Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

• the present invention relates to new and improved pharmaceutical compositions and dosage forms comprising recombinant human parathyroid hormone (rhPTH(l-84)) having improved in-use stability.
• rhPTH(l-84) recombinant human parathyroid hormone
• Parathyroid hormone is a secreted, 84 amino acid product of the mammalian parathyroid gland that controls serum calcium levels through its action on various tissues, including bone. Studies in humans with certain forms of PTH have demonstrated an anabolic effect on bone, and have prompted significant interest in its use for the treatment of osteoporosis and related bone disorders.
• PTH is particularly sensitive to various forms of degradation. Unlike other proteins that have been formulated successfully, PTH is particularly sensitive to oxidation, and further requires that its N-terminal sequence remain intact in order to preserve bioactivity. For example, oxidation can occur at methionine residues at positions 8 and 18, giving rise to the oxidized PTH species ox-M(8)-PTH and ox-M(l8)-PTH, while deamidation can occur at asparagine in position 16, giving rise to dl6- PTH. The polypeptide chain becomes truncated by breakage of peptide bonds, both at the N- and C-terminals. Furthermore, PTH may also be adsorbed to surfaces, form unspecific aggregates and/or precipitate, thus reducing the available concentration of the drug. All these degradation reactions, and combinations thereof, leads to partial or complete loss of PTH bioactivity.
• parathyroid hormone requires a formulation that is acceptable in terms of storage and in-use stability and ease of preparation and reconstitution. Because it is a protein and thus far more labile than the traditional small molecular weight drugs, the formulation of parathyroid hormone presents challenges not commonly encountered by the pharmaceutical industry.
• a full-length rhPTH(l-84) has recently been approved as a safe and effective treatment for hypoparathyroidism (sold by Shire Pharmaceuticals under the brand name NATPARA ® /NATPAR ® ). It is the first specific hormone replacement for hypoparathyroidism, and is a once-daily subcutaneous injectable, to be taken as an adjunct to calcium and vitamin D.
• NATPARA ® is currently supplied as a multiple dose, dual-chamber glass cartridge containing a sterile lyophilized powder and diluent in various dose strengths.
• the sterile lyophilized powder contains either 0.40 mg, or 0.80 mg, or 1.21 mg, or 1.61 mg of parathyroid hormone depending on dose strength and 4.5 mg sodium chloride, 30 mg mannitol, and 1.26 mg citric acid monohydrate.
• the weight of the sterile diluent is 1.13 g and the diluent contains a 3.2 mg/mL aqueous solution of m-cresol.
• each dose consists of a solution of rhPTH(l- 84) at a pH between 5 and 6.
• the disposable NATPARA ® medication cartridge is designed for use with a reusable mixing device for product reconstitution and a reusable Q-Cliq pen for drug delivery.
• the Q-Cliq pen delivers a fixed volumetric dose of 71.4 pL.
• each NATPARA ® dual- chamber cartridge delivers 14 doses of NATPARA. ®
• NATPARA ® solutions may form protein particulates during the in-use period.
• greater robustness of the NATPARA ® formulation against physical and chemical stresses encountered during the normal processing conditions, product shelf- and in-use life is desired.
• a stable, liquid pharmaceutical formulation comprising recombinant human parathyroid hormone (rhPTH(l-84)) is provided.
• This formulation is designed for use directly as a liquid for injection, without the step of reconstituting a powder.
• the pharmaceutical formulation comprises: (a) a therapeutically effective amount of recombinant human parathyroid hormone (rhPTH(l-84));
• said pharmaceutical formulation is formulated as a liquid for injection, and wherein the formulation is physically and chemically stable and remains clear, colorless, and free of visible particles for at least 48 hours.
• the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 72 hours. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 96 hours. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 7 days. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 14 days. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 21 days.
• the surfactant is a poloxamer. In one embodiment, the surfactant is Poloxamer-l88. In one embodiment, the surfactant is Poloxamer-l88 present at about 0.03 to about 3% w/v of the formulation.
• the tonicity agent is selected from sodium chloride, sucrose, and glycerol, or combinations thereof.
• the tonicity agent is sodium chloride present at about 0.2 to about 20% w/v of the formulation.
• the tonicity agent is sucrose present at about 0.2 to about 20% w/v of the formulation.
• the tonicity agent is glycerol present at about 0.2 to about 20% w/v of the formulation.
• 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.
• 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.
• the antioxidant is methionine and it 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.
• the pharmaceutical formulation has a pH of about 3.8 to about 6.2, or about 5.5.
• the pharmaceutical formulation comprises:
• said pharmaceutical formulation is formulated as a liquid for injection, and wherein the formulation is physically and chemically stable and remains clear, colorless, and free of visible particles for at least 48 hours.
• the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 72 hours. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 96 hours. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 7 days. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 14 days. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 21 days. [00021] In another aspect, a pharmaceutical formulation comprising recombinant human parathyroid hormone (rhPTH(l-84)) is provided as a lyophilized powder to be reconstituted prior to injection. In one embodiment, the pharmaceutical formulation comprises:
• said pharmaceutical formulation is formulated as a lyophilized powder to be reconstituted prior to injection, and wherein the formulation is physically and chemically stable and remains clear, colorless, and free of visible particles for at least 48 hours after reconstitution.
• the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 72 hours. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 96 hours. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 7 days. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 14 days. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 21 days.
• the bulking agent is mannitol. In one embodiment, the bulking agent is mannitol present at about 0.3% to about 30% w/v of the formulation.
• the cryoprotectant is sucrose. In one embodiment, the cryoprotectant is sucrose present at about 0.2 to about 20% w/v of the formulation.
• the pharmaceutically acceptable buffer is 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. In one embodiment, the pharmaceutically acceptable buffer is L-Histidine buffer. In one embodiment, the pharmaceutically acceptable buffer is succinate buffer.
• the pharmaceutical formulation further comprises an antioxidant.
• the antioxidant is methionine.
• the antioxidant is methionine and it is present at a concentration of about 0.015% to about 1.50% w/v of the formulation.
• the antioxidant is methionine present at about 0.15% w/v or 10 mM.
• the pharmaceutical formulation further comprises a surfactant.
• the surfactant is a poloxamer.
• the surfactant is Poloxamer-l88.
• the surfactant is Poloxamer-l88 present at about 0.03 to about 3% w/v of the formulation.
• the pharmaceutical formulation has a pH of about 3.8 to about 6.2, or about 4.3, or about 5.5.
• the pharmaceutical formulation comprises:
• said pharmaceutical formulation is formulated as a lyophilized powder to be reconstituted prior to injection, and wherein the formulation is physically and chemically stable and remains clear, colorless, and free of visible particles for at least 48 hours after reconstitution.
• the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 72 hours. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 96 hours. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 7 days. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 14 days. In one embodiment, the pharmaceutical formulation remains clear, colorless, and free of visible particles for at least 21 days.
• Figure 1 shows a comparison of opalescence of reference suspensions (RS).
• Figure 2 shows the appearance for rhPTH formulated in different buffers upon agitation in 2R glass vials at ambient conditions (220 rotations per minute (rpm), orbital shaking).
• Figures 3A-3C show RP-HPLC data for the main peak of rhPTH for the pH screen samples stored at 40, 25, and 5 °C, respectively, for up to 6 months.
• Figures 4A-4C show RP-HPLC data for the oxidized Met8 rhPTH impurity for the pH screen samples stored at 40, 25, and 5 °C, respectively, for up to 6 months.
• Figures 5A-5C show RP-HPLC data for the oxidized Metl8 rhPTH impurity for the pH screen samples stored at 40, 25, and 5 °C, respectively, for up to 6 months.
• Figures 6A-6C show RP-HPLC data for the IsoAsp33 rhPTH for the pH screen samples stored at 40, 25, and 5 °C, respectively, for up to 6 months.
• Figures 7A-7C show RP-HPLC data for the rhPTH((l-30)+(l-33)) impurities for the pH screen samples stored at 40, 25, and 5 °C, respectively, for up to 6 months.
• Figures 8A-8C show RP-HPLC data for the rhPTH(l-45) fragment impurity for the pH screen samples stored at 40, 25, and 5 °C, respectively, for up to 6 months.
• Figures 9A-9C show RP-HPLC data for the main peak of rhPTH for samples formulated in pH 5.5 acetate buffer containing 50 mM NaCl with different excipients and stored at 40, 25, and 5 °C, respectively.
• Figures 10A-10C show RP-HPLC data for oxidized Met8 rhPTH impurity for samples formulated in pH 5.5 acetate buffer containing 50 mM NaCl with different excipients and stored at 40, 25, and 5 °C, respectively.
• Figures 11A-11C show RP-HPLC data for oxidized Metl8 rhPTH impurity for samples formulated in pH 5.5 acetate buffer containing 50 mM NaCl with different excipients and stored at 40, 25, and 5 °C, respectively.
• Figures 12A-12C show RP-HPLC data for IsoAsp33 rhPTH impurity for samples formulated in pH 5.5 acetate buffer containing 50 mM NaCl with different excipients and stored at 40, 25, and 5 °C, respectively.
• Figure 13 shows the appearance of the lyophilized cakes for rhPTH formulations according to various embodiments of the disclosure.
• the terms“about” and“approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.
• the term“approximately” or“about,” as applied to one or more values of interest refers to a value that is similar to a stated reference value.
• the term“approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
• the terms“carrier” and“diluent” refers to a pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) carrier or diluting substance useful for the preparation of a pharmaceutical formulation.
• exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
• the terms“treat” or“treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.
• the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
• the subject is a human.
• the term“effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof.
• the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if 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 or drugs employed, the mode of administration, and the like.
• compositions of the invention refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human).
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
• compositions according to the invention possess improved in-use stability of rhPTH(l-84) as compared to commercially available rhPTH(l-84) formulations.
• the term“in- use,” as used herein, refers to the period of time during which a multidose formulation can be used while retaining quality within an accepted specification, once the multidose container is opened.“In-use stability,” therefore, refers to the stability of a multidose formulation during the in-use period.
• the in-use period is 7 days.
• the in-use period is 14 days.
• the in-use period is 21 days.
• the in-use period is one month.
• compositions disclosed herein incorporate as the active ingredient the full length, 84 amino acid form of human parathyroid hormone, obtained either recombinantly, by peptide synthesis or by extraction from human fluid.
• the recombinant human form of PTH is abbreviated rhPTH(l-84), which has the amino acid sequence reported by Kimura et al, Biochem Biophys Res Comm, 114 (2):493.
• compositions of the invention may incorporate those homologues, fragments, or variants of human PTH that have human PTH activity as determined in the ovarectomized rat model of osteoporosis reported by Kimmel et al, Endocrinology, 1993, 32(4): 1577 and incorporated herein by reference.
• the parathyroid hormone compositions of the present invention are provided in a single-unit or multi-unit liquid dosage form, as an aqueous hormone solution for injection that does not require any reconstitution, dilution, or mixing.
• the parathyroid hormone compositions of the present invention are provided in a lyophilized powder dosage form containing not more than 3% water by weight, that results from the freeze-drying of a sterile, aqueous hormone solution prepared by mixing the selected parathyroid hormone, a non-volatile buffering agent and an excipient.
• the PTH compositions of the present invention incorporate PTH in a therapeutically effective amount, a term used with reference to amounts useful either therapeutically or in medical diagnosis.
• the particular amount of parathyroid hormone incorporated in the preparation can be pre-determined based on the type of PTH selected and on the intended end-use of the preparation.
• the compositions are exploited for therapeutic purposes, and particularly for the treatment of osteoporosis and related bone disorders, as well as hypoparathyroidism.
• such therapy entails administration of the liquid and/or reconstituted lyophilized composition by injection, e.g., a sub-cutaneous injection, in unit doses that reflect the prescribed treatment regimen.
• the treatment regimen may include administering recombinant human PTH(l-84) within the range from about 0.01 mg PTH/mL of injected solution to 5 mg PTH/mL of injected solution per patient, with injection volumes being e.g., from about 0.3 mL to about 2.3 mL, or from about 0.5 mL to about 2 mL, or from 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.
• injection volumes being e.g., from about 0.3 mL to about 2.3 mL, or from about 0.5 mL to about 2 mL, or from 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 m
• the purified and sterile-filtered PTH is incorporated with the buffering agent and excipients to form an aqueous solution containing PTH in a concentration range from 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.
• PTH is incorporated with the buffering agent and excipients to form an aqueous solution containing PTH in 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.
• Molar equivalents of the substantially equipotent forms of PTH such as the PTH(l-84) variants and fragments, can be similarly incorporated in place of the human PTH(l- 84), if desired.
• compositions of the invention further comprise a pharmaceutically acceptable excipient and/or carrier.
• 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 hereby incorporated by reference in their entirety. Non-limited examples of suitable excipients are presented below.
• formulations disclosed herein further comprise a surfactant.
• the surfactant may be selected from poloxamer (e.g., Poloxamer-l88), 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 monoethaolamine (MEA), cocamide diethanolamine (DEA), dodecyldimethylamine oxide, or any combination thereof.
• the surfactant is selected from Poloxamer- 188, Polysorbate 20, Polysorbate 80, and polyethylene glycol, and combinations thereof.
• the surfactant is a poloxamer. In one embodiment, the surfactant is Poloxamer-l88.
• 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%.
• the surfactant is Poloxamer-l88, and it is present at about 0.3% w/v of the composition.
• compositions of the present disclosure further comprise a tonicity agent.
• Tonicity is a measure of the effective osmotic pressure gradient (as defined by the water potential of two solutions) of two solutions separated by a semipermeable membrane. Tonicity is commonly used when describing the response of cells immersed in an external solution. In other words, tonicity is the relative concentration of solutions that determine the direction and extent of diffusion. Body fluids normally have an osmotic pressure that corresponds to that of a 0.9% solution of sodium chloride.
• a composition e.g., solution or gel
• a composition is isotonic with a body fluid solution when the magnitude of the salts is equal between the composition and the physiologic solution. Tonicity equilibrium is reached in physiologic solutions by water moving across the membranes, but the salts staying in their solution of origin.
• a solution is isotonic with a living cell if there is no net gain or loss of water by the cell, or other changes in the cell, when it is in contact with that solution.
• a tonicity agent used in the compositions disclosed herein is an electrolyte, mono- or disaccharide, inorganic salt (e.g., sodium chloride, calcium chloride, sodium sulfate, magnesium chloride), a polyol, or a combination thereof.
• a tonicity agent is glucose, sucrose, sodium chloride, potassium chloride, calcium chloride, sodium sulfate, magnesium chloride, dextrose, mannitol, glycerol, or any combination thereof.
• the tonicity agent is selected from sodium chloride, sucrose, and glycerol, or combinations thereof.
• the tonicity agent is sucrose.
• the tonicity agent is sodium chloride.
• the tonicity agent is glycerol.
• the tonicity agent may be present at any concentration necessary to achieve isotonic conditions.
• 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.
• the tonicity agent is sucrose, and it is present at about 0.2% to about 20% of the composition, or at about 8.5% w/v of the composition. In one embodiment, the tonicity agent is glycerol, and it is present at about 0.2% to about 20% of the composition, or at about 2.3% w/v of the composition. In one embodiment, the tonicity agent is sodium chloride, and it is present at about 0.2% to about 20% of the composition, or at about 0.8% w/v of the composition.
• compositions of the present disclosure are sterile and preservative-free.
• compositions of the present disclosure optionally comprise a preservative.
• a preservative is a paraben-free preservative.
• Parabens are a series of parahydroxybenzoates or esters of parahydroxybenzoic acid and are known to cause cytokine release and irritation and have been linked to several types of cancer. Examples of parabens include methyl paraben, ethyl paraben, propyl paraben, butyl paraben, heptyl paraben, isobutyl paraben, isopropyl paraben, benzyl paraben, and their sodium salts.
• Exemplary paraben-free preservatives include methylphenol (cresol), including 3- methylphenol (meta-cresol or m-cresol), phenol, phenethyl alcohol, caprylyl glycol, phenoxy ethanol, a sorbate, potassium sorbate, sodium sorbate, sorbic acid, sodium benzoate, benzoic acid, acemannan, oleuropein, carvacrol, cranberry extract, gluconolactone, green tea extract, Helianthus annuus seed oil, Lactobacillus ferment, ETsnea barbata extract, polyaminopropyl biguanide, polyglyceryl-3 palmitate, polyglyceryl-6 caprylate, pomegranate extract, Populus tremuloides bark extract, resveratrol, Rosmarinus officinalis leaf extract, benzyl alcohol, or any combination thereof.
• a preservative is selected from m-cresol, phenol, benzyl alcohol, sodium benzoate, and propyl paraben, and combinations thereof. In one embodiment, a preservative comprises m-cresol.
• compositions of this disclosure may comprise a preservative at a concentration of about 0.005% to about 10% by weight, about 0.005% to about 5%, about 0.01% to about 5%, about 0.02% to about 4%, about 0.03% to about 3%, about 0.05% to about 2%, about 0.1% to about 1%, about 0.2% to about 0.5%, about 0.01% to about 10%, about 0.01% to about 5%, about 0.01% to about 2.5%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.1% to about to about 10%, about 0.1% to about 5%, about 0.1% to about 2.5%, about 0.1% to about 1%, about 0.1% to about 0.5%, or about 0.1%, or about 0.2%, or about 0.3%, or about 0.4%, or about 0.5% w/v of the composition.
• the preservative is m-cresol present at about 0.03% to about 3% of the composition. In one embodiment, m-cresol is present at 0.
• compositions of the present invention may comprise a pharmaceutically acceptable buffer by incorporating a buffering agent.
• buffering agents incorporated in the present compositions are selected from those capable of buffering the preparation to a pH within a physiologically acceptable range.
• a pH that is physiologically acceptable is that which causes either no, or minimal, patient discomfort when the formulation is administered, and can thus vary depending on the mode of administration.
• the pH of the preparation per se can vary widely, e.g., from about pH 3 to about pH 9.
• the PTH preparation is buffered to within the pH range from 3.5 to 7.5.
• Suitable buffers are accordingly those pharmaceutically acceptable agents that can buffer the pH of the preparation to within the target pH range, and include acetate buffers, phosphate buffers, L-Histidine buffers, succinate buffers.
• any pharmaceutically acceptable buffers may be suitable for formulations according to the invention, it has been surprisingly found that the nature of the buffering agent has a large effect on the stability of rhPTH solutions.
• citrate buffer which is currently used in NATPARA ® , results in rhPTH protein particulate formation at as little as 24 hours of agitation at ambient conditions.
• solutions of rhPTH prepared with acetate, phosphate, and L-Histidine buffers remain clear, colorless and free of visible particles for 24 hours of agitation.
• the selected buffering agent is incorporated to yield a final pH within the range from 3.5 to 6.5, and the buffer is present at a concentration of about 5 mM to about 50 mM.
• the pH rendered by the buffering agent is in the range from 3.8 to 6.2, and the buffer concentration about lOmM to about 30 mM.
• the pH of the formulation is 5.5.
• the pH of the formulation is 4.3.
• the buffer is acetate buffer present at a concentration of about 20 mM.
• the buffer is L-Histidine buffer present at a concentration of about 20 mM.
• the buffer is succinate buffer present at a concentration of about 20 mM.
• the formulations of the invention may further comprise one or more antioxidants to provide oxidative stability to the rhPTH protein during the in-use period.
• Antioxidants that may be suitable may include, without limitation, acetone sodium bisulfite, argon, ascorbyl palmitate, ascorbate (salt/acid), bisulfite sodium, butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), cysteine/cysteinate HC1, dithionite sodium (NA hydrosulfite, Na sulfoxylate), gentisic acid, gentisic acid ethanolamine, glutamate monosodium, glutathione, formaldehyde sulfoxylate sodium, metabisulfite potassium, methionine, monothioglycerol (thioglycerol), nitrogen, propyl gallate, sulfite sodium, tocopherol alpha, alpha tocopherol hydrogen succinate, thi
• the antioxidant may be present at any concentration necessary to achieve oxidative stability of the formulation.
• 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%, about 0.1% to about 1% w/v.
• the antioxidant is methionine present in an amount from 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.
• the parathyroid hormone compositions of the present invention are provided in a lyophilized powder form containing not more than 3% water by weight, that results from the freeze-drying of a sterile, aqueous hormone solution prepared by mixing the selected parathyroid hormone, a non-volatile buffering agent and an excipient.
• the lyophilized compositions are provided in a form that yields a unit dose of about 0.05 mg/mL to about 0.15 mg/mL recombinant human PTH(l-84) upon reconstitution into about 1 to 1.5 mL (0.7-1.8 mL) of the reconstitution vehicle, and the vials are accordingly loaded with about 1 to 1.5 mL of the aqueous PTH preparation, for subsequent freeze-drying.
• the PTH preparation subjected to freeze- drying comprises from 25 to 250 pg/mL of human PTH(l-84), about 0.3% to about 30% w/v bulking agent, about 0.2% to about 20% w/v tonicity agent, and a physiologically acceptable buffering agent in an amount capable of buffering the preparation to within the range from 3.5 to 6.5 upon reconstitution in sterile water.
• the buffering agent is incorporated in an amount sufficient to buffer the pH to 5.5 ⁇ 0.3, or 4.3 ⁇ 0.3.
• novel lyophilized formulations may further comprise one or more bulking agent for optimal cake structure and appearance.
• Bulking agents that may be suitable include compatible carbohydrates, polypeptides, amino acids or combinations thereof.
• Suitable carbohydrates may include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2- hydroxypropyl-P-cyclodextrin; polysaccharides, such as raffmose, maltodextrins, dextrans, and the like; and alditols, such as mannitol, xylitol, and the like.
• Suitable polypeptides include aspartame.
• Amino acids include alanine and glycine.
• novel lyophilized formulations may comprise one or more bulking agent selected from mannitol, glycine, poly(ethylene glycols), ammonium sulfate, sucrose, trehalose, and combinations thereof.
• novel lyophilized formulations may comprise mannitol.
• the bulking agent may be present at any concentration necessary to achieve the optimal structure and appearance of the lyophilized powder.
• the bulking agent may be present at a concentration of about 0.01% to about 50% by weight, 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.
• the bulking agent is mannitol, and it is present at about 0.2% to about 20% of the composition, or at about 2% to about 8% of the composition, or at about 3% w/v of the composition, or at about 4% of the composition.
• novel lyophilized formulations may further comprise one or more cryoprotectants to provide stability to the rhPTH protein during the freeze-drying process and product storage.
• cryoprotectants that may be suitable 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.
• novel lyophilized formulations may comprise one or more cryoprotectants selected from sucrose, glycine, mannitol, disaccharides, polyethylene glycols) and combinations thereof.
• novel lyophilized formulations may comprise sucrose.
• the cryoprotectant may be present at any concentration necessary to achieve stability of the lyophilized powder.
• the cryoprotectant may be present at a concentration of about 0.01% to about 50% by weight, 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.
• the cryoprotectant is sucrose, and it is present at about 0.2% to about 20% of the composition, or at about 1% to about 8% of the composition, or at about 2% w/v
• compositions may be provided in single or multiple dose injectable form, for example in the form of a pen.
• the compositions may, as already mentioned, be prepared by any suitable pharmaceutical method which includes a step in which the active ingredient and the carrier (which may consist of one or more additional ingredients) are brought into contact.
• the pharmaceutical composition may be provided together with a device for application, for example together with a syringe, an injection pen or an auto- injector, e.g., a Q-cliq pen.
• a device for application for example together with a syringe, an injection pen or an auto- injector, e.g., a Q-cliq pen.
• Such devices may be provided separate from a pharmaceutical composition or prefilled with the pharmaceutical composition.
• the formulations were prepared in the following manner: rhPTH(l-84) drug substance (active pharmaceutical ingredient) was exchanged against the respective base formulation buffer using a dialysis method commonly known by those of skill in the art. Solution pH adjustment was further made, if needed, with acid or base stock solutions. Stock solutions of excipients were prepared separately in the base buffer, and were mixed with the dialyzed peptide solution to achieve the final formulations with the desired peptide and excipient concentrations. The formulations were sterile filtered and filled either in glass vials or in glass cartridges. Liquid formulations were stoppered and crimped, followed by storage. Formulations meant for lyophilization were exposed to a pre-programmed lyophilization cycle consisting of freezing, annealing, primary drying and secondary drying steps, followed by stoppering and crimping.
• EXAMPLE 1 Compositions of Novel Liquid Formulations of rhPTH
• Liquid Formulations #1 to #3 have the following composition:
• EXAMPLE 2 Compositions of Novel Lyophilized Powder Formulations of rhPTH
• Lyophilized Formulations #1 to #3 have the following composition:
• Table 3, below, and Figure 2 show appearance data from agitation studies where rhPTH was formulated with different buffers.
• rhPTH was formulated in different buffers (10 mM) with sodium chloride (140 mM) in 2R glass vials.
• the agitation studies were performed using an orbital shaker at 220 rpm, with vials in horizontal position, at ambient conditions for at least 48 hours.
• the visual appearance of agitated samples were compared to reference suspensions (RSI-IV) and Standard of Opalescence (SOP) according to standard procedure outlined, e.g., in European Pharmacopoeia 5.0, 2.2.1. Clarity and Degree of Opalescence in Liquids.
• Figure 1 shows opalescence of reference suspensions RSI-IV and SOP. Water is provided for comparison.
• Figure 2 shows appearance of the suspensions of rhPTH formulated in different buffers.
• acetate buffer shows the best stability against agitation induced particulate formation in rhPTH, followed by phosphate buffer, followed by L- Histidine buffer. All three buffers show better stability than citrate buffer, which is currently used in the NATPARA ® formulation.
• Table 4 shows appearance data from agitation studies for various liquid formulations of rhPTH according to the invention.
• Liquid Formulations #l-#3 were formulated in dual-chamber cartridges, and agitation studies were performed at ambient conditions (220 rpm, orbital shaking). On regular agitation intervals, visual appearance of agitated samples was compared to reference suspensions (RSI-IV) and Standard of Opalescence (SOP) according to standard procedure. Data for the commercial NATPARA ® formulation is also provided for comparison. The opalescence at different time points is summarized in Table 4.
• EXAMPLE 5 Agitation Studies for rhPTH in Novel Lyophilized Formulations
• Table 5 shows appearance data from agitation studies for various reconstituted lyophilized formulations of rhPTH according to the invention. Lyophilized Formulations #l-#3 were formulated in dual-chamber cartridges, and agitation studies were performed at ambient conditions (220 rpm, orbital shaking). On regular agitation intervals, visual appearance of agitated samples was then compared to reference suspensions (RSI-IV) and Standard of Opalescence (SOP) according to standard procedure. Data for the commercial NATPARA ® formulation is also provided for comparison. The opalescence at different time points is summarized in Table 5.
• novel lyophilized formulations significantly improve the physical stability of rhPTH.
• novel formulations of rhPTH stay 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.
• EXAMPLE 6 In-Use Stability Studies for Liquid and Lyophilized Formulations [000162] Table 6, below, shows appearance data from in-use studies for a liquid formulation according to an embodiment of the invention, as exemplified by Liquid Formulation #2, and a reconstituted lyophilized formulation of rhPTH according to an embodiment of the invention, as exemplified by Lyophilized Formulation #2.
• EXAMPLE 7 Solution pH Screening for rhPTH for Optimal Physicochemical Stability
• Recombinant human parathyroid hormone was formulated in 10 mM citric acid buffer with 140 mM sodium chloride in a solution pH range of 3.5 to 7.5 with a 0.5 unit pH interval.
• the samples were aliquoted in 2 mL Type I borosilicate glass vials and placed on stability at temperature conditions of 5 ⁇ 3 °C (5 °C), 25 ⁇ 2 °C (25 °C), and 40 ⁇ 2 °C (40 °C). At pre-defmed intervals, samples were pulled, observed for appearance, and analyzed for rhPTH stability using the chromatographic assays in place (Size Exclusion Chromatography (SEC) and Reversed-Phase Chromatography (RP-HPLC)) with some modifications.
• SEC Size Exclusion Chromatography
• RP-HPLC Reversed-Phase Chromatography
• the supplied drug substance material was thawed and dialyzed against respective pH buffer solutions in 2 kDa molecular weight cut off (MWCO) dialysis cassette.
• the dialysis was performed at 5 ⁇ 3 °C and included at least 3 cycles of buffer exchanges over a period of ⁇ 24 hours.
• Post dialysis samples were assayed for pH and adjusted if necessary with 0.2 N sodium hydroxide.
• A280 measurements were performed and rhPTH concentration was calculated using an extinction coefficient of 0.584 (mL.mgf'cm 1 Final solution preparation was done aseptically in a laminar flow hood.
• rhPTH was prepared at a concentration of 1.0 mg/mL by using the respective buffer as the dilution medium.
• the prepared sample was filtered via a 0.22 pm PVDF filter, filled at 1.5 mL volume in 2 mL Type I borosilicate glass vials, followed by stoppering/crimping.
• each vial was observed for solution appearance in a light box. Baseline samples were separated, aliquoted in polypropylene tubes, and stored at -80 °C. Remaining vials were incubated at 5, 25, and 40 °C. At a predefined interval, sample vials were pulled from each incubation condition, observed for appearance, aliquoted in polypropylene tubes, and stored at - 80 °C until analysis. Samples were tested for physical and chemical changes using assays validated for Natpara ® , including SEC and RP-HPLC with some modifications in injection volume and injection sequence.
• tables 7 and 8 show the appearance results for the rhPTH stability samples stored at 40 and 25 °C, respectively, for 6 months.
• Opalescence against a reference suspension is noted where measured.
• White flocculant like particles were visible in pH 7.0 and 7.5 samples within 2 weeks of storage at 40 °C. This particle formation appeared to progress from the basic to acidic side of the solution pH with time. By 3 months, most of the samples stored at 40 °C had particles.
• Samples stored at 25 °C showed the same trend of particle formation as observed at 40 °C but with slower kinetics. It was also noticed that the size of particles was different depending on the solution pH. Samples formulated in the pH range of 6.5-7.5 had flocculants, while those at lower pH had fine particles.
• Samples stored at 5 °C had an initial appearance of clear, colorless, free of visible of particles which did not change over the course of 6 months.
• Table 9 provides the protein concentration data for the stability samples stored at 40 and 25 °C, respectively. Samples were thoroughly centrifuged (l7,000g for 5 minutes) and supernatants were used for A280 measurements. Appropriate light scattering corrections were made (A320 subtraction). At 40 °C, a drop in protein concentration roughly correlated with sample tendency 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.
• Figures 3-8 shows the RP-HPLC data for the rhPTH and associated impurities for the pH screen samples stored at 40, 25, and 5 °C for up to 6 months.
• For the 40 °C storage condition only data for up to 1 month is presented, as the samples were too degraded afterwards to perform peak integration.
• Oxidized Met8 Oxidation of Met8 was also observed to follow a bell shape trend (as with the main peak), where the maximum Met8 oxidation was seen in the pH range of ⁇ 4.0- 5.5 upon storage at 40 and 25 °C. At 5 oC, no trend was observed up to 6 months of storage, as shown in Figures 4A-4C.
• Oxidized Met 18 Metl8 oxidation rates were found to be maximum towards the basic solution pH range and decrease gradually as the solution pH became acidic. This trend was mainly visible at both 40 and 25 °C storage conditions, as shown in Figures 5A-5C.
• IsoAsp33 Formation of isoaspartate from asparagine33 was observed to be minimum towards the acidic side of formulated pH and was observed to increase significantly as the solution pH increases past ⁇ 5.5. This trend was clear at all storage temperatures, as shown in Figures 6A-6C.
• rhPTH((l-30)+(l-33 )) were rhPTH impurities increased significantly upon storage in samples formulated below pH 5.0 and above pH 6.0.
• the increase in impurities above pH 6.0 occurred to a significantly lesser extent than that observed at lower pH values. This increase in impurity was observed to be minimum in the pH range of 5.0-6.0, as shown in
• the present example demonstrates the impact of solution pH on the physicochemical stability of rhPTH when formulated in a pH range of 3.5 to 7.5 and exposed to thermal stress.
• Physical stability attributes as monitored using appearance (visible particle formation) and SEC (aggregates and fragments formation), and chemical stability attributes, as monitored using RP-HPLC (oxidation, deamidation, and fragmentation), suggest that a solution pH range of 5.0-6.0 is optimal for physical and chemical stability of rhPTH.
• EXAMPLE 8 Excipient Screening for rhPTH for Optimal Physicochemical Stability
• rhPTH Recombinant human parathyroid hormone
• NaCl sodium chloride
• This base formulation was spiked with excipient stock to achieve desired target levels of a given excipient.
• the samples were placed on stability at temperature conditions of 5 ⁇ 3 °C (5 °C), 25 ⁇ 2 °C (25 °C), and 40 ⁇ 2 °C (40 °C). At pre-defmed intervals, samples were pulled, observed for appearance, and analyzed for rhPTH stability using Reversed-Phase Chromatography (RP- HPLC). Baseline (time 0) samples were also exposed separately to multiple freeze-thaw cycles and orbital agitation and observed for solution appearance.
• RP- HPLC Reversed-Phase Chromatography
• the supplied drug substance material was thawed and dialyzed against the base buffer solution in 2 kDa MWCO dialysis cassette. The dialysis was performed at 5 ⁇ 3 °C and included at least 3 cycles of buffer exchanges over a period of ⁇ 30 hours. Post dialysis, samples were tested for pH and adjusted if necessary with 0.2 N sodium hydroxide. A280 measurement was performed and rhPTH concentration was calculated based on an extinction coefficient of 0.584 (mL.mg) 1 cm 1 . Final solution preparation was done aseptically in a laminar flow hood.
• rhPTH was prepared at a concentration of 1.0 mg/mL by using the base buffer as the dilution medium and spiking the excipient stock to achieve the desired excipient concentration. Additionally, m-cresol was added at a level of 0.3% (v/v) in each of the formulations.
• Table 10 provides the description of the different formulations used for the excipient screen study.
• the prepared samples were filtered via a 0.22 pm PVDF filter, filled at 1.5 mL volume in 2R Type I glass vials, followed by stoppering/crimping. Each vial was observed for solution appearance in a light box.
• Baseline samples were aliquoted in polypropylene tubes and stored at -80 °C. Remaining vials were incubated at 5, 25, and 40 °C. At predefined intervals, sample vials were pulled from each incubation condition, observed for appearance, aliquoted in polypropylene tubes, and stored at -80 °C until further analysis. Samples were tested for physical and chemical changes using assays validated for Natpara, including SEC and RP-HPLC with some modifications in injection volume and injection sequence.
• Baseline samples were exposed to repetitive freeze/thaw cycles (freeze at -80 °C for 5-12 hours and thaw at room temperature) and observed for solution appearance in a lightbox.
• a different set of baseline samples in vials were agitated horizontally under ambient temperature conditions using an orbital shaker at 220 rpm and observed for solution appearance on regular intervals in a lightbox.
• Preliminary results from agitation studies were used to select additional formulations which were further exposed to orbital agitation in 2R vials and dual- chamber cartridges.
• Tables 11-13 shows the appearance results for the rhPTH stability samples stored at 40, 25, and 5 °C, respectively, for up to 6 months. Opalescence against a reference suspension is noted where measured. Upon storage at 40 °C, samples containing arginine (150 mM) showed a significant presence of proteinaceous particles which appeared at 2 weeks and kept on increasing over time. Samples with other excipients, when stored at 40 °C, had appearance comparable to baseline for up to 3 months. By the end of 6 months storage at 40 °C, most of the samples had visible particles present with varying color and opalescence.
• Figures 9-12 show the RP-HPLC data for rhPTH and associated impurities for samples formulated in pH 5.5 acetate buffer containing 50 mM NaCl with different excipients and stored at 40, 25, and 5 °C. Results are presented for the samples where reasonable peak integration was possible without a shift in the reported relative retention times.
• IsoAsp33 Upon 25 and 40 °C storage, although samples with 150 and 300 mM NaCl showed slightly lower rate of IsoAsp33 formation, no significant differences among the excipients were noticeable (significantly lower and inconsistent IsoAsp33 levels observed with glycine, lysine, and arginine samples can be attributed to the issues in integrating missing peak/peak with slightly shifted retention time in chromatograms of these samples), see Figures 12A-12C.
• Table 14 shows the solution appearance of different formulations upon repetitive freeze-thaw performed in 2R vials. Visible particles, if observed, are reported alongside the opalescence. Samples with 150 mM NaCl or higher were significantly impacted by repetitive F/T and were found to contain white, fibrous protein-like particles. Samples with 0.02% PS20 showed granular appearance because of sand-like (non-proteinaceous) particles from the beginning. Solutions with 8% glycerol showed worsening opalescence after each F/T cycle without any visible particulate formation. [000199] Agitation studies
• Table 15 shows the appearance results from the agitation studies performed on triplicate 2R vials in horizontal position at 220 rpm under ambient conditions. All samples were clear, colorless, and free of visible particles at baseline, except PS20, which had sand-like particles present. Samples with NaCl showed the earliest sign of particle formation, the rate of which increased with increasing NaCl content. By 24 hours, samples with 150 mM NaCl and PS20 had turbid appearance. All samples except the ones with Poloxamer-l88 (P-188) developed turbid appearance by the end of 48 hours. Samples with P-188 maintained their baseline appearance until the end of study (72 hours).
• Table 16 presents the results from horizontal agitation studies performed at 220 rpm under ambient conditions in 2R vials where NaCl was removed from the base formulation. Presence of m-cresol resulted in opalescence formation significantly earlier than non m-cresol formulations. Despite the removal of 50 mM NaCl from the base formulation, all the solutions still resulted in a turbid appearance by the end of 48 hours shaking except the ones with Poloxamer-l88. These formulations were also exposed to shaking in the container/closure currently used for commercial Natpara ® (1 mL siliconized cartridges with siliconized middle and end rubber stoppers and aluminum seal using a 1.1 mL formulation fill). Appearance results similar to that obtained with shaking in 2R vials (Table 15) were observed, where Poloxamer- 188 significantly prevented/delayed the particulate formation.
• sodium chloride, sucrose, mannitol, and glycerol are suitable excipients to provide stabilization to rhPTH.
• Methionine exhibits high potential to significantly inhibit peptide oxidation.
• Poloxamer-l88 has been found to be critical to prevent visible particulate formation upon agitation.
• EXAMPLE 9 Formulation Optimization Studies for rhPTH Targeting a Liquid Dosage Form
• Recombinant human parathyroid hormone was formulated in pH 5.5 with 20 mM acetate buffer and 0.3% w/v m-cresol.
• This base formulation was prepared with varying levels of methionine (antioxidant) and Poloxamer-l88 (surfactant), excipients identified critical for rhPTH stability during early formulation screening (see Example 7).
• methionine antioxidant
• Poloxamer-l88 surfactant
• the supplied drug substance material was thawed and dialyzed against the buffer solution in 2 kDa MWCO dialysis cassette.
• the dialysis was performed at 5 ⁇ 3 °C and included at least 3 cycles of buffer exchanges over a period of -24-48 hours.
• Post dialysis samples were tested for pH and pH was adjusted, if necessary, with 0.2 N sodium hydroxide.
• A280 measurement was performed and rhPTH concentration was calculated based on an extinction coefficient of 0.584 (mL.mg) -1 cm -1 .
• Final solution preparation was done aseptically in a laminar flow hood.
• rhPTH solution was prepared at a concentration of 1.0 mg/mL by using the base buffer as the dilution medium and spiking the excipient stocks to achieve the desired excipient concentration. Additionally, m-cresol was added at a level of 0.3% w/v in each of the formulations.
• Table 17 provides the description of different formulations used for methionine and P-188 concentration optimization studies.
• Table 17 Formulations used in methionine and Poloxamer-188 (P-188) concentration optimization
• Table 18 provides the description of different formulations used for evaluating the impact of stabilizer/tonicity agents on rhPTH stability.
• Table 19 shows the appearance results for the rhPTH stability samples with different levels of methionine concentrations when stored at 40, 25, and 5 °C for up to 6 months. All samples stayed clear, colorless, and free of visible particles over the studied duration.
• Tables 20- 22 show the visual appearance results for the samples with different concentrations of Poloxamer-l88 (formulated with different methionine content - Table 17) in 2R vials that underwent horizontal orbital agitation at 220 rpm under ambient conditions.
• Sodium chloride (NaCl), sucrose, glycerol, and mannitol were selected as suitable excipients during the rhPTH excipient screening studies (Example 8).
• concentrations to be used in future formulations were selected based on the osmolality target of 250-350 mOsm/kg.
• control samples and samples containing sucrose and glycerol showed the best visual appearance profile upon agitation.
• the target concentrations of rhPTH in these formulations range between 0.35 mg/mL to 1.4 mg/mL.
• EXAMPLE 10 Development of a Multi -Dose Lyophilized rhPTH Drug Product for Subcutaneous Delivery
• RP-HPLC reversed phase-high- performance liquid chromatography
• SEC Size-exclusion chromatography

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