peak between the various methionine concentrations examined.

[0154] Optimizing the concentration of Poloxamer 188 Agitation studies were used to optimize the concentration of poloxamer 188. Tables 20-22 show the appearance results of samples containing various concentrations of poloxamer 188 (formulated with various methionine contents - Table 17) in 2R vials subjected to horizontal orbital agitation at 220 rpm under ambient conditions.

[0155] [Table 20]

[0156] [Table 21]

[0157] [Table 22]

[0158] Selection of stabilizer / tonicity agent Sodium chloride (NaCl), sucrose, glycerol, and mannitol were selected as suitable excipients during rhPTH excipient screening studies (Example 8). The concentrations to be used in future formulations were selected based on an osmolality goal of 250-350 mOsm / kg.

[0159] All samples were clear to minimal opalescence over the test period without the presence of visible particles (Table 23).

[0160] [Table 23]

[0161] RP-HPLC data for rhPTH formulated in 20 mM acetate buffer containing 25 mM methionine, 0.3% P-188, and 0.3% m-cresol with various stabilizers / isotonicity agents upon storage at 40°C, 25°C, and 5°C show no significant changes in the levels of oxidized Met8 and oxidized Met18 among the various excipients used at any incubation temperature. The same trend was observed upon storage at 5°C. The rate of IsoAsp33 formation was similar for all excipients examined, except for NaCl, which contained significantly less IsoAsp33. The NaCl formulations exhibited significantly less formation of an unidentified tailing peak, as well as lower levels of IsoAsp33, resulting in the highest recovery of the main peak compared to the other excipients.

[0162] Agitation Testing: Formulations containing various stabilizers (Table 18) were subjected to orbital agitation at 220 rpm in 2R vials and siliconized cartridges under ambient conditions. Table 24 shows appearance results from one exemplary test day with multiple replicates in 2R vials. In some cases, the three replicates of an individual vial did not exhibit the same appearance profile during agitation; the worst appearance observation is reported.

[0163] All of these formulations remained clear and free of visible particles at the end of 72 hours when stirred horizontally at 220 rpm under ambient conditions in a siliconized cartridge.

[0164] [Table 24]

[0165] Of the formulations tested, the control sample and the samples containing sucrose and glycerol exhibited the best visual appearance profiles when agitated.

[0166] In conclusion, heat stress did not reveal any differences in the oxidation profile of rhPTH between formulations containing 50 mM, 25 mM, and 10 mM methionine. The formation of visible particles in rhPTH solutions upon agitation was not observed to be dependent on the concentration of poloxamer 188. Stabilizers / tonics in the form of NaCl, sucrose, and glycerol were selected from a combination of chemical and physical changes in molecules observed upon heat and agitation stress during preliminary excipient screening (see Example 7). The concentrations of the stabilizers / tonics were selected to render the solution isotonic. Based on the overall data from heat and agitation stress, the top three formulation matrices targeted for liquid dosage forms were identified: a) pH 5.5, 20 mM acetate buffer, 10 mM methionine, 0.3 w / v% poloxamer 188, 130 mM sodium chloride, 0.3 w / v% m-cresol b) pH 5.5, 20 mM acetate buffer, 10 mM methionine, 0.3 w / v% poloxamer 188, 8.5 w / v% sucrose, 0.3 w / v% m-cresol c) pH 5.5, 20 mM acetate buffer, 10 mM methionine, 0.3 w / v% poloxamer 188, 2.3 v / v% glycerol, 0.3 w / v% m-cresol

[0167] The target concentration of rhPTH in these formulations ranges from 0.35 mg / mL to 1.4 mg / mL.

[0168] Example 10: Development of a multi-dose lyophilized rhPTH pharmaceutical formulation for subcutaneous delivery A reformulation study was conducted to understand the effects of pH, buffers, surfactants, and stabilizers / bulking agents on the chemical and physical stability of lyophilized rhPTH(1-84). The chemical stability of rhPTH(1-84) was significantly affected by solution pH, with optimal stability observed in the pH range of 5.0 to 6.5. Lower pH (4.0 to 4.5) significantly increased the fragmentation of rhPTH(1-84) but improved its stability against shaking-induced particle formation. At higher pH (above 6.5), reconstituted lyophilized formulations of rhPTH(1-84) became increasingly susceptible to particle formation.

[0169] At an optimal solution pH of 5.5, formulations containing L-histidine and phosphate buffers showed significant improvement in the resistance to visible particle formation when shaken in 2R vials and siliconized cartridges compared with formulations containing citrate buffer. The addition of poloxamer 188 to the L-histidine formulation at pH 5.5 further improved the stability of rhPTH(1-84) against shaking-induced particle formation.

[0170] Succinate buffer at pH 4.0–4.3 was also identified as another buffer candidate, as it appears to offer complete protection against shaking-induced particle formation, although it has poorer chemical stability compared to other buffers at pH 5.5.

[0171] Overall, the results of these screening studies helped identify three lead lyophilized rhPTH(1-84) formulation candidates for further evaluation in the current commercially available dual-chamber cartridge. The selection of these formulations was primarily based on the results of real-time, accelerated, and severe storage stability studies, as well as shaking-induced stress tests after reconstitution with 0.3% (v / v) m-cresol in water. The three lyophilized formulation candidates consisted of the following, which required reconstitution with WFI containing 0.3% (w / v) m-cresol prior to use: (1) 1 mg / mL rhPTH(1-84) in 20 mM L-histidine, pH 5.5, containing 4% (w / v) mannitol and 2% (w / v) sucrose (2) 1 mg / mL rhPTH(1-84) in 20 mM L-histidine at pH 5.5 containing 4% (w / v) mannitol, 2% (w / v) sucrose, and 0.3% (w / v) poloxamer 188 (3) 1 mg / mL rhPTH(1-84) in 20 mM succinate at pH 4.3 containing 3% (w / v) mannitol and 3% (w / v) sucrose

[0172] To monitor the chemical stability of rhPTH(1-84), reverse-phase high-performance liquid chromatography (RP-HPLC) was used to quantify impurities associated with oxidation, deamidation, fragmentation, and other degradation pathways. Size-exclusion chromatography (SEC) was used to quantify the primary rhPTH(1-84) molecule as well as all high- and low-molecular-weight species.

[0173] To assess the physical stress of rhPTH(1-84), orbital agitation was used, and the results were evaluated visually. All formulations were reconstituted with 0.3% (v / v) m-cresol in water and shaken at room temperature in a horizontal position using an orbital shaker set at 220 rpm.

[0174] The water content measured using the Karl Fischer method is summarized in Table 25. All formulations had a water content of less than 2%, except for the formulation containing 100 mM sodium chloride and 5% sucrose. A water content of less than 2% is significantly lower than the water content specifications for commercial pharmaceuticals and therefore does not pose a stability problem for rhPTH(1-84).

[0175] [Table 25]

[0176] Based on the collective results, a formulation consisting of 30 mM sodium chloride and 5% (w / v) sucrose with a Tg' of approximately -39°C was selected for the initial freeze-drying studies because it produced excellent cake appearance at low moisture content.

[0177] A formulation study of lyophilized rhPTH(1-84) identified an optimal pH range of 5.0–6.0, which minimized chemical degradation of rhPTH(1-84). Although rhPTH(1-84) degraded significantly faster at lower pH conditions and elevated temperatures of 25°C and 40°C, the study also confirmed that a pH of 4.0–4.3 for rhPTH(1-84) formulations maintained chemical stability for up to 6 months at 5°C storage and significantly reduced shaking-induced particle formation, making it still viable. Following extensive lyophilized formulation screening, coupled with concurrent liquid formulation development of rhPTH(1-84), three primary lyophilized formulations of rhPTH(1-84) were selected for evaluation in the current commercially available silicone-coated dual-chamber cartridge. The formulations selected were based on their 3-month stability at accelerated and harsh conditions at 25°C and 40°C, respectively, and their effect on the stability of rhPTH(1-84) against shaking-induced particle formation after reconstitution with 0.3% (v / v) aqueous m-cresol. The three formulations selected were:

[0178] Further optimization studies showed that the addition of 10 mM methionine significantly improved the stability of rhPTH(1-84) against oxidation of Met8 and Met18 residues in Formulation 2, and against aggregation in Formulation 3 above.

[0179] Since various changes can be made in the above subject matter without departing from the scope and spirit of the invention, it is intended that all subject matter contained in the above description or set forth in the appended claims be considered illustrative and exemplary of the invention. Many modifications and variations of the present invention are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications, and variations that fall within the scope of the appended claims.

[0180] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein. Finally, preferred embodiments of the present invention are described in sections.

[0181] [Embodiment 1] 1. A pharmaceutical formulation comprising: (a) a therapeutically effective amount of recombinant human parathyroid hormone (rhPTH(1-84)); (b) surfactants; (c) tonicity agent; (d) antioxidants; (e) preservatives; (f) a physiologically acceptable buffer; and (g) water wherein the pharmaceutical formulation is formulated as an injectable solution and remains physically stable, clear, and free of visible particles for at least 48 hours.

[0182] [Embodiment 2] 2. The pharmaceutical formulation of embodiment 1, which is physically stable for at least 72 hours.

[0183] [Embodiment 3] 2. The pharmaceutical formulation of embodiment 1, which is physically stable for at least 96 hours.

[0184] [Embodiment 4] 2. The pharmaceutical formulation of embodiment 1, which is physically stable for at least 7 days.

[0185] [Embodiment 5] 2. The pharmaceutical formulation of embodiment 1, which is physically stable for at least 14 days.

[0186] [Embodiment 6] 2. The pharmaceutical formulation of embodiment 1, which is physically stable for at least 21 days.

[0187] [Embodiment 7] 2. The pharmaceutical formulation of embodiment 1, wherein the surfactant is selected from poloxamer 188, and polyethylene glycol, and combinations thereof.

[0188] [Embodiment 8] 2. The pharmaceutical formulation of embodiment 1, wherein the tonicity agent is selected from sodium chloride, sucrose, and glycerol, and combinations thereof.

[0189] [Embodiment 9] 2. The pharmaceutical formulation of embodiment 1, wherein the preservative is m-cresol, phenol, benzyl alcohol, sodium benzoate, propylparaben, or a combination thereof.

[0190] [Embodiment 10] 2. The pharmaceutical formulation of embodiment 1, wherein the physiologically acceptable buffer is an acetate buffer, a phosphate buffer, an L-histidine buffer, or a succinate buffer.

[0191] [Embodiment 11] 2. The pharmaceutical formulation of embodiment 1, further comprising an antioxidant.

[0192] [Embodiment 12] 12. The pharmaceutical formulation of embodiment 11, wherein the antioxidant is methionine, N-acetyl-methionine, thiosulfate, N-acetyltryptophan, or a combination thereof.

[0193] [Embodiment 13] 2. The pharmaceutical formulation of embodiment 1, having a pH of about 4 to about 6.

[0194] [Embodiment 14] 2. The pharmaceutical formulation of embodiment 1, having a pH of about 5.5.

[0195] [Embodiment 15] 2. The pharmaceutical formulation of embodiment 1, wherein the formulation is in a single-dose vial, a multi-dose vial, a cartridge, a pre-filled syringe, or an injection pen.

[0196] [Embodiment 16] 1. A pharmaceutical formulation comprising: (a) about 0.2 to about 2.0 mg / mL recombinant human parathyroid hormone (rhPTH(1-84)); (b) about 0.03 to about 3.0 w / v % surfactant; (c) about 0.2 to about 20 w / v % of an isotonic agent; (d) about 0.015 to about 1.50 w / v % antioxidant; (e) about 0.03 to about 3% preservative; (f) about 5 mM to about 50 mM of a physiologically acceptable buffer, and (g) water wherein the pharmaceutical formulation is formulated as an injectable solution and remains physically stable, clear, colorless, and free of visible particles for at least 48 hours.

[0197] [Embodiment 17] 17. The pharmaceutical formulation of embodiment 16, which is physically stable for at least 72 hours.

[0198] [Embodiment 18] 17. The pharmaceutical formulation of embodiment 16, which is physically stable for at least 96 hours.

[0199] [Embodiment 19] 17. The pharmaceutical formulation of embodiment 16, which is physically stable for at least 7 days.

[0200] [Embodiment 20] 17. The pharmaceutical formulation of embodiment 16, which is physically stable for at least 14 days.

[0201] [Embodiment 21] 17. The pharmaceutical formulation of embodiment 16, which is physically stable for at least 21 days.

[0202] [Embodiment 22] 1. A pharmaceutical formulation comprising: (a) a therapeutically effective amount of recombinant human parathyroid hormone (rhPTH(1-84)); (b) bulking agents; (c) cryoprotectants, and (d) a pharmaceutically acceptable buffer wherein the pharmaceutical formulation is formulated as a lyophilized powder that is reconstituted prior to injection, and wherein the pharmaceutical formulation remains physically stable, clear, colorless, and free of visible particles for at least 48 hours after reconstitution.

[0203] [Embodiment 23] 23. The pharmaceutical formulation of embodiment 22, which is physically stable for at least 72 hours.

[0204] [Embodiment 24] 23. The pharmaceutical formulation of embodiment 22, which is physically stable for at least 96 hours.

[0205] [Embodiment 25] 23. The pharmaceutical formulation of embodiment 22, which is physically stable for at least 7 days.

[0206] [Embodiment 26] 23. The pharmaceutical formulation of embodiment 22, which is physically stable for at least 14 days.

[0207] [Embodiment 27] 23. The pharmaceutical formulation of embodiment 22, which is physically stable for at least 21 days.

[0208] [Embodiment 28] 23. The pharmaceutical formulation of embodiment 22, wherein the bulking agent is mannitol.

[0209] [Embodiment 29] 23. The pharmaceutical formulation of embodiment 22, wherein the cryoprotectant is sucrose.

[0210] [Embodiment 30] 23. The pharmaceutical formulation of embodiment 22, wherein the pharmaceutically acceptable buffer is an acetate buffer, a phosphate buffer, an L-histidine buffer, or a succinate buffer.

[0211] [Embodiment 31] 23. The pharmaceutical formulation of embodiment 22, wherein the pharmaceutically acceptable buffer is an L-histidine buffer.

[0212] [Embodiment 32] 32. The pharmaceutical formulation of embodiment 31, having a pH of about 5.5.

[0213] [Embodiment 33] 23. The pharmaceutical formulation of embodiment 22, wherein the pharmaceutically acceptable buffer is a succinate buffer.

[0214] [Embodiment 34] 34. The pharmaceutical formulation of embodiment 33, having a pH between about 4 and about 4.5.

[0215] [Embodiment 35] 23. The pharmaceutical formulation according to embodiment 22, further comprising an antioxidant and / or a surfactant.

[0216] [Embodiment 36] 36. The pharmaceutical formulation of embodiment 35, wherein the antioxidant is methionine and the surfactant is poloxamer 188.

[0217] [Embodiment 37] 1. A pharmaceutical formulation comprising: (a) about 0.02 to about 2.0 mg / mL recombinant human parathyroid hormone (rhPTH(1-84)); (b) about 0.3 to about 30 w / v % bulking agent; (c) about 0.2 to about 20 w / v % cryoprotectant, and (d) about 5 mM to about 50 mM of a pharmaceutically acceptable buffer wherein the pharmaceutical formulation is formulated as a lyophilized powder that is reconstituted prior to injection, and wherein the pharmaceutical formulation remains physically stable, clear, colorless, and free of visible particles for at least 48 hours after reconstitution.

[0218] [Embodiment 38] 38. The pharmaceutical formulation of embodiment 37, which is physically stable for at least 72 hours.

[0219] [Embodiment 39] 38. The pharmaceutical formulation of embodiment 37, which is physically stable for at least 96 hours.

[0220] [Embodiment 40] 38. The pharmaceutical formulation of embodiment 37, which is physically stable for at least 7 days.

[0221] [Embodiment 41] 38. The pharmaceutical formulation of embodiment 37, which is physically stable for at least 14 days.

[0222] [Embodiment 42] 38. The pharmaceutical formulation of embodiment 37, which is physically stable for at least 21 days.

[0223] [Embodiment 43] 43. A kit comprising: a first container containing the pharmaceutical formulation of any of embodiments 22 to 42; a second container containing sterile water for reconstituting the pharmaceutical formulation; and a sheet with instructions for preparing a reconstituted formulation from the formulation.

[0224] [Embodiment 44] 44. The kit of embodiment 43, further comprising a device for injecting the reconstituted rhPTH(1-84) solution.

[0225] [Embodiment 45] A method for administering a therapeutically effective amount of rhPTH(1-84) to a subject in need thereof, comprising subcutaneously, intravenously, or intramuscularly injecting the pharmaceutical formulation of any of embodiments 1 to 21 into the subject.

[0226] [Embodiment 46] 46. ​​The method of embodiment 45, wherein the injection is performed using a syringe, an auto-injector, an injection pen, or a combination thereof.

[0227] [Embodiment 47] 1. A method for administering a therapeutically effective amount of rhPTH(1-84) to a subject in need thereof, comprising: (i) reconstituting the pharmaceutical formulation of any of embodiments 22 to 42 with sterile water; and (ii) injecting the reconstituted formulation subcutaneously, intravenously, or intramuscularly into the subject; A method comprising:

[0228] [Embodiment 48] 48. The method of embodiment 47, wherein the injection is performed using a syringe, an auto-injector, an injection pen, or a combination thereof.

Claims

1. A pharmaceutical formulation in the form of a lyophilized powder to be reconstituted prior to injection, comprising: (a) a therapeutically effective amount of recombinant human parathyroid hormone (rhPTH(1-84)); (b) bulking agents; (c) a cryoprotectant; and (d) a pharmaceutically acceptable buffer wherein the bulking agent comprises mannitol, the cryoprotectant comprises sucrose, and the pharmaceutically acceptable buffer is selected from an L-histidine buffer and a succinate buffer, and wherein the pharmaceutical formulation is physically stable and remains clear, colorless, and free of visible particles for at least 48 hours after reconstitution.

2. 2. The pharmaceutical formulation of claim 1, wherein the pharmaceutically acceptable buffer is an L-histidine buffer.

3. 2. The pharmaceutical formulation of claim 1, wherein the pharmaceutically acceptable buffer is a succinate buffer.

4. The pharmaceutical formulation according to any one of claims 1 to 3, further comprising an antioxidant and / or a surfactant.

5. 5. The pharmaceutical formulation of claim 4, wherein the antioxidant is methionine and the surfactant is poloxamer 188.

6. A pharmaceutical formulation in the form of a lyophilized powder to be reconstituted prior to injection, comprising: (a) about 0.02 to about 2.0 mg / mL recombinant human parathyroid hormone (rhPTH(1-84)); (b) about 0.3 to about 30 w / v % bulking agent; (c) about 0.2 to about 20 w / v % cryoprotectant; and (d) about 5 mM to about 50 mM of a pharmaceutically acceptable buffer wherein the bulking agent comprises mannitol, the cryoprotectant comprises sucrose, and the pharmaceutically acceptable buffer is selected from an L-histidine buffer and a succinate buffer, and wherein the pharmaceutical formulation is physically stable and remains clear, colorless, and free of visible particles for at least 48 hours after reconstitution.

7. The pharmaceutical formulation of claim 6, wherein the pharmaceutically acceptable buffer is an L-histidine buffer and the aqueous solution has a pH of about 5.

5.

8. The pharmaceutical formulation of claim 6, wherein the pharmaceutically acceptable buffer is a succinate buffer and the aqueous solution has a pH between about 4 and about 4.

5.

9. The pharmaceutical formulation of claim 1, which is physically stable for at least 72 hours after reconstitution.

10. The pharmaceutical formulation of any one of claims 1 to 8, which is physically stable for at least 96 hours after reconstitution.

11. The pharmaceutical formulation of any one of claims 1 to 8, which is physically stable for at least 7 days after reconstitution.

12. The pharmaceutical formulation of any one of claims 1 to 8, which is physically stable for at least 14 days after reconstitution.

13. The pharmaceutical formulation of any one of claims 1 to 8, which is physically stable for at least 21 days after reconstitution.

14. 14. A kit comprising a first container containing the pharmaceutical formulation of any one of claims 1 to 13, a second container containing sterile water for reconstituting the pharmaceutical formulation, and a sheet with instructions for preparing the reconstituted formulation therefrom.

15. 15. The kit of claim 14, further comprising a device for injecting the reconstituted rhPTH(1-84) solution.

16. The pharmaceutical formulation of any one of claims 1 to 13, which is administered to a subject in need of rhPTH(1-84) by subcutaneous, intravenous, or intramuscular injection.

17. 14. The pharmaceutical formulation of any one of claims 1 to 13, which is reconstituted with sterile water and administered by subcutaneous, intravenous, or intramuscular injection to a subject in need of rhPTH(1-84).

18. 18. The pharmaceutical formulation of claim 16 or 17, wherein the injection is performed using a syringe, an auto-injector, an injection pen, or a combination thereof.

Images