WO2007095288A2

WO2007095288A2 - Methionine-containing protein or peptide compositions and methods of making and using

Info

Publication number: WO2007095288A2
Application number: PCT/US2007/003905
Authority: WO
Inventors: Thomas E. Tarara; Cynthia Stevenson; Stewart Thompson; Reinhard Vehring; Negar Sadrzadeh
Original assignee: Nektar Therapeutics
Priority date: 2006-02-13
Filing date: 2007-02-13
Publication date: 2007-08-23
Also published as: EP1986674A4; WO2007095288A3; EP1986674A2

Abstract

Methionine-containing protein or peptide compositions may include at least one methionine-containing protein or peptide and at least one antioxidant that inhibits oxidation of the at least one methionine-containing peptide or protein, wherein the compositions are inhalable. Particles may include a surface enriched with a surface excipient having a glass transition temperature and a core enriched with at least one methionine-containing protein or peptide. Methods of making, methods of treating and unit dosage forms are also included.

Description

Methionine-Containing Protein or Peptide Compositions and Methods of Making and Using

BACKGROUND

Cross reference to Related Applications

[001] This application relates to U.S. Provisional Application No. 60/773,384, filed February 13, 2006, from which priority is claimed under 35 USC §119(e), the entire disclosure of which is incorporated herein by reference. Field of the Invention

[002] The present invention relates to formulations of methionine-containing peptides or proteins, such as parathyroid hormones, formulations comprising parathyroid hormones, unit doses of the same, and methods of making and methods of using the same. More particularly, the present invention relates to formulations of parathyroid hormones, such as cyclized parathyroid hormones, parathyroid hormone analogs, parathyroid hormone derivatives, parathyroid hormone fragments, formulations comprising them, unit doses of the same, and methods of making and using the same. Related Art

[003] Peptides and proteins pose many problems for the pharmaceutical formulator. The complex structure - both primary and secondary - of these agents generally needs to be maintained in the formulation to guarantee activity. Amino acid residues that are involved in, or important to, the pharmacological activity of the agent may need to be protected. Amino acids that tend toward reactivity may need to be neutralized. The structural complexity of these agents makes formulating a difficult and often unpredictable task.

[004] As these agents are often "naturally" found in some sort of aqueous environment, formulating these agents in solution is a natural first choice for maintenance of activity. However, because of the greater molecular mobility in solution, stabilizing excipients frequently need to be added to an aqueous formulation to prevent oxidation or other degradation of the active agent. Even with added stabilizers, aqueous formulations may be undesirable because of their generally shorter shelf life than a comparable solid-state formulation.

[005] Adding to the complexity is the fact that solution and solid-state chemistry of these agents frequently differs. Many peptides and proteins are stabilized by their hydrogen bonding with water and removal of the water creates instability. Additionally, excipients that have a stabilizing effect in solution may be ineffective in the solid-state, and vice versa.

[006] OSTABOLIN-C™ is a cyclized 31-amino acid analog of parathyroid hormone (PTH) with chemical and physical properties similar to other PTH analogs, such as PTH 1-34. OSTABOLIN-C™ contains two methionine residues in the 8 and 18 positions, which can both undergo degradation. In some instances, this is via oxidation to form methionine sulfoxide. The mechanisms of oxidation of methionine to methionine sulfoxide are thought to be either molecular oxygen-dependent, which is mediated through a transition metal, or peroxide-mediated oxidation with an alkyl peroxide as the oxidizer. Another mode of degradation for these peptides is deamidation of either or both glutamine (positions 6 and 29) and asparagine (positions 10 and 16) residues fPharm Res 12, 2049-2052, 1995).

[007] Because methionine-containing peptides and proteins, such as cyclized

PTH analogs like OSTABOLIN-C™, may undergo undesirable degradation, such as oxidation, upon storage, there is a need for formulations of these agents having greater stability and shelf life, especially when in the solid, or dry, state.

SUMMARY OF THE INVENTION

[008] Accordingly, one or more embodiments of the present invention satisfy one or more of these needs. Thus, one or more embodiments of the present invention include compositions comprising methionine-containing peptides and proteins, methods of making and using methionine-containing peptides and proteins compositions, and systems for using methionine-containing peptides and proteins compositions. Accordingly, the present invention provides inhalable methionine-containing peptides or protein compositions, comprising at least one methionine-containing peptide or protein, such as a parathyroid hormone, and at least one antioxidant. [009] Other features and advantages of embodiments of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. Embodiments of the invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

[010] In one aspect, one or more embodiments of the present invention include compositions comprising methionine-containing peptides and proteins, such as compositions with reduced amounts of degradants, such as oxidative degradants, methods of making and using methionine-containing peptide and protein compositions, and systems for using methionine-containing peptide and protein compositions. [011] In one aspect, one or more embodiments of the present invention include compositions comprising methionine-containing peptides and proteins comprising less than about 10 wt% of degradants thereof.

[012] In some embodiments, the methionine-containing peptides or protein comprises a cyclized parathyroid hormone, a parathyroid hormone derivative, a parathyroid hormone fragment, a parathyroid hormone analog, or combinations thereof. In some embodiments, the methionine-containing peptides or protein compositions, such as cyclized parathyroid hormone compositions are in particulate or powder form. In some embodiments, the methionine-containing peptides or protein compositions, such as cyclized parathyroid hormone compositions are in liquid form. In some embodiments, the antioxidant comprises at least one of thioethers, histidine, cysteine, tryptophan, tyrosine, ascorbic acid, vitamin E, and mixtures thereof. Thioethers include, but are not limited to, methionine.

[013] In some embodiments, the methionine-containing peptides or protein compositions of the invention comprise, by weight: from about 1 % to about 25% of at least one methionine-containing peptide or protein, such as a cyclized parathyroid hormone analog; and from about 0.1% to about 10% of at least one antioxidant. The inventive compositions may further comprise, by weight: from about 1% to about 10% of at least one buffering agent; from about 10% to about 60% of at least one dispersibility- enhancing excipient; and from about 20% to about 90% of at least one glass-stabilizing excipient. In some embodiments, the compositions comprise, by weight: from about 3% to about 6% of at least one parathyroid hormone analog, derivative or fragment; from about 4% to about 6% of at least one buffering agent; from about 20% to about 50% of at least one dispersibility-enhancing excipient; from about 40% to about 80% of at least one glass-stabilizing excipient; and from about 0.2% to about 1 % of at least one antioxidant.

[014] In some embodiments, the parathyroid hormone comprises a cyclized parathyroid hormone analog; the buffering agent comprises sodium citrate, citric acid, and mixtures thereof; the dispersibility-enhancing excipient comprises an amino acid, such as leucine, dileucine, trileucine, norleucine, and mixtures thereof; the glass- stabilizing excipient comprises sucrose, rafflnose, trehalose, and mixtures thereof; and the antioxidant comprises thioethers, and mixtures thereof. In some embodiments, the compositions of the invention comprise, by weight: about 3-20% [LeU²⁷JCyCIo(GIu²²- Lys²⁶)hPTH-(1 -3I)NH₂; about 0-6% sodium citrate; about 1-40% trileucine; about 1- 70% trehalose; and about 0-5% methionine. In some embodiments, the compositions of the invention comprise, by weight: about 10-18% [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-(1- 3I)NH₂; about 1-4% sodium citrate; about 10-30% trileucine; about 20-60% trehalose; and about 0.1-1% methionine.

[015] One or more embodiments of the invention also provide methods of stabilizing formulations comprising at least one methionine-containing peptide or protein, such as at least one cyclized parathyroid hormone analog, the methods comprising formulating at least one methionine-containing peptide or protein, such as a cyclized parathyroid hormone analog with at least one antioxidant. In the methods, at least one cyclized parathyroid hormone analog may be combined with at least one antioxidant in a liquid, which may be aqueous. In some embodiments, the methods involve forming a liquid composition comprising at least one methionine-containing peptide or protein, such as at least one cyclized parathyroid hormone analog, and at least one antioxidant; and drying the liquid to form a powder comprising particles. In some embodiments, the liquid further comprises at least one surface excipient; wherein the particles comprise a core and a surface; and wherein a concentration of the surface excipient is greater on the surface of the particles than in the core. At least some of the surface excipient is exposed on the surface of the particle. Thus, the particles may comprise a shell and a core. At least one cyclized parathyroid hormone analog may comprise, for example, [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-( 1-31)NH₂.

[016] In some embodiments, the liquid composition further comprises a pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutically acceptable excipient or carrier may comprise a carbohydrate such as a trehalose, a raffinose, and mixtures thereof. In some embodiments the excipient or carrier may comprise a salt of an organic acid, such as sodium citrate. In some embodiments the excipient or carrier may comprise an oligomeric or polymeric material, such as a cellulose or cellulosic material. In some embodiments, the liquid composition further comprises at least one antioxidant, such as at least one of thioethers, histidine, tyrosine, cysteine, tryptophan, and mixtures thereof. Thioethers include, but are not limited to, methionine. In some embodiments, the method of making the composition further comprises a solvent removal or drying step such as lyophilization, spray drying, freeze drying, or spray-freeze drying.

[017] One or more embodiments of the invention also provide particles comprising a surface and a core (for example, a shell and a core), produced by the methods of the invention. Also provided are powders comprising at least one particle according to the invention.

[018] One or more embodiments of the invention comprise a formulation comprising about 10-18% [Leu²⁷]cyclo(Glu²²-Lys²⁶)h PTH-(I -31)NH₂; about 1-4% sodium citrate; about 10-30% trileucine; about 20-60% trehalose; and about 0.1-1% methionine.

[019] One or more embodiments of the invention comprise a formulation comprising about from about 3% to about 6% of at least one cyclized parathyroid hormone analog; from about 4% to about 7% of at least one buffering agent; from about

20% to about 50% of at least one dispersibility-enhancing excipient; from about 40% to about 80% of at least one glass-stabilizing excipient; and from about 0.2% to about 1 % of at least one antioxidant.

[020] One or more embodiments of the invention comprise a formulation comprising about from about 1 % to about 25% of at least one parathyroid hormone, analog, fragment or derivative, from about 0% to about 10% of at least one buffering agent; from about 10% to about 60% of at least one dispersibility-enhancing excipient; and from about 10% to about 90% of at least one glass-stabilizing excipient. [021] One or more embodiments of the invention comprise a formulation comprising about from about about 10-18% [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-(1 -3I)NH₂; about 1-4% sodium citrate; about 10-30% trileucine;about 20-60% trehalose; and about 0.1-1% methionine.

[022] One or more embodiments of the invention comprise a method of delivering a powder to the lungs of a mammalian patient, comprising administering by inhalation a composition, wherein the composition comprises particles comprising at least one methionine-containing peptide and at least one surface excipient wherein the particles comprise a core and a surface; and wherein the concentration of the surface excipient is greater on the surface of the particles than in the core. [023] In some embodiments, a particulate composition comprises a methionine- containing peptide or protein comprising a cyclized parathyroid hormone, a parathyroid hormone derivative, a parathyroid hormone fragment, a parathyroid hormone analog, or combinations thereof, and trileucine, wherein the particulate is respirable. [024] On or more embodiments of the invention also provide particles comprising a surface enriched with a surface excipient having a glass transition temperature (T₉) generally above a T₉ of a material which is desired to be stabilized, such as a methionine-containing peptide or protein, and a core enriched with at least one methionine-containing peptide or protein, such as a cyclized parathyroid hormone analog. In some embodiments, the cyclized parathyroid hormone analog comprises [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-(1-31)NH₂. In some embodiments, the glass transition temperature of the surface excipient is at least about 70⁰C, such as at least about 75°C, at least about 80⁰C, or at least about 85°C. The surface excipient may comprise at least one of trileucine, leucine, isoleucine, norieucine, long-chain saturated phospholipids, e.g., distearoyl phosphatidylcholine (DSPC) and dipalmitoyl phosphatidylcholine (DPPC), and mixtures thereof.

[025] Further embodiments comprise a method for treating therapeutically or prophylatically a patient at risk of developing a condition or disease treatable by administering a methionine containing peptide or protein, such as a cyclized parathyroid hormone analog, and at least one antioxidant, the method comprising treating a patient prior to the onset of substantial symptoms, or prior to the expected occurrence of a disease or condition which may create the risk of such condition.

[026] Further embodiments comprise a method for administering therapeutically or prophylatically to a patient at risk of developing a condition or disease treatable by administering a methionine containing peptide or protein, such as a cyclized parathyroid hormone analog, the method comprising treating a patient prior to the onset of symptoms, or prior to exposure to a disease, or prior to the expected development condition, any of which may be treatable by such administration. [027] Further embodiments comprise a kit comprising a composition comprising at least one cyclized parathyroid hormone analog, and at least one antioxidant, and a delivery device.

[028] Further embodiments comprise a kit comprising a composition and a delivery device, wherein the device comprises a metered dose inhaler having a HFA propellaπt.

[029] The particles of one or more embodiments of the invention may further comprise at least one additional active ingredient. In some embodiments, an additional active may be selected from vitamin D, estrogen, calcitonin, bisphosphonates, analogs thereof and mixtures thereof. If present, the additional active ingredient may be in the core or in the surface, or both. In some embodiments, the core is further enriched in at least one core excipient. Core excipients comprise glass stabilizers, surface modifiers, dispersibility enhancers, and combinations thereof. In one or more embodiments, glass stabilizers may comprise sugars, such as trehalose and raffinose, organic acids (or salts) such as citrate, and mixtures thereof. In one or more embodiments, surface modifiers may comprise materials which are non-hygroscopic, such as being hydrophobic, and which impart a degree of non-hygroscopicity to the particle. Surface modifiers may comprise, for example, surfactants. Dispersibilty enhancers may also comprise surfactants. In some embodiments, particles according to the invention further comprise at least one antioxidant, which may be enriched in the core or surface. Antioxidants comprise thioethers, which include, but are not limited to, methionine. Methionine may be present in an amount of from about 0.1% to about 10% by weight of the particle, or from about 0.3% to about 5% by weight of the particle, or about 0.5% by weight of the particle.

[030] The particles according to one or more embodiments of the invention may be in a powder formulation. In some embodiments of the invention, the particles (as bulk powder) when stored at room temperature (such as about 25°C) and 60% relative humidity (RH), exhibit a degradation of the cyclized parathyroid hormone analog of not more than about 5% after 38 months, or exhibit an oxidation of not more than 1% after 38 months, or both. In some embodiments of the invention, the particles, when filled into capsules (such as HPMC) and stored at about 5°C, exhibit a degradation of the cyclized parathyroid hormone analog of not more than about 10% after 24 months, or exhibit an oxidation of not more than about 2% after 24 months, or both. In some embodiments of the invention, the particles, when filled into capsules (such as HPMC) and stored at room temperature and about 60% RH, exhibit a degradation of the cyclized parathyroid hormone analog of not more than about 10% after 19 months, or exhibit an oxidation of not more than about 2% after 19 months, or both. In some embodiments, the particles of the invention further comprise at least one amino acid In some embodiments, an amino acid comprises histidine, tryptophan, tyrosine, cysteine, and mixtures thereof.

[031] One or more embodiments of the invention also provide inhalers containing the dry powder formulations according to the invention. [032] One or more embodiments of the invention also provide blisters or polymer capsules containing the dry powder formulation according to the invention. The polymer may be hydroxypropylmethylcellulose (HPMC), or any other polymer used in pharmaceutical and/or inhalation grade capsules.

[033] A dry powder formulation according to the invention may have particles having an MMD of less than about 20 microns (μm,), or less than about 10, or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 microns. The dry powder formulation according to the invention may have particles having an MMAD of less than about 10 microns, or less than about 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 microns.

[034] One or more embodiments of the invention also provide a unit dose of at least one methionine-containing peptide or protein, such as a cyclized parathyroid hormone analog, comprising a receptacle and particles contained within the receptacle, wherein the particles comprise a surface enriched with a surface excipient having a glass transition temperature above that of the methionine-contaiπiπg peptide or protein, especially above that of a cyclized parathyroid hormone analog.

[035] One or more embodiments of the invention also provide a unit dose of at least one methionine-containing peptide or protein, such as a cyclized parathyroid hormone analog, and a delivery device, wherein the particles comprise a surface enriched with a surface excipient having a glass transition temperature above that of the methionine-containing peptide or protein, especially above that of a cyclized parathyroid hormone analog.

[036] One or more embodiments of the invention also provide a unit dose comprising a dispersible powder composition for delivery to the lungs of a patient by inhalation from a dry powder inhalation device, the composition including particles comprising a solid state matrix of a methionine containing peptide or protein, such as a cyclized parathyroid hormone analog, the particles having a MMAD of less than about

10 microns.

[037] In one or more embodiments the particles comprise a core enriched with at least one methionine-containing peptide or protein, e.g., at least one cyclized parathyroid hormone analog, and a surface excipient with a glass transition temperature above at least about 50⁰C.

[038] One or more embodiments of the invention comprise a particulate composition comprising at least one methionine-containing peptide or protein, and an oxygen scavenger, proximate to the particulate.

[039] One or more embodiments of the invention comprises a particulate comprising at least one methionine-containing peptide or protein, and antioxidant, and including an oxygen scavenger, proximate to the particulate.

[040] One or more embodiments of the invention comprise a particulate composition comprising a parathyroid homone, fragment thereof, analog thereof, derivative thereof, cyclized parathyroid hormone analog, or mixtures thereof, and an oxygen scavenger, proximate to the particulate. [041] One or more embodiments of the invention comprise a particulate composition comprising a parathyroid homone, fragment thereof, analog thereof, derivative thereof, cyclized parathyroid hormone analog, or mixtures thereof, and an antioxidant.

[042] One or more embodiments of the invention comprise a particulate composition comprising a parathyroid homone, fragment thereof, analog thereof, derivative thereof, cyclized parathyroid hormone analog, or mixtures thereof, and wherein the particulate composition is chemically or physically stable, or both, at room temperature for at least about 19 months, or at 5°C for at least about 150 months, or both.

[043] One or more embodiments of the invention comprise a particulate composition comprising a parathyroid homone, fragment thereof, analog thereof, derivative thereof, cyclized parathyroid hormone analog, or mixtures thereof, and at least one of an antioxidant or an oxygen scavenger, and wherein the particulate composition is chemically or physically stable, or both, at room temperature for at least about 22 months, or at 5°C for at least about 150 months, or both.

[044] One or more embodiments of the invention comprise a unit dosage form wherein the unit dose comprises a particulate comprising at least one methionine- containing peptide or protein, such as a cyclized parathyroid hormone analog and an oxygen scavenger, proximate to the particulate. The unit dose may be packaged in a capsule, blister or the like, or may be in bulk powder form.

[045] . One or more embodiments of the invention comprise a unit dosage form wherein the unit dose comprises a particulate comprising at least one methionine- containing peptide or protein, such as a cyclized parathyroid hormone analog, and antioxidant, and including an oxygen scavenger, proximate to the particulate. The unit dose may be packaged in a capsule, blister or the like, or may be in bulk powder form.

[046] Other embodiments of the present invention comprise any two of more features, aspects functions or embodiments set forth herein.

[047] Other features and advantages of the present invention will be set forth in the description of invention that follows, and will be apparent, in part, from the description or may be learned by practice of the invention. The invention will be realized and attained by the devices and methods particularly pointed out in the written description and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[048] The present invention is further described in the description of invention that follows, in reference to the noted plurality of non-limiting drawings, wherein: [049] Figure 1 shows the amino acid sequence of one embodiment of a cyclized parathyroid hormone analog marketed as OSTABOLIN-C™. [050] Figure 2 shows representative RP-HPLC chromatograms of a 1 % methionine formulation at room temperature and 50⁰C.

[051] Figure 3 shows percent peak area of oxidation peak (RRT 0.83 - 0.84) in one non-methionine and three methionine solution formulations at room temperature. [052] Figure 4 shows percent peak area of oxidation peak (RRT 0.83 - 0.84) in three methionine solution formulations at 50⁰C.

[053] Figures 5A, 5B, and 5C show percent peak area of oxidation peak for methionine and non-methionine formulations as a function of time and temperature. Figure 5A is at 25°C; Figure 5B is 40⁰C; and Figure 5C is 50⁰C. [054] Figure 6 shows an Arrhenius plot of OSTABOLIN-C™ oxidative degradation rate constants for methionine and non-methionine formulations. [055] Figure 7 shows values of T₉ versus weight fraction of water in accordance with the Gordon-Taylor equation, for a formulation according to Example 3. [056] Figure 8 is an HPLC stability analysis of two formulations (with and without an antioxidant) and stored at different temperatures, according to Example 3, showing the percent peak area of oxidation.

[057] Figure 9 is an HPLC stability analysis for three different formulations and three different storage conditions, for formulations made according to Example 4, showing, stability (as percentage oxidation) over the time indicated. [058] Figure 10 is an HPLC chromatagram for a formulation according to

Example 6, with and without an oxygen scavenger.

[059] Figure 11 is an HPLC stability analysis showing percent peak area of oxidative degradation peak in OCIP bulk powder (4% OCIP) and 5.0 mg fill weight capsules with and without an oxygen scavenger at 50°C as a function of storage duration.

[060] Figure 12 is an HPLC stability analysis showing the purity of Ostabolin-

C™ stored as bulk powder or 5.0 mg fill weight capsules with and without an oxygen scavenger at 50°C as a function of storage duration.

DESCRIPTION OF THE INVENTION

[061] Unless otherwise stated, a reference to a compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.

[062] As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise.

[063] Reference herein to "one embodiment", "one version" or "one aspect" shall include one or more such embodiments, versions or aspects, unless otherwise clear from the context.

[064] Except where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[065] Additionally, the recitation of numerical ranges within this specification is considered to be a disclosure of all numerical values within that range. For example, if a range is from about 1 to about 50, it is deemed to include, for example, 1 , 7, 34, 46.1 , 23.7, or any other value within the range.

[066] Before further discussion, a definition of the following terms will aid in the understanding of the present invention. [067] "Amino acid" refers to any compound containing both an amino group and a carboxylic acid group. Although the amino group most commonly occurs at the position adjacent to the carboxy function, the amino group may be positioned at any location within the molecule. The amino acid may also contain additional functional groups, such as amino, thio, carboxyl, carboxamide, imidazole, etc. An amino acid may be synthetic or naturally occurring, and may be used in either its racemic or optically active (D-, or L-) form.

[068] "Leucine," whether present as a single amino acid or as an amino acid component of a peptide, refers to the amino acid leucine, which may be a racemic mixture or in either its D- or L- form, as well as modified forms of leucine (i.e., where one or more atoms of leucine have been substituted with another atom or functional group) in which the dispersibility-enhancing effect of the modified amino acid or peptide is substantially unchanged or unimproved over that of the unmodified material. [069] "Dipeptide," also referred to herein as a dimer, refers to a peptide including two amino acids.

[070] "Tripeptide," also referred to herein as a trimer, refers to a peptide including three amino acids.

[071] A "surface active" material is one having surface activity (measured, e.g., by surface tensiometry), as characterized by its ability to reduce the surface tension of the liquid in which it is dissolved. Surface tension, which is associated with the interface between a liquid and another phase, is that property of a liquid by virtue of which the surface molecules exhibit an inward attraction. [072] Typically, in the context of the present invention, a surface active dipeptide or tripeptide is identified by preparing solutions of varying concentrations (for example, from approximately 0.01% wt/vol (0.1 mgfml) to approximately 2% wt/vol (20 mg/ml)) of the subject peptide in water, and measuring the surface tension of each of the solutions. A surface-active peptide is one that, when present at any concentration in solution, though typically present in an amount greater than 0.25 mg/ml, is effective to lower the surface tension of water. A peptide that is more surface-active than another peptide is one that decreases the surface tension of water to a greater extent, when present in the liquid at the same concentration and measured under the same set of experimental conditions.

[073] "Dry powder" refers to a powder composition that typically contains less than about 20 wt% moisture, or less than 10 wt% moisture, or contains less than about 5-6 wt% moisture, or contains less than about 3 wt% moisture, depending upon factors, such as the particular formulation.

[074] A dry powder that is "suitable for pulmonary delivery" refers to a composition comprising solid (i.e., non-liquid) or partially solid particles that are capable of being (i) readily dispersed in/by an inhalation device and (ii) inhaled by a subject so that a portion of the particles reach the lungs to permit penetration into the alveoli. Such a powder is considered to be "respirable."

[075] "Aerosolized" or "aerosolizable" particles are particles which, when dispensed into a gas stream by either a passive or an active inhalation device, remain suspended in the gas for an amount of time sufficient for at least a portion of the particles to be inhaled by the patient, so that a portion of the particles reaches the lungs.

[076] "Emitted Dose" or "ED" provides an indication of the delivery of a drug formulation from a suitable inhaler device after a firing or dispersion event. More specifically, for dry powder formulations, the ED is a measure of the percentage of powder which is drawn out of a unit dose package and which exits the mouthpiece of an inhaler device. The ED is defined as the ratio of the dose delivered by an inhaler device to the nominal dose (i.e., the mass of powder per unit dose placed into a suitable inhaler device prior to firing). The ED is an experimentally-determined parameter, and is typically determined using an in-vitro device set up which mimics patient dosing. To determine an ED value for a powder, as used herein, a nominal dose of dry powder (as defined above) is placed into a Turbospin® DPI device (PH&T, Italy), described in U.S. Patent Nos. 4,069,819 and 4,995,385, which are incorporated herein by reference in their entireties. The Turbospin® DPI is actuated, dispersing the powder. The resulting aerosol cloud is then drawn from the device by vacuum (30 L/min) for 2.5 seconds after actuation, where it is captured on a tared glass fiber filter (Gelman, 47 mm diameter) attached to the device mouthpiece. The amount of powder that reaches the filter constitutes the delivered dose. For example, for a capsule containing 5 mg of dry powder that is placed into an inhalation device, if dispersion of the powder results in the recovery of 4 mg of powder on a tared filter as described above, then the ED for the dry powder composition is 80% [= 4 mg (delivered dose)/5 mg (nominal dose)]. For non- homogenous powders, ED values provide an indication of the delivery of drug from an inhaler device after firing rather than of dry powder, and are based on amount of drug rather than on total powder weight. Similarly, for MDI and nebulizer dosage forms, the ED corresponds to the percentage of drug which is drawn from a dosage form and which exits the mouthpiece of an inhaler device. Table 21 presents ED measurements taken with an Anderson Cascade lmpactor (a standard analytical tool). [077] "Fine particle dose" or "FPD" is defined as the mass percent of powder particles having an aerodynamic diameter less than a predetermined value, such as 3.3 μm. The FPD may be determined by measurement in an Andersen cascade impactor. This parameter provides an indication of the percent of particles having the greatest potential to reach the deep lung of a patient for systemic uptake of a drug substance. [078] A "dispersible" or "dispersive" powder is one having an ED value of at least about 30%, such as at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or more.

[079] As used herein, "passive dry powder inhaler" refers to an inhalation device that relies upon a patient's inspiratory effort to disperse and aerosolize a pharmaceutical composition contained within the device in a reservoir or in a unit dose form and does not include inhaler devices which comprise a means for providing energy, such as pressurized gas and vibrating or rotating elements, to disperse and aerosolize the drug composition.

[OSO] As used herein, "active dry powder inhaler" refers to an inhalation device that does not rely solely on a patient's inspiratory effort to disperse and aerosolize a pharmaceutical composition contained within the device in a reservoir or in a unit dose form and does include inhaler devices that comprise a means for providing energy to disperse and aerosolize the drug composition, such as pressurized gas and vibrating or rotating elements. [081] As used herein, "mass median diameter" or "MMD" refers to the median diameter of a plurality of particles, typically in a polydisperse particle population, i.e., consisting of a range of particle sizes. MMD values as reported herein are determined by laser diffraction (Sympatec Helos, Clausthal-Zellerfeld, Germany), unless the context indicates otherwise. Typically, powder samples are added directly to the feeder funnel of the Sympatec RODOS dry powder dispersion unit. This can be achieved manually or by agitating mechanically from the end of a VIBRI vibratory feeder element. Samples are dispersed to primary particles via application of pressurized air (2 to 3 bar), with vacuum depression (suction) maximized for a given dispersion pressure. Dispersed particles are probed with a 632.8 nm laser beam that intersects the dispersed particles' trajectory at right angles. Laser light scattered from the ensemble of particles is imaged onto a concentric array of photomultiplier detector elements using a reverse-Fourier lens assembly. Scattered light is acquired in time-slices of 5 ms. Particle size distributions are back-calculated from the scattered light spatial/intensity distribution. [082J As used herein, "mass median aerodynamic diameter" or "MMAD" refers to the median aerodynamic size of a plurality of particles, typically in a polydisperse population. The "aerodynamic diameter" is the diameter of a unit density sphere having the same settling velocity in air as a powder and is therefore a useful way to characterize an aerosolized powder or other dispersed particle in terms of its settling behavior. The aerodynamic diameter encompasses particle or particulate shape, density, and physical size of the particle or particulate. As used herein, MMAD refers to the median of the aerodynamic particle or particulate size distribution of an aerosolized powder determined by cascade impaction, unless the context indicates otherwise. [083] "Pharmaceutically acceptable salt" includes, but is not limited to, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate salts, or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, ^{^} potassium, calcium, aluminum, lithium, and ammonium (including alkyl substituted ammonium).

[084] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention, and taken into the lungs with no significant adverse toxicological effects to the subject, and particularly to the lungs of the subject.

[085] "Pharmacologically effective amount" or "physiologically effective amount of a bioactive agent" is the amount of an active agent present in an aerosolizable composition as described herein that is needed to provide a desired level of active agent in the bloodstream or at the site of action (e.g., the lungs) of a subject to be treated to give an anticipated physiological response when such composition is administered pulmonarily. The precise amount will depend upon numerous factors, e.g., the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e., the number of doses administered per day), patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.

[086] "Polymer" refers to a high molecular weight polymeric compound or macromolecule built by the repitition of small, simple chemical units. A polymer may be a naturally occurring (e.g., proteins, carbohydrates, nucleic acids) or a non-naturally occurring polymer (e.g., polyethylene glycols, polyvinylpyrrolidones, Ficolls, and the like), as well known in the art.

[087] As used herein, "effective amount" refers to an amount which is operative to achieve the desired result. "Effective amount" encompasses both therapeutically effective amounts and prophylactically effective amounts.

[088] As used herein, "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic result. A therapeutically effective amount of a given active agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the patient.

[089] As used herein, "prophylactically effective amount" refers to an amount that is effective to achieve the desired prophylactic result. Because a prophylactic dose is administered in patients prior to onset of disease (or prior to the onset of significant symptoms thereof) the prophylactically effective amount typically is less than the therapeutically effective amount.

[090] The present invention is generally directed to compositions comprising cyclized parathyroid hormone (PTH) analogs, and methods of making the same. The compositions of the present invention can be liquid or dry. Liquid compositions include, but are not limited to, solutions, suspensions and dispersions. Dry compositions include lyophillizates, powders, etc.

[091] Unless otherwise noted or clear from the context, a parathyroid hormone, cyclized parathyroid hormone analog, parathyroid hormone fragment or parathyroid hormone derivative are deemed to be interchangeable for formulation purposes herein.

A methionine containing proteins or peptide is deemed to comprise each and all of the foregoing, individually or collectively.

Methionine-Containinp Proteins or Peptides

[092] Initially, it is noted that no distinction is intended between proteins and peptides, and that the two terms may be used interchangeably herein. Generally, on a relative scale, peptides will be smaller and proteins will be larger.

[093] One or more methionine-containing proteins or peptides may be included in the compositions. Methionine-containing proteins and peptides according to the present invention include, but are not limited to, parathyroid hormone, insulin-like growth factor, epidermal growth factor, nerve growth factor, transforming growth factor alpha precursor, transforming growth factor beta, transforming growth factor beta precursor, fibroblast growth factor, vaccinia growth factor, platelet derived growth factor, interleukins, e.g., IL-2, interferons, e.g., interferon beta-1b, tissue-factor-pathway inhibitor, factor VIII, growth hormone, prolactin, and granulocyte colony-stimulating factor, to name only a few. Any of the foregoing methionine-containing protein or peptides can be mutated or modified, and such variants are within the scope of the present invention. Parathyroid hormones include, for example, parathyroid hormone analogs.

[094] Exemplary PTH peptide analogs may be selected from the group consisting of PTH-(I -31 )NH2; PTH-(1-30)NH2; PTH-(I -29)NH2; PTH-(I -28)NH2; Leu27PTH-(1-31)NH2; Leu27PTH-(1-30)NH2; Leu27PTH-(1-29)NH2; Leu27cyclo(22- 26)PTH-(1-31 )NH2; Leu27cyclo(22-26)PTH-(1-34)NH2; Leu27cydo(Lys26-Asp30)PTH- (1-34)NH2; Cyclo(Lys27-Asp30)PTH-(1-34)NH2; Leu27cyclo(22-26)PTH-(1-31 )NH2; Ala27 or Nle27 orTyr27 or Ile27 cyclo(22-26)PTH-(1-31)NH2; Leu27cyclo(22-26)PTH- (1-32)NH2; Leu27cyclo(22-26)PTH-(1-31)OH; Leu27cyclo(26-30)PTH-(1-31)NH2; Cys22Cys26Leu27cyclo(22-26)PTH-(1 -31 )NH2; Cys22Cys26Leu27cyclo(26-30)PTH- (1-31 )NH2; Cyclo(27-30)PTH-(1-31)NH2; Leu27cyclo(22-26)PTH-(1-30)NH2; Cyclo(22- 26)PTH-(1-31 )NH2; Cyclo(22-26)PTH-(1-30)NH2; Leu27cyclo(22-26)PTH-(1-29)NH2; Leu27cydo(22-26)PTH-(1-28)NH2; Glu17,Leu27cydo(13-17)(22-26)PTH-(1-28)NH2; and Glu17,Leu27cyclo(13-17)(22-26)PTH-(1-31)NH2.

[095] Additionally, or alternatively, the present invention may comprise the full length, 84 amino acid form of parathyroid hormone, particularly the human form, hPTH (1-84), obtained either recombinantly, by peptide synthesis or by extraction from human fluid. See, for example, U.S. Pat. No. 5,208,041 , incorporated herein by reference. The amino acid sequence for hPTH (1-84) is reported by Kimura et al. in Biochem. Biophys. Res. Comm., 114(2):493. The composition of the present invention may also comprise fragments or variants of fragments of human PTH or of rat, porcine or bovine PTH that have human PTH activity as determined, for example, in the ovarectomized rat model of osteoporosis reported by Kimmel et al., Endocrinology, 1993, 32(4): 1577. [096] The parathyroid hormone fragments may in some embodiments incorporate at least the first 34 N-terminal residues, such as PTH(I -34), PTH(1-37), PTH(I -38) and PTH(I -41). Alternatives in the form of PTH variants incorporate from 1 to 5 amino acid substitutions that improve PTH stability and half-life, such as the replacement of methionine residues at positions 8 and/or 18 with leucine or other hydrophobic amino acid that improves PTH stability against oxidation and the replacement of amino acids in the 25-27 region with trypsin-insensitive amino acids such as histidine or other amino acid that improves PTH stability against protease. The hormones may be obtained by known recombinant or synthetic methods, such as described in U.S. Pat. No. 4,086,196, incorporated herein by reference. [097] As noted, parathyroid hormone is an 84-amino acid peptide, but analogs

(and derivatives or fragments) include, for example, a number of variants, such as truncated variants. PTH analogs include, but are not limited to, PTH(1-11), PTH(1-14), PTH(1-34), and PTH(3-34). PTH analogs within the scope of the invention comprise, for example, those disclosed in U.S. Patent Nos. 5,001,223; 5,093,233; 5,420,242; 5,814,603; 5,814,607; 5,955,425; 6,080,721; 6,146,852; 6,417,333; 6,472,505; 6,495,662; 6,537,965; 6,541,220; 6,770,623; 6,803,213; 6,962,796; and 6,977,077; and U.S. Published Application Nos. 20030144209; 20030171282; 20030228665; 20040005668; 20040014150; 20050026839; 20050060764; 20050215476; and 20060019902; WO05009358; WO04093902; WO04067021; WO0214466; EP1592434; EP0672057; and EP0656784, the entire disclosures of which are incorporated herein by reference. PTH analogs also include, but are not limited to, cyclized PTH analogs. [098] Cyclized PTH analogs falling within the scope of the invention include, but are not limited to, those described in, for example, U.S. Patent Nos. 5,556,940; 6,110,892; 5,955,425; 6,316,410; and 6,541,450, which are incorporated herein by reference. Examples of such cyclized PTH analogs include, but are not limited to, [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-(1 -3I)NH₂ (OSTABOLI N-C™, such as that supplied by Zelos). The compositions of the present invention may include one or more such cyclized PTH analogs. For example, the composition may include, but is not limited to, one, two, three, or more such analogs.

[099] The amount of the methioniπe-containing protein or peptide in the composition is an amount that is sufficient to achieve a desired effect by administration of a desired dose. In practice, this will vary widely depending upon the particular agent, its activity, the severity of the condition to be treated, the patient population, dosing requirements, and the desired therapeutic effect. The amount may be such that two or more doses are needed to achieve the desired effect. The compositions of the invention are particularly useful for active agents that are delivered in doses of from 0.001 mg/day to 100 mg/day, such as in doses from 0.01 mg/day to 75 mg/day, or in doses from 0.10 mg/day to 50 mg/day. It is to be understood that more than one cyclized PTH analog may be incorporated into the formulations described herein and that the use of the term "analog" in no way excludes the use of two or more such analogs. [0100] Generally, the methionine-containing protein or peptide in the composition will comprise from about 1% to about 50%, by weight of the composition. In some embodiments, the amount will range from about 2% to about 40%, or from about 3% to about 25%, or from about 4% to about 10%, by weight of the composition. The amount can range from about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%, to about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or more, by weight of the composition.

[0101] It should be noted that reference to numeric ranges throughout this specification is intended to encompass all numbers falling within the disclosed ranges. Thus, for example, the recitation of the range of about 1 % to about 50% includes 1 %, 2%, 3%, 4%, 5%,.6%, 7%, 8%, or 9%, 10%, 12%, 14%, 16%, 20%. 25%, 30%, 35%, 40%, 45%, and 50%, as well as, for example, 21.3%, 7.9%, and 44.5%. [0102] Additional active ingredients can be included in the present compositions.

Choices are not limited, but may be chosen for a desired combined therapeutic effect. For example, active ingredients that may be added for a complementary therapeutic effect include, but are not limited to, vitamin D and analogs, estrogen, calcitonin, bisphosphonates, and mixtures thereof. A particularly desirable choice is calcitonin. Antioxidants

[0103] The compositions of the present invention may additionally include one or more antioxidants. Antioxidants according to the invention prevent or reduce the oxidation of the methionine-containing protein or peptide of the compositions. In some embodiments, a suitable antioxidant is a pharmacetucially-acceptable antioxidant. In some embodiments, an antioxidant is a material which reduces the oxidation potential of the methionine-containing protein or peptide active material. In one or more embodiments, an antioxidant is any material that reduces the oxidation of the methionine-containing protein or peptide active agent, such as cyclized parathyroid hormone analog, by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95% or 97% or 98% or 99%.

[0104] In some embodiments, the antioxidants prevent or reduce the oxidation of amino acid residues in methionine-containing protein or peptide. Amino acids that are particularly sensitive to oxidation include, but are not limited to, methionine. Thus, agents that act to prevent or reduce oxidation of methionines are considered antioxidants within the scope of this invention.

[0105] In one example, OSTABOLIN-C™, a cyclized PTH analog, contains oxidation-sensitive amino acids, including two methionine residues in the 8 and 18 positions, which can both undergo degradation via oxidation to form methionine sulfoxide. See Figure 1. The mechanisms of oxidation of methionine to methionine sulfoxide are believed to be either molecular oxygen dependent mediated through a transition metal, or peroxide-mediated oxidation with an alkyl peroxide as the oxidizer. Regardless of the mechanism by which the oxidation occurs, antioxidants may be included in the inventive compositions to reduce or prevent oxidation, at either or both the methionine site(s), as well as that of any other any other oxidizable residue. [0106] In some embodiments of the invention, the antioxidant comprises at least one thioether. Examples of thioether antioxidants include, but are not limited to, those disclosed in U.S. Patent No. 6,989,138, which is incorporated herein by reference in its entirety. While the particular choice of thioether is not limited, one example is methionine. Other antioxidants that can be included in the compositions of the invention include, but are not limited to other amino acids, such as histidine, cysteine, tryptophan, and tyrosine, vitamin E, and ascorbic acid. The composition may comprise one or more antioxidants.

[0107] The amount of the antioxidant in the composition is an amount that is sufficient to achieve a desired antioxidant effect. The amount may be such that oxidation of the methionine-containing protein or peptide is slightly reduced or an amount sufficient to essentially completely prevent oxidation. In one or more embodiments, the amount of antioxidant may be sufficient to provide a shelf life of a product, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24, 25, 50, 60, 70. 80, 90, 100, 120, 150, 160 or more months, whether measured at room temperature (such as about 25°C), elevated temperature (such as about 40⁰C) or low temperature (such as about 5°C).

[0108] In one or more embodiments, the amount of antioxidant may be sufficient to extend shelf life of a product, such as by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or 50 or 100 or 150 or more months, whether measured at room temperature (such as about 25°C), elevated temperature (such as about 40⁰C) or low temperature (such as about 5°C). By extending the shelf life it is meant in comparison to a shelf life of the same formulation without the anti-oxidant.

[0109] Generally, the antioxidant in the composition will comprise from about

0.1 % to about 10%, by weight of the composition. In some embodiments, the amount will range from about 0.2% to about 9%, or from about 0.3% to about 8%, or from about 0.4% to about 7%, or about 0.5% to about 5%, by weight of the composition. The amount can be about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10%, by weight of the composition. The amount chosen will depend on its desired effect on the composition and can be varied as needed. Other Excipients

[0110] The compositions of the invention may further include one or more buffering, or pH-adjusting or -controlling, agents. These agents are generally a salt prepared from an organic acid or base. Representative buffers include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride, betaine, or phosphate buffers. Suitable amino acids, which may also function in a buffering capacity, include alanine, glycine, arginine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, tyrosine, tryptophan, and the like.

[0111] These agents, if present, are generally present in amounts of from about

1 % to about 10%, by weight, of the composition. In some embodiments, the amount ranges from about 2% to about 9%, or from about 3% to about 8%, or from about 4% to about 7%, or from about 5% to about 6%, or about 5%, by weight, of the composition. The amount chosen will depend upon its desired effect on the composition and can be varied as needed.

[0112] Some embodiments of the invention include excipients that are designed to impart desired physical characteristics to the end product, which may further impart desired or improved actions on the treated subject. Thus, the inventive compositions may comprise a pharmaceutically acceptable excipient or carrier, which may be taken into the lungs with no significant adverse toxicological effects to the subject, and particularly to the lungs of the subject.

[0113] Generally, such excipients will, at least in part, serve to further improve the features of the active agent composition, for example by providing more efficient and/or reproducible delivery of the active agent, improve the handling characteristics of powders, such as flowability and consistency, and/or facilitate manufacturing and/or filling of unit dosage forms. In particular, excipient materials can often function to further improve the physical and chemical stability of the active agent, minimize the residual moisture content and hinder moisture uptake, and to enhance particle size, degree of aggregation, particle surface properties, such as rugosity, ease of inhalation, and the targeting of particles to the lung. One or more excipients may also be provided to serve as bulking agents when it is desired to reduce the concentration of active agent in the formulation.

[0114] One particular type of formulation-enhancing excipient that may be included in the formulation is dispersibility-enhancing excipients. These excipients generally provide more efficient and/or reproducible delivery of the methionine- containing protein or peptide, by improving the physical characteristics of the dry formulation. Dispersibility-enhancing agents may comprise amino acids and polypeptides that function as dispersing agents. Amino acids falling into this category include, but are not limited to, hydrophobic amino acids such as leucine, norleucine, valine, isoleucine, tryptophan, alanine, methionine, phenylalanine, tyrosine, histidine, and proline. Dispersibility-enhancing peptide excipients include dimers, trimers, tetramers, and pentamers comprising one or more hydrophobic amino acid components such as those described above. Examples include, but are not limited to, dimers, trimers, tetramers, and pentamers having at least two leucines in any position, such as dileucine and trileucine, as disclosed in U.S. Patent Nos. 6,518,239, and 6,835,372, both to Kuo et al, both of which are incorporated herein by reference their entireties. [0115] These excipients, if present, are generally present in the composition in amounts ranging from about 0.01% to about 95%, such as about 0.5% to about 80%, or about 1 % to about 60%, by weight. The amount can be from about 10% to about 60%, or from about 20% to about 50%, or from about 30% to about 40%, or about 35%, by weight of the composition. The amount chosen will depend on its desired effect on the composition and can be varied as needed.

[0116] Some formulations may benefit from the addition of a component that stabilizes the glass transition temperature of the composition. In some embodiments, this component will have a higher glass transition temperature than the methionine- containing protein or peptide. In some embodiments, the excipient may have a glass transition temperature (Tg) above about 35°C, such as above about 40⁰C, above about 45°C, above about 55°C, above about 60⁰C, above about 65°C, above about 70⁰C, above about 75°C, above about 80⁰C, above about 85°C, or above about 90⁰C, as measured by differential scanning calorimetry (DSC).

[0117] Glass transition stabilizing excipients may comprise carbohydrates, excipients suitable for use in the invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like, and combinations of any of the foregoing. [0118] In one or more embodiments, stabilizing excipients comprise polyvinylpyrrolidone (PVP), polyvinyl acetate (PVA), vinylpyrrolidone/vinyl acetate copolymer (PVP-VA), poly ethylene oxide (PEO), cellulose, starch, polyethylene glycol (PEG), hydroxypropyl cellulose (HPC), hydroxyl propyl methyl cellulose (HPMC), and their copolymers and derivatives; carbohydrates; polyols; sugars; oligo saccharides such as cyclodextrins; proteins, peptides and amino acids', lipids and modified lipids such as lipid-PEG and lipid-sugar esters; salts; citric acid; citrates; known glass formers; and combinations thereof.

[0119] The glass transition stabilizing excipients, if present, will generally be present in an amount by weight of from about 10% to about 90% of the composition. In some embodiments, they are present in an amount from about 20% to about 80%, or from about 30% to about 70%, or from about 40% to about 60%, or about 50%, by weight of the composition. The amount may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% by weight of the composition. The amount chosen will depend upon its desired effect on the composition and can be varied as needed.

[0120] Other pharmaceutical excipients and additives useful in the present pharmaceutical formulation include, but are not limited to, amino acids, peptides, proteins, non-biological polymers, biological polymers, carbohydrates, such as sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and sugar polymers, which may be present singly or in combination. Exemplary protein excipients include albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like. Suitable excipients are those provided in U.S. Patent No. 6,136,346 and WO 96/32096, which are incorporated herein by reference in their entireties.

[0121] The inventive compositions may also include polymeric excipients/additives, e.g., polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethylstarch, dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin.

[0122] The inventive compositions may further include flavoring agents, taste- masking agents, inorganic salts (for example sodium chloride), antimicrobial agents (for example benzalkoπium chloride), sweeteners, antioxidants, antistatic agents, surfactants (for example polysorbates such as "TWEEN 20" and "TWEEN 80"), sorbitan esters, lipids (for example phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines), fatty acids and fatty esters, steroids (for example cholesterol), and chelating agents (for example EDTA, zinc and other such suitable cations). Other pharmaceutical excipients and/or additives suitable for use in the compositions according to the invention are listed in "Remington: The Science & Practice of Pharmacy", 21^st ed., Lippincott, Williams & Wilkins, (2005), and in the "Physician's Desk Reference", 60^th ed., Medical Economics, Montvale, NJ (2006), both of which are incorporated herein by reference in their entireties. Formulations [0123] The compositions described herein may be in powdered form (e.g., including particles of the invention) or may be liquids. In one or more embodiments liquid formulations comprise solutions in which the active drug is dissolved in a solvent (e.g., water, ethanol, ethanol-water, saline). In other embodiments, liquid formulations comprise colloidal suspensions, or dispersions. The liquid formulation may also be a solution, suspension or dispersion of the methionine-containing protein or peptide in a low boiling point propellant.

[0124] Liquid formulations containing the disclosed dileucyl-containing peptides are also highly dispersible, possessing high ED values.

[0125] Some embodiments of the invention include particles that have physical characteristics that allow for their delivery to the deep lung. In one embodiment, a powdered or liquid formulation for use in the present invention includes an aerosol having a particle size selected to permit penetration into the alveoli of the lungs. In one or more embodiments, the size is typically less than about 10 μm mass median diameter (MMD), such as less than about 7.5 μm, or less than 5 μm, and may be in the range of about 0.1 μm to 5 μm, or about 0.5 μm to 4 μm in diameter, or any combination of the foregoing. When in a dry powder form, the pharmaceutical formulation may have a moisture content below about 10 wt%, such as below about 5 wt%, or below about 3 wt%. Such powders are described in WO 95/24183, WO 96/32149, WO 99/16419, and WO 99/16422, ail of which are incorporated herein by reference in their entireties. [0126] In some embodiments, the composition comprises particles having a mass median aerodynamic diameter from about 0.5 μm to about 10 μm, such as about 1 μm to about 9 μm, or about 1.5 μm to about 8 μm, or about 2 μm to about 6 μm or about 2.5 μm to about 5 μm, or any combination of the foregoing. [0127] Some particles according to some aspects of the invention are formed in such a manner that the compositions are uniformly distributed throughout the particle. That is, the methionine-containing protein or peptide, as well as other elements of the composition, which may include antioxidant, buffer, dispersibility-enhancing agent, and glass-stabilizing agent, are uniformly distributed throughout the particle. [0128] Other particles according to other aspects of the invention are formed in such a way as to enrich particular elements of the formulation in particular sections of the particle. For example, a particle may generally be described as including a core at its center and a surface around its periphery. With regard to the heterogeneity within the particle, the transition from core to surface may be gradual or abrupt, or any variation thereof. Particles may be manufactured such that a core is enriched with one element and a surface is enriched with another.

[0129] This heterogeneity may be achieved by forming the core and surface in separate preparation steps, using different compositional elements during the different steps. Alternatively, the heterogeneity may be achieved by introducing into a homogeneous mixture a component that has an affinity for a particular section of a particle or which migrates during a drying phase, for example. Examples of such methods are described in U.S. Patent No. 6,518,239, the entire disclosure of which is incorporated herein by reference.

[0130] In one or more embodiments of the invention, heterogeneous particles are formed by forming a liquid composition comprising a methionine-containing protein or peptide and an antioxidant. The liquid composition additionally includes at least one surface excipient, which is an agent that has a tendency to migrate to the surface of the particle. Such surface excipients may be "surface active agents," as described in U.S. Patent No. 6,518,239. Examples of such agents include, but are not limited to, di-and tripeptides containing at least two leucines.

[0131] Unit dose pharmaceutical compositions may be contained in a container.

Examples of containers include, but are not limited to, capsules, blisters, vials, ampoules, or container closure systems made of metal, polymer (e.g., plastic, elastomer), glass, or the like.

[0132] The container may be inserted into an aerosolization device. The container may be of a suitable shape, size, and material to contain the pharmaceutical composition and to provide the pharmaceutical composition in a usable condition. For example, the capsule or blister may comprise a wall which comprises a material that does not adversely react with the pharmaceutical composition. In addition, the wall may comprise a material that allows the capsule to be opened to allow the pharmaceutical composition to be aerosolized. In one version, the wall comprises one or more of gelatin, hydroxypropyl methylcellulose (HPMC), polyethyleneglycol-compounded HPMC, hydroxyproplycellulose, agar, aluminum foil, or the like. In one version, the capsule may comprise telescopically adjoining sections, as described for example in U.S. Patent No. 4,247,066, which is incorporated herein by reference in its entirety. The size of the capsule may be selected to adequately contain the dose of the pharmaceutical composition. The sizes generally range from size 5 to size 000 with the outer diameters ranging from about 4.91 mm to 9.97 mm, the heights ranging from about 11.10 mm to about 26.14 mm, and the volumes ranging from about 0.13 ml_ to about 1.37 ml_, respectively. Exemplary standard capsule sizes, and their corresponding volumes are shown in Table 1 below: Table 1

[0133] Suitable capsules are available commercially from, for example, Shionogi

Qualicaps Co. in Nara, Japan and Capsugel in Greenwood, South Carolina. After filling, a top portion may be placed over the bottom portion to form a capsule shape and to contain the powder within the capsule, as described in U.S. Patent Nos. 4,846,876 and 6,357,490, and in WO 00/07572, which are incorporated herein by reference in their entireties. After the top portion is placed over the bottom portion, the capsule can optionally be banded. Preparing Compositions

[0134] The compositions of one or more embodiments of the present invention may be made by any of the various methods and techniques known and available to those skilled in the art.

[0135] Dry powder formulations may be prepared by solvent removal processes appropriate to remove solvent from a liquid solution, suspension or dispersion. Example of suitable solvent removal processes comprise spray drying, freeze drying, and spray-freeze drying. Spray drying of the formulations is carried out, for example, as described generally in the "Spray Drying Handbook", 5^th ed., K. Masters, John Wiley & Sons, Inc., NY, N. Y. (1991), and in WO 97/41833, the contents of which are incorporated herein by reference.

[0136] The methionine-containing protein or peptides can be spray-dried from an aqueous solution. Utilizing this approach, the methionine-containing protein or peptide is first dissolved in water, optionally containing a physiologically acceptable buffer or other excipient as describe above. The pH range of active agent-containing solutions is generally between about 2 and 11, with approximately neutral pHs being preferred, since such pHs may aid in maintaining the physiological compatibility of the powder after dissolution of powder within the lung. The aqueous formulation may optionally contain additional water-miscible solvents, such as acetone, alcohols, and the like. Representative alcohols are lower alcohols such as methanol, ethanol, propanol, isbpropanol, and the like. The pre-spray dried solutions will generally contain solids dissolved at a concentration from 0.01% (weight/volume) to about 20% (weight/volume), usually from 0.1% to 3% (weight/volume). [0137] The solutions are then spray dried in a spray drier, such as those available from commercial suppliers such as Niro A/S (Denmark), Buchi (Switzerland) and the like, resulting in a dispersible, dry powder. Optimal conditions for spray drying the solutions will vary depending upon the formulation components, and are generally determined experimentally. The gas used to spray dry the material is typically air, although inert gases such as nitrogen or argon are also suitable. Moreover, the temperature of both the inlet and outlet of the gas used to dry the sprayed material is such that it does not cause decomposition of the active agent in the sprayed material. Such temperatures are typically determined experimentally, although generally, the inlet temperature will range from about 50⁰C to about 200⁰C while the outlet temperature will range from about 30⁰C to about 150⁰C.

[0138] Variations of the above may be utilized. One such process is described in

U.S. Patent No. 5,985,248, assigned to Nektar Therapeutics, which document is incorporated herein by reference in its entirety. In this method, a hydrophobic drug is dissolved in an organic solvent or co-solvent system, and the hydrophilic components (e.g., the leucyl-containing peptides and optional other excipients) are at least partially dissolved in the same organic solvent or co-solvent system. The resulting solution is then spray-dried to form particles. Typically, the solubility of the active agent and the hydrophilic component will govern the selection of the organic solvent system. The organic solvent is selected to provide a solubility for the hydrophilic component of at least 1 mg/ml, and preferably at least 5 mg/ml, and a solubility for the hydrophobic drug of at least 0.01 mg/ml, preferably at least 0.05 mg/ml.

[0139] Alternatively, the composition may be prepared by spray-drying a suspension, as described in U.S. Patent No. 5,976,574, assigned to Nektar Therapeutics, which document is incorporated herein by reference in its entirety. In this method, the hydrophobic drug is dissolved in an organic solvent, e.g., methanol, ethanol, isopropanol, acetone, heptane, hexane chloroform, ether, followed by suspension of the hydrophilic excipient in the organic solvent to form a suspension. The suspension is then spray-dried to form particles. Preferred solvents, for both of the above spray-drying methods include alcohols, ethers, ketones, hydrocarbons, polar aprotic solvents, and mixtures thereof.

[0140] The dry powders of the invention may also be prepared by combining aqueous solutions or suspensions of the formulation components and spray-drying them simultaneously in a spray-dryer, as described in U.S. Patent No. 6,001 ,336, assigned to Nektar Therapeutics, which document is incorporated herein by reference in its entirety. Alternatively, the dry powders may be prepared by preparing an aqueous solution of a hydrophilic excipient or additive, preparing an organic solution of a hydrophobic drug, and spray drying the aqueous solution and the organic solution simultaneously through a nozzle, e.g., a coaxial nozzle, to form a dry powder, as described in WO 98/29096, which is incorporated herein by reference in its entirety. [0141] As shown in the Examples, solution stability studies did not show a significant difference in the oxidation rate when comparing solutions with and without methionine. When the same formulations were spray dried, a surprising difference in oxidation was observed between compositions with and without methionine. [0142] While not bound by theory, it is believed that during spray drying, a shell phase and a core phase are created. The shell phase is created via trileucine which provides added stability to the core phase by encapsulating it. The role of the shell phase is to stabilize the core phase by encapsulating it and improving the dispersibility and aerosol character due to its hydrophobic nature. This is done by formulating in such a manner that trileucine is much closer to its saturation solubility as compared with the core components. In this mechanism trileucine comes out of solution first as a precipitate, which leads to the formation of a skin on the surface of the particle. At the same time, there is a concentration and viscosity gradient in the drying droplet. At the surface, the viscosity is high and has little water since it solidifies here first. The center of the particle is still liquid and at lower relative viscosity at this phase of drying and the core components can diffuse to the center due to the solubility and concentration gradient in the droplet. The particle completely solidifies with trileucine enriched on the surface or shell and the core components include Ostabolin-C™, trehalose, citrate and methionine. All of which have a much greater solubility than trileucine. [0143] Thus, in one or more embodiments, a phase where Ostabolin-C™ resides

(i.e., the core) where methionine is present and affords unexpected stability. This could be due to interaction between methionine and Ostabolin-C™.

[0144] Alternatively, powders may be prepared by lyophilization, vacuum drying, spray-freeze drying, super critical fluid processing, air drying, or other forms of evaporative drying. In some instances, it may be desirable to provide the dry powder formulation in a form that possesses improved handling/processing characteristics, e.g., reduced static, better flowability, low caking, and the like, by preparing compositions composed of fine particle aggregates, that is, aggregates or agglomerates of the above- described dry powder particles, where the aggregates are readily broken back down to the fine powder components for pulmonary delivery, as described, e.g., in U.S. Patent No. 5,654,007, which is incorporated herein by reference in its entirety. [0145] In another approach, dry powders may be prepared by agglomerating the powder components, sieving the materials to obtain agglomerates, spheronizing to provide a more spherical agglomerate, and sizing to obtain a uniformly-sized product, as described, e.g., in WO 95/09616, which is incorporated herein by reference in its entirety.

[0146] Dry powders may also be prepared by blending, grinding, sieving or jet milling formulation components in dry powder form. [0147] Once formed, the dry powder compositions are preferably maintained under dry (i.e., relatively low humidity) conditions during manufacture, processing, and storage. Irrespective of the drying process employed, the process will preferably result in respirable, highly dispersible particles comprising the methionine-containing protein or peptide, an antioxidant, and a dileucyl-containing dimer or trimer. Stability

[0148] The methionine-containing protein or peptide compositions according to the invention typically have improved shelf stability. For example, after storage at about 25°C and 60% RH for 38 months, one or more embodiments of the formulations of the invention (such as a bulk powder) may exhibit a degree of degradation and/or oxidation of the methionine-containing protein or peptide of less than about 10%, or 8%, or 5% or 4% or 3% or 2% or 1 %. In other embodiments, after storage at about 25°C and 60% RH for 19 months, one or more embodiments of the formulations of the invention (such as powder packaged in appropriate capsules) may exhibit a degree of degradation and/or oxidation of the methionine-containing protein or peptide of less than about 10%, or 8%, or 5% or 4% or 3% or 2% or 1 %. In other embodiments, after storage at about 5°C for 24 months, one or more embodiments of the formulations of the invention (such as powder packaged in appropriate capsules) may exhibit a degree of degradation and/or oxidation of the methionine-containing protein or peptide of less than about 10%, or 8%, or 5% or 4% or 3% or 2% or 1 %.

[0149] Considered another way, the compositions of the present invention increase the shelf-life, depending upon storage temperature, by at least about 2, 4, 6, 8, 10, 12, or more months, as compared to methionine-containing protein or peptide compositions excluding an antioxidant and or other excipients of the inventive formulations. In some embodiments the shelf life is greater by at least 22 or 24 or 26 or 28 or 30 months at 25°C, or greater by at least 100, or 120, or 130, or 140, or 150 or 160 or 170 months at 5°C, or both.

[0150] In some embodiments the shelf life is of the product, either as bulk powder or in capsules is at least 20 or 25 or 30 or 35 or 40 or 45 or 50 or 60 or 70 or 80 months at 25°C, or greater by at least 120, or 130, or 140, or 150 or 160 or 170 or 180 or 190 or 200 months at 5°C, or both. Administration of the Compositions

[0151] The compositions of one or more embodiments of the present invention may be administered by any of the various methods and techniques known and available to those skilled in the art.

[0152] For example, in one or more embodiments, the compositions described herein may be delivered using any suitable dry powder inhaler (DPI), i.e., an inhaler device that utilizes the patient's inhaled breath as a vehicle to transport the dry powder drug to the lungs. Such an inhaler device is described in US 4,069,819 and 4,995,385, the disclosures of which are incorporated herein by reference in their entireties. [0153] The compositions described herein may be delivered using any suitable dry powder inhaler (DPI), i.e., an inhaler device that utilizes the patient's inhaled breath as a vehicle to transport the dry powder drug to the lungs. When administered using a device of this type, the powdered medicament is contained in a receptacle having a puncturable lid or other access surface, preferably a blister package or cartridge, where the receptacle may contain a single dosage unit or multiple dosage units. Convenient methods for filling large numbers of cavities with metered doses of dry powder medicament are described in U.S. Pat. No. 5,826,633, incorporated herein by reference.

[0154] Also suitable for delivering the powders described herein are dry powder inhalers of the type described, for example, in U.S. Pat. Nos. 3,906,950 and 4,013,075, 4,069,819, and 4,995,385, incorporated herein by reference, wherein a premeasured dose of aminoglycoside dry powder for delivery to a subject is contained within a capsule such as a hard gelatin capsule or HPMC capsule. HPMC capsules are preferred, sucha as size #2 capsules containing up to 50 mg powder, preferably 20-40 mg. It is to be understood that other sized capsules, such as 00, 0, No. 1, or No. 3 sized capsules are also suitable for use with the present invention and their suitability depends, among other factors, upon the inhalation device used to administer the powders

[0155] In some embodiments, a preferred device is Nektar Therapeutics¹ dry powder inhalation devices as described in U.S. Patent Nos. 5,458,135; 5,740,794; and 5,785,049, the disclosures of which are incorporated herein by reference in their entireties. Nektar Therapeutics' T-326 Dry Powder Inhaler (DPI) is a hand-held, manually operated, passive device. A single primary drug package (capsule) containing a dry powder for inhalation is Inserted into the DPI, and the DPI plunger is depressed, puncturing the capsule. When the subject inhales through the DPI, the airflow agitates the capsule and disperses the powder into an aerosol, which is carried into the respiratory tract. This device is described generally in the patents noted above. [0156] Another preferred device is disclosed in USSN 60/854,601 , filed October

25, 2006, assigned to Nektar Therapeutics, the entire disclosure of which is incorporated herein by reference.

[0157] When administered using a device of this type, the powder is contained in a receptacle having a puncturable lid or other access surface, preferably a blister package or cartridge, where the receptacle may contain a single dosage unit or multiple dosage units. Convenient methods for filling large numbers of cavities (i.e., unit dose packages) with metered doses of dry powder medicament are described, e.g., in WO 97/41031 (1997), which is incorporated herein by reference in its entirety. [0158] Also suitable for delivering the powders described herein are dry powder inhalers of the type described, for example, in U.S. Patent Nos. 3,906,950 and 4,013,075, which are incorporated herein by reference in their entireties, wherein a premeasured dose of dry powder for delivery to a subject is contained within a hard gelatin capsule.

[0159] Other dry powder dispersion devices for pulmonarily administering dry powders include those described, for example, in EP 129985; EP 472598; EP 467172; and U.S. Patent No. 5,522,385, which are incorporated herein by reference in their entireties. Also suitable for delivering the dry powders of the invention are inhalation devices such as the Astra-Draco "TURBUHALER". This type of device is described in detail in U.S. Patent Nos. 4,668,281 ; 4,667,668; and 4,805,811, all of which are incorporated herein by reference in their entireties. Other suitable devices include dry powder inhalers such as the ROTAHALER™ (Glaxo), Discus™ (Glaxo), Spiros™ inhaler (Dura Pharmaceuticals), and the Spinhaler™ (Fisons). Also suitable are devices which employ the use of a piston to provide air for either entraining powdered medicament, lifting medicament from a carrier screen by passing air through the screen, or mixing air with powder medicament in a mixing chamber with subsequent introduction of the powder to the patient through the mouthpiece of the device, such as described in U.S. Patent No. 5,388,572, which is incorporated herein by reference in its entirety.

[0160] Dry powders may also be delivered using a pressurized, metered dose inhaler (MDI), e.g., the Ventolin™ metered dose inhaler, containing a solution or suspension of drug in a pharmaceutically inert liquid propellant, e.g., a chlorofluorocarbon or fluorocarbon, as described in U.S. Patent Nos. 5,320,094 and 5,672,581 , which are both incorporated herein by reference in their entireties. [0161] The compositions of the present invention are suitable for use with metered-dose inhalers (MDIs). In some embodiments, the composition may comprise a suspension, made by using, for example, surface-active agents. See U.S. Patent No. 5,118,494, and WO 91/11173 and WO 92/00107.

[0162] A particularly useful class of MDIs are those which use hydrofluoroalkane

(HFA) propellants. The HFA propellants are further particularly well suited to be used with stabilized dispersions of an active agent such as formulations and composition of methionine-containing protein or peptide. Suitable propellants, formulations, dispersions, methods, devices and systems comprise those disclosed in US 6,309,623, the disclosure of which is incorporated by reference in its entirety. The various embodiments of the compositions, formulations, systems and methods of the present invention are suited to, and often optimal for, combination with such HFA-propellant based MDIs. In part, this is due to physical properties of the various embodiments of the particles and/or composition of methionine-containing protein or peptide, especially a cyclized PTH analog, such as the density, specific surface area, MMD and/or MMAD, as well as due in part to the methods of administration and methods of treatment herein.

[0163] Prior to use, dry powders may be stored under ambient conditions, and preferably are stored at temperatures at or below about 25°C, and relative humidities (RH) ranging from about 30 to 60%. More preferred relative humidity conditions, e.g., less than about 30%, may be achieved by the incorporation of a dessicating agent in the secondary packaging of the dosage form. [0164] The time for closing is typically short. For a single unit dose, or capsule

(e.g., 5 mg dose), the total closing time is normally less than about 1 minute. A 2 capsule dose (e.g., 10 mg) usually takes about 1 min. A 5 capsule dose (e.g., 25 mg) may take about 3.5 min to administer. Thus, the time for dosing may be less than about

5 min, such as less than about 4 min, less than about 3 min, less than about 2 min, or less than about 1 min. Dosing may comprise administration of a single unit dose, or capsule, or multiple unit doses, or capsules.

[0165] Additionally or alternatively, the compositions described herein may be administered by nebulization. For example, a dry powder may be dissolved or suspended in a solvent, e.g., water, ethanol, or saline, and delivered as an aerosolized solution using a nebulizer.

[0166] Liquid formulations can be atomized by any of a variety of procedures, such as a two-fluid nozzle, a pressure nozzle, or a spinning disc, or atomized with an ultrasonic nebulizer or a vibrating orifice aerosol generator (VOAG), jet nebulizer, vibrating porous plate nebulizer, or a thermal vaporizing device. In one or more embodiments, a liquid formulation is atomized with a pressure nozzle, such as a BD

AccuSpray nozzle. Thus, aerosolization apparatuses may be based on condensation aerosolization, an impinging jet technique, electrospray techniques, thermal vaporizing, or a Peltier device.

[0167] Vibrating porous plate nebulizers work by using a sonic vacuum produced by a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate.

See, e.g., U.S. Patent Nos. 5,758,637; 5,938,117; 6,014,970; 6,085,740; and

6,205,999, which are incorporated herein by reference in their entireties.

[0168] Jet nebulizers involve use of air pressure to break a liquid solution into aerosol droplets. In one or more embodiments, a jet nebulizer generates droplets as a mist by shattering a liquid stream with fast moving air supplied by tubing from an air pump. Droplets that are produced by this method typically have a diameter of about 2-5 μm.

[0169] In one or more embodiments, an ultrasonic nebulizer that uses a piezoelectric transducer to transform electrical current into mechanical oscillations is used to produce aerosol droplets. The resulting droplets typically have an MMAD in the range of about 1 to about 5 microns.

[0170] In one or more embodiments, the aerosol generator is the commercially available Aerogen (available from Nektar Therapeutics, San Carlos, CA) aerosol generator which comprises a vibrational element and dome-shaped aperture plate with tapered holes. When the plate vibrates several thousand times per second, such as about 100 kHz to about 150 kHz, a micro-pumping action causes liquid to be drawn through the tapered holes, creating a low-velocity aerosol with a precisely defined range of droplet sizes. The Aerogen aerosol generator does not require propellant. [0171] In condensation aerosol generators, the aerosol is formed by pumping drug formulation through a small, electrically heated capillary. Upon exiting the capillary, the formulation is rapidly cooled by ambient air, and a gentle aerosol is produced that is relatively invariant to ambient conditions and the user inhalation rate. See, e.g., U.S. Patent No. 6,701,922 and WO 03/059413, which are incorporated herein by reference in their entireties. In one or more embodiments, the condensation aerosol generator comprises one disclosed by Alexza Molecular Delivery Corporation. See, e.g., U.S. Published Application No. 2004/0096402, which is incorporated herein by reference in its entirety.

[0172] Another apparatus for delivery of a metered quantity of a liquid pharmaceutical composition for inhalation is described for example in WO 91/14468 and WO 97/12687, which are incorporated herein by reference in their entireties. The nebulizers described therein are known by the name Respimat®. [0173] One or more electrosprays may be used to nebulize liquid formulations.

The term electrostatic spray (also known as electrohydrodynamic spray or electrospray) refers to systems in which the dispersion of the liquid relies on its electric charging, so that nebulization and gas flow processes are relatively uncoupled. Examples of electrospray devices are disclosed in U.S. Patent Nos. 6,302,331; 6,583,408; and 6,803,565, which are incorporated herein by reference in their entireties. [0174] lExamples of foregoing devices are disclosed in U.S. Published

Application No. 2004/0262513, which is incorporated herein by reference in its entirety. [0175] In one or more embodiments, the aerosol generator comprises a thin film, high surface area boiler that relies on capillary force and phase transition. By inducing phase transition in a capillary environment, pressure is imparted onto the expanding gas, which is ejected. This technology has been disclosed by Vapore, Inc., and is known as Vapore-Jet CFV technology. See, e.g., U.S. Patent Nos. 5,692,095; 5,870,525; 6,162,046; 6,347,936; 6,585,509; and 6,634,864, and U.S. Application No. 10/691,067, which are all incorporated herein by reference in their entireties. [0176] Active agents may be delivered simultaneously, some preferred order, and/or providing one agent in an aerosol of a certain size to target one region of the lung while providing another in another size to target another region. Thus, purposeful variation of the aerosol size can cause some aerosol to deposit more proximally near the endotracheal tube to treat that area, while also sending in small aerosol to penetrate more deeply.

Utility

[0177] The compositions of the invention are useful, when administered pulmonarily in a therapeutically effective amount to a mammalian subject, for treating or preventing any condition responsive to the administration of methionine-containing protein or peptide. In one or more embodiments, the compositions of the invention comprising a parthyroid homone analog, derivative or fragment, such as a cyclized parathyroid hormone, are useful, when administered pulmonarily in a therapeutically effective amount to prevent and/or treat osteoporosis. Compositions of the invention may be used, for example, to stimulate new bone formation and significantly increase bone mineral density.

[0178] In one or more embodiments, the methionine-containing peptide or protein composition, such as cyclized parathyroid hormone analog, is formulated with trileucine or with a di-mer or trimer containing one or two leucyl residues for good aerosol performance as taught, for example by the Kuo et al patents, supra. The result is a particle with good aerosol characteristics and good dispersibility, suitable for respiration, as well as good physical and chemical stability. Such a formulation is highly compatible with the Nektar Therapeutics T-326 Inhaler device. In some embodiments, a composition comprising trileucine or with a di-mer or trimer containing one or two leucyl residues desirably lowers the powder density, and/or increases drug loading and/or lowers fill mass, or combinations thereof.

[0179] The following examples are illustrative of the present invention, and are not to be construed as limiting the scope of the invention. Variations and equivalents of this example will be apparent to those of skill in the art in light of the present disclosure, the drawings, and the claims herein. Unless otherwise stated, all percentages are by weight of the total composition.

EXAMPLES

Example 1. Effect of Methionine on Solution Stability of OSTABOLI N-C ™

[0180] This Example shows the impact of the addition of an antioxidant such as methionine, on the solution stability of OSTABOLIN-C™. Thus this Example demonstrates the effect of varying levels of methionine (1 %, 5% and 10%) on the oxidative stability of OSTABOLIN-C™ in solution (4% OSTABOLIN-C™, 20% trileucine, 71% trehalose, 5% citrate) as a function of storage temperature and duration. OSTABOLIN-C™ purity was determined using reverse-phase high performance liquid chromatography (RP-HPLC)

[0181J Solution stability studies of an OSTABOLIN-C™ solution formulation (4%

OSTABOLIN-C™, 20% trileucine, 71% trehalose, 5% citrate) have demonstrated that oxidation is a primary degradation product. The basic formulation solution was found to be stable for approximately 24 hours at 2 — 8°C and approximately 8 hours at 15^CC and 25°C. Methionine (Met) 8 and 18 of OSTABOLIN-C™ are the primary sites of oxidation (see Figure 1 ). Met residues are susceptible to oxidation, resulting in the formation of methionine sulfoxide. Peroxides, such as hydrogen peroxide react with metal ions to form free radicals that can initiate oxidation of proteins and peptides. Met can also be photooxidized by a free radical pathway or via singlet oxygen intermediate formation. [0182] The materials used in this Example include Ostabolin-C, L-trileucine, trehalose dehydrate, sodium citrate dehydrate, citric acid monohydrate, methionine, bottled water, HCI, NaOH, sodium perchlorate anhydrous and phosphoric acid. [0183] The equipment used in this Example include a top loading balance, pH meter, pH buffer solutions at pH 4 and pH 7, a Mettler AT261 balance and a sterilizing low extractable membrane 0.2 μm nylon filter system.

[0184] Solutions were prepared using stock solutions: 30 mg/ml sodium citrate,

50 mg/ml trehalose, 1 mg/ml methionine and 3 mg/ml trileucine. The trileucine solution was pH adjusted to pH 5.0 with 5 M HCI. The sodium citrate solution was pH adjusted to pH 5.0 using the citric acid stock solution. All the stock solutions were filtered through a 0.2 μm nylon filter. Formulation components containing water content were corrected for weight. All formulations were prepared from common excipient stock solutions.

[001] Varying amounts of methionine stock solution were added to citrate buffer

(5%) trileucine (20%) and trehalose stock solutions by weight to achieve the target concentration. OSTABOLIN-C™ (4%) and water were added to the excipient solutions to achieve the target solution concentration. The pH was adjusted with 0.5M NaOH to achieve a target pH of 5.0. Methionine formulations containing 1 %, 5% and 10% were made, with the formulations shown in Table 2. Table 2. Methionine formulations

[0185] 1.0 mL aliquots of the OSTABOLIN-C™ basic formulation solution were transferred into 2 mL amber glass vials and sealed. These samples were stored at either room temperature or 50⁰C. Samples were collected at t = 0, 2, 4, 8, 24, 30, 48, 96 and 168 hours and stored at -20⁰C. Samples were removed from -20⁰C and thawed to 5°C prior to analysis by HPLC. Methods [0186] Each sample preparation was analyzed using the HPLC method described in below.

HPLC Method

[0187] The standard range for the samples was approximately between 20 to

120 μg/ml of OSTABOLI N-C ™ in water. Mobile phase A contained 0.1 M sodium perchlorate. The solution was adjusted to pH 3.10 ± 0.05 with phosphoric acid. The mobile phase was filtered through 0.2 μm nylon filter and stored at room temperature.

Mobile phase B contained acetonitrile. The ratio of mobile phase A/B varied during the run according to the gradient specified below. The following conditions were used for the analysis:

Parameter Specification

Column: YMC ODS-AQ 150mm x 6.0mm ID, 5 μm, 12θA Guard Column: YMC ODS-AQ Column Temperature: 25°C Sample Temperature: 5°C Flow rate: 1.5 mL/min Injection Volume: 10O mL Run Time: 40 minutes Detector wavelength: 210 nm

Mobile Phase A/B gradient:

^aOSTABOLIN-C™ peak retention time of approximately 15 minutes. Main degradant peak, "oxidation" retention time of 13.3 minutes.

Results for all HPLC analyses are reported as relative retention times (RTT) and/or % area under the curve. The latter value is the area under the peak associated with the corresponding RTT. Results and Discussion

[0188] To study the inhibitory effect of antioxidants on oxidation of OSTABOLIN-

C™, varying amounts of methionine (1%, 5% and 10%) were added to the base formulation. The amount of oxidative degradant was determined as a function of temperature and storage duration. The results of the RP-HPLC analyses for

OSTABOLIN-C™ purity are listed in Tables 3 and 4, with a representative chromatogram shown in Figure 2. The other degradant peak area levels remained unchanged within the instrumental and method variability throughout the duration of the study.

Table 3. Effect of 1%, 5% and 10% methionine on OSTABOLIN-C™ solution formulation at room temperature

Table 4. Effect of 1%, 5%, and 10% methionine on OSTABOLIN-C™ solution formulation at 50⁰C

[002] The initial levels of the oxidative degradant peak (Oxidation #1 RRT 0.83

— 0.84) were found to decrease with increasing amounts of methionine in the formulation (0.29% with 1 % methionine to 0.19% with 10% methionine). (Tables 3 and 4) This was an improvement relative to a formulation containing no methionine, with initial oxidative degradation values of 0.63% at room temperature. [0189] After 30 hours of storage at room temperature, the oxidative degradation peak increased by a similar amount for all three methionine formulations (0.14% - 0.19%), which is lower than the increase observed in the formulation containing no methionine. This is an indication that the presence of methionine in the formulation not only decreases the initial amount of oxidation, but also leads to a lower rate of degradant growth (See Figure 3).

[0190J As expected, at 50⁰C, the level of oxidation was higher than at room temperature. However, at 50°C, a lower rate of oxidative degradant growth was observed with increasing amount of methionine in the formulation (Table 4, Figure 4). Peak area levels of Oxidation #1 RRT 0.83 - 0.84 increased by 1.45% over initial values for the 1 % methionine formulation, 1.30% for the 5% methionine formulation, and by 1.12% over initial values for the 10% methionine formulation. Based on these results, it is clear that the presence of Met in the formulation inhibited oxidation degradation.

[0191] Increasing the methionine concentration in the formulation from 1 % to

10% resulted in a decrease in the initial levels of Oxidation #1 RRT 0.83 — 0.84 peak at room temperature. The rate of the oxidative degradation peak growth did not change with increasing methionine concentration at room temperature. However, the presence of methionine was found to decrease the growth in comparison to formulations containing no methionine. Increasing the amount of methionine in the formulations at 50⁰C did result in a corresponding decrease in the rate of oxidative degradation. Inclusion of methionine in the 4% formulation results in an initial reduction in the oxidation levels as well as a reduction in the rate of oxidation growth at elevated temperature.

[0192] Example 2. Effect of Methionine on Solid-State Stability of

OSTABOLIN-C™ [0193] Example 1 shows that the addition of methionine resulted in a reduction in oxidation in the solution state. This Example shows the inhibitory effect of the addition of an antioxidant such as methionine, on oxidative degradation in the solid-state.

[0194] This Example demonstrates the chemical stability of selected

OSTABOLIN-C™ (OSTAB OL I N-C ™) formulations containing trileucine, trehalose, sodium citrate, citric acid, and methionine. Formulations were assessed for chemical stability using reverse-phase high performance liquid chromatography (RP-HPLC) after storage under accelerated conditions.

[0195] The materials used in this study are the same as those listed in Example

1.

[0196] The equipment used in this Example are the same as those listed in

Example 1 with the addition of a Buchi mini spray dryer and an atomizer liquid nozzle.

[0197] Solutions were prepared according to the procedures outlined in Example

1. Five different formulation solutions (Table 5) were prepared and subsequently spray dried using the following conditions:

Parameter Value

Inlet T (Ti_n) (⁰C) 105 ± 3

Outlet T (T_0U,) (⁰C) 62 ± 3

Atomizer Pressure (psi) 60 ± 5

Atomizer gas flow, Rotameter (mm) < 40 mm

Solution flow rate (mL/miπ) 5.0 ± 0.5

Dryer manifold pressure (in H₂O) 2.5 ± 0.5

Total gas flow rate (Venturi) (in H₂O) 1.6 ± 0.2

Drying gas N₂

Table 5. Formulation targets at 1.0% solids (w/w)

Packaging and Testing Schedule

[0198] Powders were weighed into 1.5 ml HPLC vials. Pouches and rayon balls were equilibrated for 24 hours at 11 % RH. Fifteen capped vials and one rayon ball were placed in a small inner foil pouch and sealed with a hand sealer inside a glovebox equilibrated to approximately 11% RH. This smaller pouch was then placed inside a larger foil pouch and sealed. The powders were stored under accelerated conditions and assayed by RP-HPLC for impurities at various times. [0199] Each sample preparation was analyzed using the HPLC method described in Example 1. The standard range for the samples was approximately between 80 to 120 μg/ml of OSTABOLIN-C™ in water. Mobile phase A contained 0.1 M sodium perchlorate. The solution was adjusted to pH 3.10 ± 0.05 with phosphoric acid. The mobile phase was filtered through 0.2 μm nylon filter and stored at room temperature. Mobile phase B contained acetonitrile. The ratio of mobile phase A/B varied during the run according to the gradient specified in Example 1.

Differential Scanning Calorimetry (DSC)

[0200] The glass transition temperature (T₉) of each powder was measured using a differential scanning calorimeter. Sample preparation was using the test method DSC- 2920 Differential Scanning Calorimeter. Samples were pre-equilibrated at 11.3% RH using saturated salt solution of lithium chloride in a vacuum dessicator for a period of at least two days before sealing the pan. Each test run consisted of heating the sample from -60⁰C to 220⁰C at a heating rate of 2°C/min. The heating rate was modulated by superimposing a sinusoidal heating profile at ±0.318°C/min. Moisture Content

[0201] The moisture content was assessed by measuring the weight loss upon drying using a TGA-2950 instrument. To increase the sample mass and sensitivity, a disc of powder was prepared using a 5/32" diameter custom-made stainless steel press. Gentle pressure was applied to produce a 3 to 13 mg disc that fits inside a DSC pan. The pan was hermetically sealed using a sample encapsulation press. In order to provide a vent, the lid of the sample pan was pierced just prior to starting the experiment. The samples were heated to 110⁰C at 2°C/min and held for 60 minutes to ensure that all the internal water molecules are dried out.

[0202] The moisture content and the glass transition temperature (T₉) of the

OSTABOLIN-C™ spray dried powder formulations were determined at 11.3% RH using

Thermo Gravimetric Analysis and Differential Scanning Calorimetry, respectively, and are shown in Table 6. No trend was observed in either the moisture content or T₉ with increasing methionine in the formulation.

Table 6. Moisture content and T₉ values of OSTABOLIN-C™ formulations

[0203] The results of the RP-HPLC analysis are listed in Table 7. The results from the oxidation peak (RRT 0.83 - 0.84) are reported and discussed. [0204] The addition of methionine to the OSTABOLIN-C™ formulation was evaluated at 0.5% and 2% (Table 7). The initial levels of the oxidative degradation peak (Table 7) were found to decrease slightly with increasing amounts of methionine (0.17 - 0.18% with no methionine and 0.14 - 0.15% with 2% methionine). A small increase in the oxidative degradation peak was observed after approximately 80 hours of storage at room temperature in both methionine formulations in comparison to the formulations containing no methionine (Figure 5A). Although an increase in the oxidation peak was observed at 40 and 50⁰C, there was a lower rate of oxidative degradant growth with increasing amount of methionine in the formulation (Table 7, Figures 5B and 5C). Based on these results, it is clear that the presence of methionine in the formulation inhibited the rate of oxidation degradation in the solid-state. In one or more embodiments, a rate of inhibition of powder (either bulk or in capsules) oxidation is reduced by about 10 times, or 12 times or 15 times or 20 times or 25 times or 30 times or more. In one or more embodiments, an initial amount of oxidation is decreased by about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or more, over a given time period. In one or more embodiments, a rate of degradant growth is decreased by about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or more, over a given time period.

Table 7. Oxidation peak (%), Reaction rate constants and shelf-life prediction results of OSTABOLlN-C ™ formulations

[0205] The reaction kinetics of chemical degradation pathways was determined as a function of temperature for oxidation peak at RRT 0.83 - 0.84. Zero order reaction kinetics were used to determine the rate constants via linear regression analysis on the timepoints. The observed zero-order rate constants were observed to increase with increasing temperature. The oxidation pathway was also evaluated using an Arrhenius model. Figure 6 shows that the OSTABOLIN-C™ oxidation degradation follows

Arrhenius kinetics below the T₉ of the formulations. Extrapolation of the degradation rate constants to 5°C and room temperature confirm that a longer shelf-life is predicted with increasing methionine concentrations in the formulation (Table 7).

Example 3 Physical and Chemical Stability of OCIP Formulations (RD 325) [0206] In this Example, long term (6 months) physical and chemical stability of

Ostabolin-C inhalation powder (OCIP) formulations were investigated. Solutions with ("Formula A") and without ("Formula B") methionine were prepared as shown in Table 8. The solutions were subsequently spray dried on a bench scale spray drier using spray drying conditions outlined in Example 2. The bulk powders were filled into size 2 HPMC capsules at a 5 mg fill weight. The capsules were subsequently placed into HDPE bottles and the bottles then placed into a labeled 6.25 in. x 7.25 in. pouch containing two conditioned rayon balls and sealed. This pouch was then placed into a labeled 7 in. x 7.25 in pouch containing a desiccant and sealed. The samples were then stored at either 5°C, 25°C/60% RH or 40°C/75% RH conditions for up to 24 months and tested periodically. Table 8 Formulation components used in Example 3

[0207] The moisture content and the glass transition temperature (T_g) of the powder formulations were determined for both the bulk powder and the filled capsules as described in Example 2 as a function of % RH using TGA, KF and DSC. The results are shown in Table 9. Table 9. Moisture content and Tg results for Formulations A and B

[0208] Increasing the relative humidity the bulk powder was exposed to resulted in a decrease in the T₉ of the FORMULA B formulation and a corresponding increase in the moisture content. The T_Q of the powder filled in capsules was lower than that of the bulk powder. The values of T₉ versus weight fraction of water along with the calculated values represented by the solid line obtained by application of the Gordon-Taylor equation, as shown in Figure 7.

[0209] The T₉ of the 0.5% methionine formulation bulk powder (Lot # 4792-13) was 75⁰C, indicating that the presence αf methionine in the formulation did not have an impact on the glass transition temperature. Although the 4% Ost-C with 0.5% methionine formulation (Lot # 4792-13) was not evaluated at different RH conditions, similar results to ZL1- 001 are expected due to the similarities in T₉ values. The particle size of the Ostabolin-C formulations was measured by Sympatec laser diffraction method (Table 10). The initial median particle diameter (x50) was 1.81 μm and 1.95 μm for FORMULA B (Lot # 4792-04) and the 4% Ost-C with 0.5% methionine formulation (Lot # 4792-13), respectively. The initial particle size distribution width (x84 — x16) was 2.29 μm and 2.68 μm for FORMULA B and 4% Ost-C with 0.5% methionine formulations, respectively. Inclusion of methionine in the 4% Ostabolin- C formulation had no significant effect on the particle size distribution. Table 10 Particle Size

[0210] The %ED values of FORMULA B (Lot # 4792-04) for filled inhalation grade capsules at 2.5 mg, 5 mg and 15 mg packaged strength were found to be 87.8%, 91.8% and 92.0%, respectively. No differences were observed in the %ED as a function of fill weight indicating the powders were physically stable. Aerosol properties of the 4% Ost-C with 0.5% methionine formulation (Lot # 4792-13) were not determined but are expected to be similar to those observed for FORMULA B.

[0211] Aerodynamic particle size distribution was measured using an Anderson

Cascade Impactor, at a flow rate of 56.6 L/min.

[0212] The effect of methionine on the Ostabolin-C chemical stability was evaluated. Oxidation levels were determined as a function of temperature and storage duration. The results of the RP-HPLC analysis are listed in Table 11. The initial level of the oxidative degradation peak was found to increase approximately five-fold when FORMULA B (Lot # 4792-04) was filled into capsules, from 0.17% to 0.84%, respectively. In contrast, the 0.5% methionine formulation (Lot # 4792-13) only showed a two-fold increase in oxidation levels between the bulk powder (0.34%) and capsules (0.51 %). FORMULA B (Lot # 4792-04) was stored at 5°C, 25°C and 40⁰C for up to 91 days. Oxidation levels in the capsules decreased from 0.84% to 0.61 % at 5°C but increased to 1.39% at 25°C and 2.65% at 4O⁰C after storage for 91 days. In contrast, oxidative degradation levels for 4% Ost-C with 0.5% methionine formulation (Lot # 4792-13) capsules stored at 25°C and 40⁰C remained, for practical purposes, unchanged after 48 days of storage (See Figure 8, Table 11). Table 11 HPLC results

[0213] Based on these results, it is clear that the presence of methionine in the formulation is inhibiting the rate of oxidative degradation in the filled capsules. These results are in agreement with those observed previously of bulk OCIP formulations containing methionine.

Example 4: Evaluation of OCIP High Strength Formulations

[003] Three high strength Ostabolin-C (Ost-C) formulations (16% Ost-C, 16%

Ost-C with 0.5% methionine, and 25% Ost-C) were manufactured using the same excipient ratios as in an Ost-C formulation designated ZL1-001. (ZL1-001 contained 4% Ost-C, 5% sodium citrate, 61% trehalose and 30% trileucine.) The composition of the three high strength formulations is shown in Table 12. Table 12

The formulations were then assessed for physical properties and chemical stability, as discussed in Example 3. No significant differences were observed in the physical properties of the three OCIP high strength formulation bulk powder. The moisture content values, as measured by the method of Example 2, for powder filled in HPLC vials and capsules were significantly higher at the initial time points in comparison to the results observed for the bulk powder (Table 13). The moisture content of the three lots decreased up to approximately 2% after storage in HPLC vials for 2 weeks. No further changes were observed in the moisture content after 4 weeks storage.

Table 13 Moisture Content of OCIP high strength formulations filled in HPLC vials and HPMC capsules as a function of storage time at 40^oC/ambient RH

[0214] The feedstock solutions of the 16% Ost-C formulations with and without methionine were found to be chemically stable (no change in the oxidation levels) after storage at room temperature and 2-8⁰C for up to 24 hours (Table 14), as measured by HPLC as described in Example 1. The oxidation product monitored in this example was the peak eluting at 13.3 minute and the relative retention time of the oxidation peak was between 0.8 and 0.9 minute (Table 14). The initial oxidation of the three feed solutions ranged from 0.13% to 0.15% at both room temperature and 2-8°C. The oxidation levels did not change after storage at 2-8°C and room temperature for up to 24 hours. The solutions were subsequently spray dried using the conditions listed in Example 2. The oxidation levels of all three bulk powders ranged from 0.13% to 0.14%, similar to the levels obtained in the feedstock solutions. The oxidation levels of all the formulations did not change upon spray drying, indicating that the spray drying process was not inducing oxidation.

Table 14. % Oxidation on OCIP high strength formulations: feedstock solution and bulk powder

[0215] The spray dried powders were hand-filled to HPLC vials (as control) as well as clear inhalation grade HPMC capsules with 5 mg fill weight. Filled vials and capsules were packaged in double pouches containing one desiccant in the secondary pouch and sealed on a commercial sealer at ambient laboratory conditions. The samples were stored at 40°C/ambient RH chamber and analyzed by RP-HPLC (as outlined in Example 1 ) at t = 0, 1 , 2, 4 and 12 weeks.

[0216] The high strength formulations filled in HPLC vials demonstrated a small increase in oxidation levels after storage at 40°C/ambient RH for up to 12 weeks (Table 15). After storage at 40°C/ambient RH for 4 weeks, the oxidation levels were 0.79% for Formulation #1 (lot # 4640-48, 16% Ost-C), 0.65% for Formulation #3 (lot #4640-49, 25% Ost-C) and 0.48% for Formulation #4 (lot # 4640-50, 16% Ost-C with 0.5% met). As shown by Figure 9, it appears that increased amount of Ostabolin-C active in the formulation also results in a decrease in the growth of the oxidation degradant. No significant changes in oxidation levels were observed upon storage for up to 4 weeks in the HPLC vials. Formulations #3, containing 16% Ost-C with 0.5% methionine showed the smallest increase in oxidation levels, indicating the ability of the methionine to retard the oxidation (see Figure 9). The methionine containing formulation demonstrated the least increase in oxidation levels after storage for 12 weeks, again indicating that the methionine is preventing the formation of the oxidation product. Table 15. % Oxidation on OCIP high strength formulations in capsules and vails after storage at 40°C/ambient RH

Example 5 (RD937) 16% Ostabolin-C Inhalation Powder (Formulation Code ZL1- 002), Development Lot #N020298, Bulk Powder Stability Results

[0217] OCIP formulation ZL1-002 contains 16% Ostabolin-C, 1.8% Citric Acid,

2.6% Sodium Citrate, 52.9% Trehalose, 26.3% Tri-leucine, and 0.5% Methionine. Material and equipment used in this Example as well as the solution preparation and spray dried procedures are those as outlined in Example 2. The objective of this Example is to demonstrate the bulk powder stability of the spray dried powder. [0218] The spray-dried powder Lot #N020298 (ZL 1-002) was hand filled into 4 mL amber glass vials. The vials and packaging materials were equilibrated in ICS at 11 ± 3%RH over 12 hours prior to filling. Two vials were filled for each stability condition. One vial was filled for moisture testing and the other vial for determination of powder morphology, content and purity determination by HPLC (as described in Example 1) and particle size distribution by Sympatec. Each vial and two rayon balls were sealed in a 6.25 in. x 7.25 in. foil pouch using a hand sealer. The sealed pouch was subsequently sealed in a 7 in. x 7.25 in. secondary pouch with a 5g desiccant using a commercial sealer at ambient condition. The stability pouches were packaged and stored in the stability chambers at either 5°C or 25°C/60% RH. The powder was characterized at initial, 4 weeks, 3 months and 6 months.

[0219] The moisture content of the bulk powder was determined using Karl

Fischer analysis as described in Example 2. See Table 16. At the initial time point, the moisture content was 2.9%. At both 5°C and 25°C/60% RH storage conditions, there was an increase in the moisture content over storage at 5 months. The moisture content increased 0.8% over initial values after storage at 5°C and 1.0% at 25^QC/60% RH.

Table 16. Moisture Content of 16% OCIP, Lot #N 020298

{0220] The particle size of the 16% OCIP formulation was measured by

Sympatec laser diffraction method (Table 17). The initial median particle diameter (x50) was 1.89 — 1.90 μm. There was no change observed in the particle size distribution on the powder storage for 6 months at both storage conditions (5⁰C and 25°C/60% RH) in comparison to the initial time point. Table 17. Particle Size results for 16% OClP, Lot #N020298

[0221] The HPLC data for up to 6 months for both storage conditions (5⁰C and

25°C/60%RH) showed no change in the oxidation level for Lot#N020298 (Table 18). The Ostabolin-C content data up to 6 months ranged from 0.15 mg/mg to 0.16 mg/mg. At the 5°C storage condition, there was no change in the Ostabolin-C content and % purity for up to 6 months. There was also no change in % total impurities after storage for 6 months. The oxidation peak at RRT 0.84 — 0.85 increased from an initial value of 0.11% to 0.14% after storage for 6 months. No changes were observed in any of the other impurities present in the bulk powder at levels >0.10%. Storage of the bulk powder at 25°C/60% RH demonstrated a small decrease in the content and % purity (98.4% - 98.0%) from the initial values. The oxidation peak increased from 0.11 % to 0.16% and % total impurities increased from 1.6% to 2.0% over storage for 6 months. Table 18. Content and Purity Results for 16% OCIP Lot #N020298

[0222] In summary, 16% OCIP (ZL1-002) bulk powder Lot #N020298 showed changes in physical and chemical properties after storage at 5⁰C and 25°C/60%RH for 6 months. The moisture content at 5°C increased 0.8% from the initial value and the oxidation peak at RRT 0.84 - 0.85 increased from an initial value of 0.11% to 0.14%. At 25°C/60% RH, the moisture value increased 1.0% over the initial value. In addition, a small decrease was found in the content and % purity (from 98.4% - to 98.0%). The oxidation peak also increased from 0.11% to 0.16% over 6 months of storage. The trend on increase moisture content and decrease on the purity over 6 months of storage are within the acceptance criteria for the bulk powder and this trend will continue to be monitored on future bulk powder stability studies.

Example 6: The effect of Oxygen Scavenger on the rate of Ostabolin-C oxidation in 4% Ostabolin-C lnahlation Powder (RD 938)

[0223] This Example evaluated the effect of an oxygen scavenger (as

PharmaKeep® oxygen scavenger) on the rate of oxidation of 4% Ostabolin-C Inhalation Powder (OCIP) (Formulation Code: ZL1-001). PharmaKeep® is a proprietary oxygen absorber marketed by Sud-Chemie Group/Mitsubishi Gas Chemical Company. The material, equipment, solution preparation and spray drying conditions used in this Example are the same as those cited in Example 2.

[0224] A 4% OCIP (Formulation Code ZL1-001 ) filled in HPLC vials at 2.5 mg fill weight and capsules at 5.0 mg fill weights were packaged in pouches. The pouches were then packaged with and without oxygen scavenger and stored at 5O⁰C for up to 52 weeks. The samples were analyzed by RP-HPLC to determine the Ostabolin-C™ content and purity, as described in Example 1. As shown in Figure 10, comparison of the HPLC chromatograms of OCIP powders in capsules, packaged with and without oxygen scavenger demonstrates that all of the samples had approximately the same number of major degradant peaks. While the number of peaks was approximately unchanged, the results demonstrated that the amount of degradation was less in both the bulk powder and capsule samples packaged with an oxygen scavenger than without oxygen scavenger.

[0225] The rate of change of the oxidation peak at 5OC storage condition, is plotted as a function of time in Figure 11 shows an increase from 0.2% to 7.7!% in the OClP powders stored in vials in the absence of oxygen scavenger. However, in the presence of oxygen scavenger, the oxidative degradation peak only increased 0.4%, from 0.4% to 0.8% over a duration of 12 months at 50⁰C. This demonstrates a significant decrease in the degradation growth due to the presence of the oxygen scavenger.

[0226] The oxidative degradation was also observed in the 4% OCIP formulation at 5.0 mg fill weight capsules. The formation of this peak in the capsules packaged without oxygen scavenger increased fromo 0.5% at the initial timepoint to 9.7% over a duration of 12 months (Figure 11). However, in the presence of oxygen scavenger, the amount of oxidative degradation increased only from 0.5% to 0.6% over a duration of 12 months at storage temperature of 50⁰C.

[0227] The change in purity of OCIP as a function of time and packaging conditions is shown in Figure 12. A 4% OCIP formulation packaged both in bulk and in capsules with oxygen scavenger resulted in improved purity of OCIP with time. This was most noticeable for OCIP packaged in capsules, where the purity decreased from 97.8% to 65.7% in the absence of oxygen scavenger, but decreased from 97.3% to 81.1 % in the presence of oxygen scavenger, approximately half the rate of change in purity observed in the absence of oxygen scavenger.

[0228] Suitable oxygen scavengers comprise any material which can reduce the amount of free or molecular oxygen to react with other species in a composition. In some embodiments, suitable oxygen scavengers are those that bind molecular oxygen. [0229] An oxygen scavenger, such as PharmaKeep®, can slow the rate of oxidation and/or degradation in a methionine-containing protein or peptide, such as OCIP, when stored in bulk and/or in capsules. In one or more embodiments, the rate of oxidatation and/or degradation is slowed by at least about 50% or 60% or 70% or 80% or 90% or 95% or 97% or 98% or 99% over 1 or 3 or 6 or 12 or 24 or 36 months or more. In one or more embodiments, the amount of impurities in the methionine- containing protein or peptide, such as OCIP is reduced by at least about 10% or 20% or 30% or 40% or 45% or 50% or more over 1 or 3 or 6 or 12 or 24 or 36 months or more.

[0230] Example 7. Stability results for 16% OCIP, 5.0 mg fill weight bulk capsules, lot #5244-13

[0231] This Example demonstrates the physical and chemical stability of 16%

OCIP, 5.0 mg bulk capsules after storage at 5°C, 25°C/60% RH and 40⁰C. The samples were evaluated for appearance, moisture content (as described in Example 2), identity, content and purity (as described in Example 1 ) and aerosol properties (as described in Example 3).

[0232] The appearance and moisture content (Table 19) of the powder and capsules remained unchanged for the duration of the study.

Table 19 Moisture Content Results for 16% OClP (Formulation Code ZL1-0021. Bulk Capsules, 5.0 mq Fill Weight. Lot 5244-13

[0233] The Ostabolin-C content decreased from an initial value of 0.726 mg

Ostabolin-C/mg capsule, to 0.700 mg Ostabolin-C/capsule when the capsules were stored at 40⁰C for two months, 0.701 mg Ostabolin-C/capsule after storage for 3 months at 5°C and 0.706 mg Ostabolin-C/capsule after storage for 6 months at 25°C/60% RH (Table 20). The total impurities increased from an initial value of 1.55% to 1.91% after storage for 3 months at 5^βC, 3.21% at 25°C/60% RH after storage for 6 months and 4.06% after storage for two months at 40⁰C. The oxidation peak value at RRT = 0.84 increased from an initial value of 0.31 % to 1.23% after storage for two months at 40⁰C, 0.34% after storage for 3 months at 5°C and 1.00% after storage for 6 months at 25°C/60% RH (Table 20). Although the total impurities for capsules at both fill weights were determined to be within the acceptance criteria, a trend of increasing impurity levels was observed with the increase in temperature from 5°C to 40⁰C. In addition, the impurity levels increased with time for all the storage conditions. These values are within the acceptance criteria for the bulk capsules. Table 20 Identity, Content and Purity Results for 16% OCtP (Formulation Code ZH -002). Bulk Capsules. 5.0 mq Fill Weight

[0234] No statistically significant change was observed in the mean emitted powder mass (Table 21) and MMAD values (Table 22) from the initial time point after storage for up to 1 month at 5°C, 6 months at 25°C/60% RH and 2 months at 40^βC. In Table 22, A= Adapter; T=Throat and F= Filter, all components of the Anderson Impactor. The numerals 1-7 refer to the various stages of the Impactor.

Table 21 Emitted Mass and Emitted Dose Results for 16% OCIP (Formulation Code ZL1-002Ϊ, Bulk Capsules, 5. 0 mg FiIi Weight

Table 22 PSD Results for 16% OCIP (ZL1 -002) at 5.0 mg Fill Weight as a Function of Storage Condition and Duration

Table 22

Time point Storage Condition

Test Article (months) 5°C 25°C/60% RH 40⁰C

MMAD (μm) Initial 0.6 0.6 0.6

1 0.6 0.7

2

3 HiHHiHi 0.5 Mu-I-I- 6 ( 3.5

Initial 72 72 72

1 74 75

% FPD_<2._{8 μn}, 2 73

3 78

6 79

A: 0.0 4: 135.8 A: 0.0 4: 135.8 A: 0.0 4: 135.8

T: 0.0 5: 1B6.5 T: 0.0 5: 186.5 T: 0.0 5: 186.5

Initial 1: 0.0 6: 194.4 1 : 0.0 6: 194.4 1: 0.0 6: 194.4

2: 0.0 7: 185.0 2: 0.0 7: 185.0 2: 0.0 7: 185.0

3: 48., 5 F: 346.9 3: 48.5 F: 346.9 3: 48.i 5 F: 346.9

A: 0.0 4: 143.9 A: 0.0 4: 157.5

T: 0.0 5: 201.7 T: 0.0 5: 238.1

1 1: 0.0 6: 205.1 1 : 0.0 6: 209.3

2: 0.0 7: 187.7 2: 6.5 7: 183.0

3: 50. 2 F: 298.0 3: 54.4 F: 295.6

A: 0.0 4: 106.5

Average Ostabolin- C per stage T: 0.0 5: 150.4 (micrograms)

2 1: 0.0 6: 167.4

2: 0.0 7: 190.8

3: 42. S F: 411.8

A: 0.0 4: 135.0

T: 0.0 5: 176.9

3 1 : 0.0 6: 192.6

2: 0.0 7: 203.0

3: 54. 8 F: 373.0

A: 0.0 4: 127.3

6 T: 0.0 5: 183.3

1 : 0.0 6: 200.5

[0235] The 16% OCIP (Formulation Code: ZL1-002), Bulk Capsules (5.0 mg Fill

Weight) were observed to be chemically and physically stable when stored at 5°C for 3 months, at 25°C/60% RH for 6 months and at 40⁰C for 2 months.

[0236] Example 8. Stability Results for 4% OCIP, bulk capsules at 2.5 mg and 5.0 mg fill weights, Lot #5244-05

[0237] This Example demonstrates the chemical and physical stability of 4%

OCIP (Formulation Code: ZL1-003), Bulk Capsules (2.5 mg and 5.0 mg Fill Weight), Lot

5244-05 after storing the samples at 5°C, 25°C/60% RH for a duration of 6 months and

40⁰C for a duration of two months.

[0238] The appearance and moisture content (Table 23) of the powder and capsules remained unchanged for the duration of the study.

Table 23 Moisture Content Results for 4% OClP (ZLI -0031, Bulk Capsules. 2.5 mg and 5.0 mg Fill Weights

[0239] The Ostabolin-C content for both the 2.5 mg and 5.0 mg capsules remained unchanged for the duration of the study. For the 2.5 mg fill weight capsules (Table 24), the total impurities increased from an initial value of 2.11% to 2.52% after storage for 6 months at 5°C, 4.96% at 25°C/60% RH and 5.55% after storage for 2 months at 40⁰C. For the 5.0 mg fill weight capsules the total impurities increased from an initial value of 2.21 % to 3.54% after storage for 6 months at 5°C, 4.45% at 25°C/60% RH and 4.67% after storage for 2 months at 40⁰C (Table 25). For the 2.5 mg fill weight capsules the oxidation peak value at RRT = 0.84 increased from an initial value of 0.45% to 2.41 % after storage for 2 months at 40⁰C, 0.53% and 1.64% after storage for 6 months at 5°C and 25°C/60% RH, respectively. For the 5.0 mg fill weight capsules the oxidation peak value at RRT = 0.84 increased from an initial value of 0.42% to 2.28% after storage for 2 months at 40⁰C, 1.42% and 1.74% after storage for 6 months at 5°C and 25°C/60% RH, respectively. Although the total impurities for capsules at both fill weights were determined to be within the acceptance criteria, a trend of increasing impurity levels was observed with the increase in temperature from 5⁰C to 40⁰C. In addition, the impurity levels increased with time for all the storage conditions.

Table 24 Identity, Content and Purity Results for 4% OCIP {ZL1 -003) 2.5 mg Fill weights as a Function of Storage Condition and Duration

Table 25 Identity, Content and Purity Results for 4% OCIP (ZL1 -003) 5.0 mg Fill Weights as a Function of Storage Condition and Duration

[0240] For capsules at both the fill weights, no statistically significant change was observed in the mean emitted powder mass and MMAD values from the initial time point after storage for up to six month at 5°C, 25°C/60% RH and two months at 40⁰C. [0241] The 4% OCIP (Formulation Code: ZL1-003), Bulk Capsules (2.5 mg and

5.0 mg Fill Weight) were observed to be chemically and physically stable when stored at 5⁰C and at 25°C/60% RH for 6 months and at 40⁰C for 2 months.

[0242] Example 9. Pharmacokinetic analysis of a dry powder formulation of

OSTABOLIN-C™

[0243] A dry powder comprising 16% w/w Ostabolin-C, 0.5% w/w methionine,

26.25% w/w trileucine, 52.875% w/w trehalose, and 4.375% w/w citrate.

Following 15-minute inhalation of 100 and 250 /yg/kg, absorption of OSTABOLIN-C™ was rapid with maximum plasma concentrations occurring between 15 and 35 minutes post-dose. OSTABOLIN-C™ was found to be subject to extensive distribution following both IV and inhaled administration. The clearance of OSTABOLIN-C™ was rapid following both inhaled and IV administration with mean terminal elimination half-lives of approximately 14 to 30 minutes. The inhaled absolute bioavailability of OSTABOLIN-

C™ was between 0.75 and 2.0 % for all animals. Exposure to OSTABOLIN-C™ increased in a greater than dose proportional manner over the 100 to 250//g/kg dose range with a 2.9-fold increase in dose resulting in an approximate 5 to 7-fold increase in exposure.

[0244] In this Example the comparative pharmacokinetic parameters of

Ostabolin-C were determined, following one intravenous and two inhalation (oral/nasal) administrations to the monkey. Ostabolin-C was supplied in two physical forms,

Ostabolin-C for intravenous dosing, and a dry powder formulation for inhalation exposure. In conclusion, administration of the test article, Ostabolin-C, to the

Cynomolgus monkey by a single intravenous bolus injection of 4.3 μg/kg and by two days of oral-nasal inhalation administration of 85.8 and 247.7 μg/kg was well tolerated and without treatment related in-life observations. Following inhalation administration of

Ostabolin-C, maximum plasma concentration was reached within 35 minutes and was rapidly and extensively distributed followed by clearance.

[0245] Although the present invention has been described in considerable detail with regard to certain versions thereof, other versions are possible, and alterations, permutations and equivalents of the version shown will become apparent to those skilled in the art upon a reading of the specification and study of the drawings. Also, the various features of the versions herein can be combined in various ways to provide additional versions of the present invention. Furthermore, certain terminology has been used for the purposes of descriptive clarity, and not to limit the present invention. Therefore, any appended claims should not be limited to the description of the preferred versions contained herein and should include all such alterations, permutations, and equivalents as fall within the true spirit and scope of the present invention. [0246] Having now fully described this invention, it will be understood to those of ordinary skill in the art that the methods of the present invention can be carried out with a wide and equivalent range of conditions, formulations, and other parameters without departing from the scope of the invention or any embodiments thereof. [0247] All patents and publications cited herein are hereby fully incorporated by reference in their entirety. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that such publication is prior art or that the present invention is not entitled to antedate such publication by virtue of prior invention.

Claims

WHAT IS CLAIMED IS:

1. A methionine-containing peptide or protein composition comprising: at least one methionine-containing peptide or protein and at least one antioxidant that inhibits oxidation of the at least one methionine-containing peptide or protein, wherein the composition is inhalable.

2. The composition of claim 1 , wherein the methionine-containing peptide comprises at least one member selected from parathyroid hormone, insulin-like growth factor, epidermal growth factor, nerve growth factor, transforming growth factor alpha precursor, transforming growth factor beta, transforming growth factor beta precursor, fibroblast growth factor, vaccinia growth factor, platelet derived growth factor, interleukin-1 , interleukin-2, interferon alpha, interferon beta, tissue-factor-pathway inhibitor, Factor VIII, growth hormone, prolactin, and granulocyte colony-stimulating factor, and analogues thereof

3. The methionine-containing peptide or protein composition according to claim 1 , wherein the methionine-containing peptide or protein comprises at least one parathyroid hormone, parathyroid hormone derivative, fragment, analog or mixtures thereof

4. The methionine-containing peptide or protein composition according to claim 1 , wherein the methionine-containing peptide or protein comprises at least one cyclized parathyroid hormone analog.

5. The methionine-containing peptide or protein composition according to claim 4, in liquid form.

6. The methionine-containing peptide or protein composition according to claim 4, in dry form.

7. The methionine-containing peptide or protein composition according to claim 6, comprising particulates.

8. The methionine-containing peptide or protein composition according to claim 6, wherein the particulates do not oxidize by more than about 10% after 19 months.

9. The methionine-containing peptide or protein composition according to claim 6, wherein the particulates do not oxidize by more than about 5% after 19 months.

10. The methionine-containing peptide or protein composition according to claim 6, wherein the particulates have a shelf life of at least about 2 months longer than a shelf life of the particulates in the absence of an antioxidant.

11. The methionine-containing peptide or protein composition according to claim 6, further comprising a unit dose comprising said particulates.

12. The methionine-containing peptide or protein composition according to claim 11 , wherein the particulates do not oxidize by more than about 10% after 19 months.

13. The methionine-containing peptide or protein composition according to claim 1 , wherein the antioxidant comprises at least one of thioethers, histidine, cysteine, tryptophan, tyrosine, and mixtures thereof.

14. The methionine-containing peptide or protein composition according to claim 13, wherein the antioxidant comprises at least one thioether.

15. The methionine-containing peptide or protein composition according to claim 14, wherein the at least one thioether comprises methionine.

16. The methionine-containing peptide or protein composition according to claim 1 , wherein the methionine-containing peptide or protein comprioses at least one cyclized parathyroid hormone analog, present in a weight percentage from about 1% to about 25% ; and the antioxidant comprises a thioether, present in a weight percentage from about 0.1% to about 10%.

17. The methionine-containing peptide or protein composition according to claim 14, further comprising, by weight: from about 1 % to about 10% of at least one buffering agent; from about 10% to about 60% of at least one dispersibility-enhancing excipient; and from about 10% to about 90% of at least one glass-stabilizing excipient.

18. The methionine-containing peptide or protein composition according to claim 17, comprising, by weight: from about 3% to about 6% of at least one cyclized parathyroid hormone analog; from about 4% to about 7% of at least one buffering agent; from about 20% to about 50% of at least one dispersibility-enhancing excipient; from about 40% to about 80% of at least one glass-stabilizing excipient; and from about 0.2% to about 1% of at least one antioxidant.

19. The methionine-containing peptide or protein composition according to claim 18, wherein: the at least one cyclized parathyroid hormone analog is chosen from cyclized parathyroid hormone analogs, and mixtures thereof; the at least one buffering agent is chosen from sodium citrate, citric acid, and mixtures thereof; the at least one dispersibility-enhancing excipient is chosen from leucine, dileucine, trileucine, norleucine, and mixtures thereof; the at least one glass-stabilizing excipient is chosen from polymers, polyols, carbohydrates, sucrose, raffinose, trehalose, and mixtures thereof; and the at least one antioxidant is chosen from thioethers and mixtures thereof.

20. The methionine-containing peptide or protein composition according to claim 1 , comprising, by weight: about 10-18% [Leu²⁷]cyclo(Glu²²-Lys²⁶)h PTH-(I -3I)NH₂; about 1-4% sodium citrate; about 10-30% trileucfne; about 20-60% trehalose; and about 0.1-1% methionine.

21. A method of stabilizing an inhalable formulation comprising at least one methionine-containing peptide or protein, the method comprising formulating the at least one methionine-containing peptide or protein with at least one antioxidant that inhibits oxidation of the at least one methionine-containing peptide or protein.

22. The method according to claim 21 , wherein the at least one methionine- containing peptide or protein comprises at least one cyclized parathyroid hormone analog.

23. The method according to claim 22, wherein the at least one cyclized parathyroid hormone analog is combined with the at least one antioxidant in a liquid.

24. The method according to claim 21 , comprising: forming a liquid composition comprising the at least one cyclized parathyroid hormone analog and the at least one antioxidant; and drying the liquid to form a powder comprising particles.

25. The method according to claim 24, wherein the liquid composition further comprises at least one surface excipient; and wherein the particles comprise a core and a surface; and wherein a concentration of the at least one surface excipient is greater on the surface of the particles than in the core.

26. The method according to claim 24, wherein the liquid composition is aqueous.

27. The method according to claim 24, wherein the cyclized parathyroid hormone analog is [Leu²⁷lcyclo(Glu²²-Lys²⁶)hPTH-(1-31)NH₂.

28. The method according to claim 24, wherein the liquid composition further comprises a pharmaceutically acceptable excipient or carrier.

29. The method according to claim 28, wherein the pharmaceutically acceptable excipient or carrier comprises at least one of trehalose, raffinose, and mixtures thereof.

30. The method according to claim 21 , wherein the at least one antioxidant comprises at least one of thioethers, histidine, tyrosine, cysteine, tryptophan, and mixtures thereof.

31. The method according to claim 30, wherein the antioxidant comprises a thioether.

32. The method according to claim 31, wherein the thioether is methionine.

33. The method of claim 21 , wherein the drying comprises lypohilization, spray- drying, freeze-drying, or spray-freeze drying.

34. A composition comprising particles, the particles comprising: at least one methionine-containing peptide and at least one surface excipient; wherein the particles comprise a core and a surface; wherein the concentration of the surface excipient is greater on the surface of the particles than in the core; and wherein the composition is inhalable.

35. The particle according to claim 34, wherein the at least one methionine- containing protein or peptide comprises at least one cyclized parathyroid hormone analog.

36. The particle according to claim 34, wherein the cyclized parathyroid hormone analog is [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-(1-31)NH₂.

37. The particle according to claim 36, wherein a glass transition temperature of the surface excipient is at least about 70⁰C.

38. The particle according to claim 34, wherein the surface excipient comprises at least one of trileucine, leucine, isoleucine, norleucine, and mixtures thereof.

39. The particle according to claim 38, wherein the methionine is at least about 0.5% by weight of the particle.

40. The particle according to claim 34, which, when stored at 5°C, for 24 months, exhibits a degree of degradation of the cyclized parathyroid hormone analog of less than about 10%.

41. The particle according to claim 34, which, when packaged into capsules and stored at 25°C and 60%RH, for 19 months, exhibits a degree of degradation of the cyclized parathyroid hormone analog of less than about 10%.

42. A nebulizer containing the composition of claim 34.

43. An inhaler containing the composition of claim 34.

44. A metered dose inhaler containing the composition of claim 34.

45. A blister containing the composition of claim 34.

46. A dry powder formulation according to claim 34, comprising particles having an MMD of less than about 10 microns.

47. The dry powder formulation according to claim 34, wherein the particles have an MMD of less than about 4 microns or a MMAD of less than about 10 microns, or both.

48. A unit dose, comprising: a receptacle; and particles contained within the receptacle, wherein the particles comprise a surface enriched with a surface excipient having a glass transition temperature, and a core enriched with at least one methionine-containing protein or peptide having a glass transition temperature lower than the glass transition temperature of the surface excipient.

49. The unit dose of claim 48 and further including an oxygen scavenger.

50. The unit dose of claim 49 wherein the oxygen scavenger decreases a rate of degradation of the methionine-containing protein or peptide by at least about 50% during storage over time.

51. The unit dose of claim 49 wherein the oxygen scavenger provides a reduction in an amount of impurity formation by at least about 10% during storage over time.

52. A method of treating or providing prophylaxis against a condition mediated by methionine-containing peptide or protein composition, comprising: pulmonarily administering an effective amount of a composition comprising at least one methionine-containing peptide or protein and at least one antioxidant that inhibits oxidation of the at least one methionine-containing peptide or protein, to a patient in need thereof.

53. The method of claim 52, wherein the condition comprises a parathyroid hormone mediated disease, and wherein the patient comprises a mammal.

54. A particle, comprising: a shell enriched with trileuciπe; and a core enriched with a parthyroid homone, analog, fragment or derivative; and further comprising methionine; and trehalose.

55. The particle of claim 54 wherein the parthyroid homone, analog, fragment or derivative is selected from the group consisting of PTH-(I -31 )NH2; PTH-(I -30)NH2; PTH-(I -29)NH2; PTH-(I -28)NH2; Leu27PTH-(1-31)NH2; Leu27PTH-(1-30)NH2; Leu27PTH-(1-29)NH2; Leu27cyclo(22-26)PTH-(1-31)NH2; Leu27cyclo(22-26)PTH-(1- 34)NH2; Leu27cyclo(Lys26-Asp30)PTH-(1-34)NH2; Cyclo(Lys27-Asp30)PTH-(1- 34)NH2; Leu27cyclo(22-26)PTH-(1-31 )NH2; Ala27 or Nle27 orTyr27 or Ile27 cyclo(22- 26)PTH-(1-31)NH2; l_eu27cyclo(22-26)PTH-(1-32)NH2; Leu27cyclo(22-26)PTH-(1- 31)OH; Leu27cyclo(26-30)PTH-(1-31 )NH2; Cys22Cys26Leu27cyclo(22-26)PTH-(1- 31)NH2; Cys22Cys26Leu27cyclo(26-30)PTH-(1-31)NH2; Cyclo(27-30)PTH-(1-31 )NH2; Leu27cyclo(22-26)PTH-(1 -30)NH2; Cyclo(22-26)PTH-(1-31 )NH2; Cyclo(22-26)PTH-(1- 30)NH2; Leu27cyclo(22-26)PTH-(1-29)NH2; Leu27cyclo(22-26)PTH-(1-28)NH2; Glu17,Leu27cyclo(13-17)(22-26)PTH-(1-28)NH2; and Glu17,Leu27cyclo(13-17)(22- 26)PTH-(1-31)NH2.

56. The particle of claim 54, wherein the particle comprises: about 2 to 25% [Leu²⁷]cyclo(Glu²²-Lys^2$)hPTH-(1 -3I)NH₂; about 2 to 10% sodium citrate; about 20 to 40% trileucine; about 40 to 70% trehalose; and about 0.1 to 5% methionine.

57. A method of delivering a powder to the lungs of a mammalian patient, comprising administering by inhalation a composition, wherein the composition comprises particles comprising: at least one methionine-containing peptide and at least one surface excipient; wherein the particles comprise a core and a surface; and wherein the concentration of the surface excipient is greater on the surface of the particles than in the core.

58. The method of claim 57 wherein a shelf life of the composition comprising methionine-containing peptide and antioxidant is increased by at least about 12 months over that of the composition absent the antioxidant.

59. A respirable particulate composition comprising: at least one parthyroid homone, analog, fragment or derivative and at least one adjunct material, and wherein the composition is chemically and physically stable for least about 12 months at 25°C, or about 150 months at 5°C, or both.

60. The respirable composition of claim 59 wherein the composition is further characterized by a particle size of less than about 5 microns, or a FPF less than about 2.8 microns of at least about 72%, or both.

61. The respirable composition of claim 59 and further including at least one of an antioxidant that inhibits oxidation of the parthyroid homone, analog, fragment or derivative, or an oxygen scavenger that reduces an oxidation potential of the parthyroid homone, analog, fragment or derivative, or both