Title:  Inhaleable spray dried 4-helix bundle protein powders having minimized aggregation

United States Patent:  6,838,075

Issued:  January 4, 2005

Inventors:  Stevenson; Cynthia (Mountain View, CA); Hastedt; Jayne E. (San Carlos, CA); Lehrman; Russ (Los Altos, CA); Chiang; Hi-Shi (San Jose, CA); Bennett; David B. (San Jose, CA); Lesikar; David (Palo Alto, CA); Yang; Bing (Redwood City, CA); Gong; David (San Mateo, CA); Cabot; Kirsten (San Francisco, CA)

Assignee:  Nektar Therapeutics (San Carlos, CA)

Appl. No.:  389628

Filed:  March 14, 2003

Abstract

The present invention provides highly dispersible spray-dried powder compositions, and in particular, inhaleable dry powder compositions for aerosolized delivery to the lungs. The powders of the invention are produced by spray drying a 4 .alpha.-helix bundle protein under conditions which both (i) protect the protein from aggregation and (ii) provide particles suitable for inhalation (i.e., demonstrating superior aerosol performance).

Description of the Invention

FIELD OF THE INVENTION

The present invention is directed to highly dispersible spray-dried powder compositions, and in particular, to inhaleable dry powder compositions for aerosolized delivery to the lungs. The powders of the invention are produced by spray drying a 4 .alpha.-helix bundle protein under conditions which both (i) protect the protein from aggregation and (ii) provide particles ideally suited for inhalation (i.e., demonstrating superior aerosol performance).

BACKGROUND OF THE INVENTION

Traditionally, inhalation therapy has played a relatively minor role in the administration of biotherapeutics and conventional pharmaceuticals when compared to more traditional drug administration routes, such as oral and intraveneous. Injection is still the customary route of delivery of biotherapeutics (e.g., peptides, proteins and nucleic acids), and due to the many drawbacks associated with injection (e.g., inconvenience, discomfort, patient aversion to needle-based delivery methods), alternative administration routes are needed.

Pulmonary delivery is one such alternative administration route which can offer several advantages over needle-based administration. These advantages include the convenience of patient self-administration, the potential for reduced drug side-effects, ease of delivery by inhalation, the elimination of needles, and the like. Many preclinical and clinical studies with inhaled proteins, peptides, DNA and small molecules have demonstrated that efficacy can be achieved both within the lungs and systemically. However, despite such results, the role of inhalation therapy in the health care field has not grown as expected over recent years, in part due to a set of problems unique to the development of inhaleable drug formulations. In particular, dry powder formulations for pulmonary delivery, while offering unique advantages over cumbersome liquid dosage forms and propellant-driven formulations, can be prone to aggregation and low flowability phenomena which considerably diminish the efficiency of dry powder-based inhalation therapies.

In recent years, driven in part by the interest in aerosol delivery of dry powders and some of the short-comings of well-known techniques for preparing dry powders (e.g., lyophilization, air-drying, and co-precipitation), spray drying has been employed as a method for preparing micron-sized powders for pulmonary administration (Platz, R., et al., International Patent Publication No. WO 96/32149). Spray drying utilizes a hot gas stream to evaporate microdispersed droplets created by atomization of a liquid feedstock to form dry powders. While spray-drying has been long employed in the food and pharmaceutical industries to prepare dry powders, its application to therapeutic proteins has been rather limited because of the concern that certain proteins may be thermally degraded during the spray drying process. While there is now a growing body of evidence to support the general utility of spray drying macromolecule-based biotherapeutic formulations to produce biologically active powders suitable for inhalation (Foster, L., et al., International Patent Publication No. WO 98/16205; Platz, R., et al., International Patent Publication No. WO 97/41833; Eljamal, M., et al., International Patent Publication No. WO 96/32152, Eljamal, M., et al., International Patent Publication No. 96/32116; Eljamal, M., et al., International Patent Publication No. 95/24183; Bennett, D., et al., International Patent Publication No. 01/00312), many peptides and proteins, when exposed to the harsh conditions of spray drying, are prone to a certain degree of aggregation (unfolding).

Certain proteins, and in particular, proteins characterized as belonging to the 4-.alpha.-helical bundle superfamily (e.g., hGH, INF-.gamma., INF-.beta., GM-CSF, M-CSF, IL-2, IL-4, IL-5). are extremely susceptible to denaturation, unfolding, aggregation and precipitation, with loss of biological activity. These proteins share extensive sequence and structural (conformational) homology, characterized by a protein core folded in an up, up, down, down, antiparallel, left handed for .alpha.-helix bundle with a double-overhand loop topology. Thus, due to their instability, spray-drying and formulating this class of proteins for inhalation presents a unique set of challenges.

Several aspects of the spray-drying process can contribute to protein unfolding for this class of proteins, such as shear stress, high temperatures, exposure of a protein in a droplet to the liquid air interface (surface effects), liquid-wall interactions, and the like, and can result in the formation of dried particles which contain a high degree of protein in aggregated form that are in a size range unsuitable (or at least non-optimal) for inhalation. Examples of 4-.alpha.-helix bundle protein instability upon processing are numerous. Recombinant consensus .alpha.-interferon (rConIFN) was shown to be destabilized by air-jet nebulization, which resulted in rapid formulation of insoluble noncovalent aggregates, with only about 25% of the initial monomeric protein remaining after 25 minutes of nebulization. (Ip, A. Y., et al., J. Pharm Sci., 84(10), 1995: 1210-1214). In an examination of the feasibility of spray-drying proteins such as hGH, 25% of the protein was found to be degraded during processing, although addition of 0.1% (w/v) polysorbate 20 reduced the formation of insoluble and soluble aggregates during spray drying by about 70-85% (Mumenthaler, M., et al., Pharm. Research, 11 (1), 1994: 12). The addition of polysorbate 20 in the presence of divalent zinc ions was found to further suppress hGH degradation upon spray-drying (Maa, Y-F., et al., J. Pharm. Sci., 87 (2), 1998: 152-159). In an investigation of the bioactivity and physical stability of interleukin-2 upon delivery by continuous infusion, transient surface association of IL-2 with the catheter tubing was identified as being responsible for the majority of the biological activity loss observed (.about.90% loss) (Tzannis, S., et al., Proc. Natl. Acad. Sci. USA, 93: 5460 (1996).

Of the 4.alpha.-helix bundle proteins, growth hormone is particularly unstable, and many approaches have been employed to date to arrive at stable therapeutic formulations. Degradation products of growth hormone include deaminated or sulfoxylated products and dimer or polymer forms. Specifically, the predominant degradation reactions of growth hormone are (i) deamidation by direct hydrolysis or via a cyclic succinimide intermediate to form various amounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH, and D-iso-asp-hGH, and (ii) oxidation of the methionine residues in positions 14 and 125. Human growth hormone is also readily oxidized in positions 14 and 125. More importantly, aggregate formation in human growth hormone is detrimental, since this can lead to reduced bioactivity and increased immunogenicity (Becker, et al., Biotech. Appl. Biochem., 9:478-487 (1987); Leppert, P., Moore, W. V., J. Clin. Endocrinol., 51: 691-697 (1980)).

Thus, protein denaturation, the formation of aggregates, and production of powders having poor flow properties and low dispersibilities continue to plague development efforts to prepare aerosolizable 4-helix bundle protein powders for inhalation therapy. Moreover, many of the approaches utilized to date are undesirable or unsuited for powder formulations for inhalation therapies, such as the use of surfactants, which are thought to interfere with the lung pathology and are epithelial irritants, or increasing the protein solids concentration of pre-spray dried solutions, which can result in particles that are too big for efficient delivery to the deep lung.

Thus, a need exists for improved inhaleable powder aerosols for the pulmonary delivery of 4-helix bundle proteins, and in particular, for spray-dried powders having excellent aerosol properties and reduced aggregation.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery of unique conditions for spray drying 4-.alpha. helix bundle proteins to provide respirable protein powder formulations, and in particular, 4 .alpha.-helix bundle protein powder formulations that are (i) resistant to protein aggregation during spray drying and upon storage, (ii) highly dispersible, and (iii) in a size range suitable for inhalation. The stabilizing conditions or excipients employed are effective to mask the protein from the extreme conditions of spray drying, such that process parameters which would otherwise (i.e., in the absence of such stabilizing conditions) lead to a large degree of protein aggregation are suitable, and often optimal, for forming particles (i) containing less than 10% total protein aggregates, and more preferably less than 7% total aggregates, and even more preferably less than 5% total aggregates, and (ii) having an emitted dose of at least about 65%, and more preferably of at least about 70%, The spray dried particles of the invention are sized appropriately for inhalation therapy, i.e., having an MMAD less than 10 microns, preferably less than 5 microns, more preferably less than 3.5 microns, and most preferably less than 3 microns.

In a preferred embodiment of the invention, the 4-.alpha. helix bundle protein comprises human growth hormone.

The spray dried formulations of the invention, in certain particular embodiments, include a stabilizing excipient effective to maintain or lower the aggregate level (i.e., either the insoluble or soluble aggregate level) of the powder in comparison to the spray-dried neat formulation. Specifically, stabilizing excipients include sugars, amino acids, and oligomers comprising 2 to 5 amino acids.

In one embodiment, the stabilizing excipient is sucrose or raffinose.

In another embodiment, the stabilizing excipient is leucine, isoleucine, or norleucine.

In yet another embodiment, the stabilizing excipient is trileucine.

In one particular embodiment of the invention, the stabilizer is present in the spray dried particles in an amount less than or equal to about 30% by weight.

In yet another particular embodiment, the spray dried powder of the invention comprises at least about 50% by weight of a 4 .alpha.-helical bundle protein such as human growth hormone.

The spray dried powder of the invention is also stable upon storage, such that, in a certain embodiment, it maintains a total aggregate content of less than about 10% after storage for one month at 40^oC.

In yet another embodiment of the invention, the 4 .alpha.-helical bundle protein is spray dried under optimized spray drying conditions such that the presence of additional stabilizers is not required. Surprisingly, the neat spray drying methodology is effective to produce spray dried particles containing minimal amounts (i.e., less than 10%) of protein aggregates (soluble and insoluble aggregates), excellent dispersibilities (greater than 65%, and preferably 70% emitted dose), and small aerodynamic particle sizes (MMADs less than about 4 microns).

In another aspect, the invention provides formulations and spray-drying conditions which minimize spray-drying induced changes in secondary structure of the 4 .alpha.-helical bundle protein, i.e., the .alpha.-helix is largely retained. In particular, the invention provides spray dried powders which exhibit a loss in .alpha.-helix of no more than about 50%, preferably no more than 40%, more preferably no more than 30%, and even more preferably no more than about 10-20% relative to the native structure prior to spray-drying, as measured by either FT IR or circular dichroism (CD). Particularly preferred conformational stabilizers and solvents for use in the spray-dried formulations of the invention are those which preserve .alpha.-helix content of the protein and disfavor .beta.-sheet formation including alcohols, in particular, ethanol. In one embodiment of this aspect of the invention for preserving .alpha.-helicity of the protein, the solids concentration in the pre-spray dried solution is less than 2 mg/mL.

DETAILED DESCRIPTION OF THE INVENTION

Formulation Components

The present invention is based in part upon the Applicants' discovery of optimal conditions for spray drying molecules, and in particular proteins such as 4 .alpha.-helical bundle proteins, to prepare stable, non-aggregated respirable dry powders. The challenge facing the inventors was to balance the factors influencing protein aggregation, deamidation (degradation), and denaturation with those affecting aerodynamic particle size and particle dispersibility, as well as storage stability. These factors often work in opposing directions, such that conditions which provide minimized protein aggregation often result in particles having properties that are unsuitable for inhalation (e.g., large particle sizes, low dispersibilities).

Described below are preferred stabilizing excipients and formulation components, which, when employed in conjunction with particular spray drying process parameters described in greater detail below, are effective to mask/shield the protein from the harsh conditions of spray drying, such that conditions which would otherwise lead to a large degree of protein aggregation are suitable, and often optimal for forming respirable particles. Not to be bound by any particular theory, the stabilizer may work in a variety of ways, e.g., by decreasing surface tension, increasing diffusion, hydrophobic/ionic association with the protein to stabilize against unfolding, buffering, etc. Thus, these stabilizers can be employed to not only minimize protein aggregation, but to increase the aerosol performance of the resulting spray-dried compositions, and in some cases, to provide aerosolizable formulations in situations where an aerosolizable formulation, and in particular an aerosolizable 4 .alpha.-helical bundle protein formulation, was previously unknown or unattainable.

The components of the spray-dried compositions of the invention will now be described.

A. 4 .alpha.-Helical Bundle Proteins

The present invention is directed but not limited to protein pharmaceutical agents and in particular, to 4 .alpha.-helical bundle proteins. Proteins belonging to this structural family include G-CSF, growth hormone, IFN-.gamma., IFN-.beta., GM-CSF, IL-2, IL-4, IL-5, and M-CSF. Although having limited homology at the sequence level, these growth factors and cytokines share a four helix topology (A-D) with overhand loop connections. More specifically, these proteins share a common fold in their conformational structure, characterized by an up-up-down-down, antiparallel, left-handed, four-.alpha.-helical bundle with a double overhand loop topology. One illustrative 4 .alpha.-helical protein, IL-2, in its native structure possesses from about 42-63% .alpha. helix, with little or no .beta.-sheets (Tzannis, S., et al., Proc. Natl. Acad. Sci. USA, 93:5460-5465 (1996). One of the aims of the present invention is to produce respirable protein powders in which the protein .alpha.-helix secondary structure is retained while .beta.-sheet formation is inhibited during spray drying.

Particularly suitable for use in the methods and compositions described herein are growth factors such as growth hormone, and in particular human growth hormone. Human growth hormone (hGH) is a protein secreted from the anterior pituitary gland in a pulsatile manner and is essential for regulation of growth. hGH is a single chain polypeptide of 191 amino acids with a molecular weight of approximately 22 kDa and a pI near 5.3. hGH stimulates skeletal growth throughout life and is essential in the normal metabolism of body nutrients, carbohydrates, proteins and lipids. It plays an important role in protein metabolism as it tends to increase protein synthesis (anabolism) and decreases protein destruction (catabolism) by using fat as a more efficient source of energy. The amount of natural hGH secreted is high in children, reaches maximal levels during adolescence and then decreases to its lowest levels during adulthood. Human growth hormone for use in the compositions of the invention will typically be recombinantly prepared (e.g., by E. coli) or pituitary-derived.

The amount of 4 .alpha.-helical bundle protein in the formulation will be that amount necessary to deliver a therapeutically effective amount of the protein per unit dose to achieve the desired result. 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 composition will generally contain anywhere from about 1% by weight to about 99% by weight active protein, typically from about 2% to about 95% by weight active protein, and more typically from about 5% to 85% by weight 4-.alpha. helix bundle protein, and will also depend upon the relative amounts of excipients/additives contained in the composition. More specifically, the spray dried composition will typically contain at least about one of the following percentages of active agent: 10%, 20%, 30%, 40%, 50% or more by weight of 4-.alpha. helix bundle protein. Preferably, the spray dried powder will contain at least about 50%, e.g., from about 50-100% by weight 4-.alpha. helix bundle protein such as human growth hormone. In specific embodiments of the invention, the spray dried powder will comprise at least about 60% by weight 4-.alpha. helix bundle protein, even more preferably at least about 70% by weight 4-.alpha. helix bundle protein (e.g., from about 70%-100% active protein), more preferably at least about 80% or more by weight 4-.alpha. helix bundle protein (80-100% by weight active protein), or from about 90-100% by weight 4-.alpha. helix bundle protein. In general, a high protein content is desired in the particles. The spray-dried compositions of the invention are particularly useful for proteins, e.g., 4-.alpha. helix bundle proteins, that are delivered in doses of from 0.001 mg/day to 100 mg/day, preferably in doses from 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10 mg/day to 50 mg/day.

B. Stability-Enhancing Excipients

Compositions of the invention will, in some instances, include one or more protective or stabilizing excipients. As discussed above, one aspect of the invention is based upon the Applicants' discovery that when employed in conjunction with optimized spray-drying conditions, certain excipients or additives (e.g., amino acids such as leucine, oligomers such as trileucine, sugars such as sucrose, mannitol, and raffinose, and salts such as sodium chloride, potassium chloride, and the like) are superior not only in their ability to minimize protein aggregation, but to also reduce the aerodynamic size and increase the dispersibility of the resulting powdered compositions, as demonstrated in the Examples using the exemplary protein, human growth hormone (hGH). Moreover, several of the exemplary compositions of the invention were stable upon extended storage, i.e., having aggregate levels that did not increase substantially upon storage.

Exemplary stabilizers include amino acids, peptides and particularly oligomers comprising 2-9 amino acids, and more preferably 2-5 mers, and polypeptides, all of which may be homo or hetero species. Amino acids for use as 4 .alpha.-helical bundle protein stabilizers include glycine (gly), alanine (ala), valine (val), norvaline (2-aminopentanoic acid), 2-aminoheptanoic acid, leucine (leu), isoleucine (ile), methionine (met), proline (pro), phenylalanine (phe), trytophan (trp), serine (ser), threonine (thr), cysteine (cys), tyrosine (tyr), asparagine (asp), glutamic acid (glu), lysine (lys), arginine (arg), histidine (his), norleucine (nor), and modified forms thereof. Preferred are amino acids such as leucine, isoleucine, norleucine, valine, norvaline, 2-aminoheptanoic acid, phenylalanine, and tryptophan. Particularly preferred stabilizing amino acids/excipients are leucine, isoleucine, and norleucine.

In considering the characteristics of preferred amino acid stabilizers, preferable are those amino acids having relatively low solubilities in water, e.g., from about 10 mg/ml to about 75 mg/ml. Not to be bound by any theory, reduced aqueous solubility lends to decreasing moisture sorption and delayed crystallization in the resulting spray dried powder, both of which are desirable characteristics for a respirable powder of the invention (e.g., in this regard, leucine is preferred over histidine which is preferred over alanine which is preferred over glycine). Also preferred are amino acids having somewhat large Van der Waals volumes, e.g., greater than about 100 A³, e.g., isoleucine, leucine, lysine, methionine and phenylalanine. Increasing Van der Waals volume tends to correlate with increased Tg of the resulting spray dried pharmaceutical powder, thus indicating greater storage stability. Also preferred are hydrophobic amino acids, such as leucine (leu), valine (val), isoleucine (isoleu), tryptophan (try), alanine (ala), methionine (met), phenylalanine (phe), tyrosine (tyr), histidine (his), and proline (pro). Another factor preferred for an amino acid stabilizer is the ability to decrease the surface tension of water, which correlates with lower MMDs and reduced protein aggregation in the resulting spray dried particles; surface active amino acids which are effective in lowering the surface tension of water include asparagine, isoleucine, phenylalanine, tryptophan, tyrosine, leucine and valine. Also preferred are amino acids having a glass transition temperature greater than 40^oC., more preferably greater than 50^oC., even more preferably greater than 60^oC., and most preferably 70^oC. or greater. The inclusion of such amino acids in spray dried powders typically improves the aerosol performance, and in particular, MMADs and EDs, by about 10-25%. One particularly preferred amino acid is leucine, which due to its surface activity, tends to concentrate on the surface of spray-dried particles, i.e., the concentration of leucine on the surface of spray dried protein particles is typically greater than in the bulk powder. Other surface active amino acids which tend to concentrate on the surface of spray dried protein particles include asparagine, isoleucine, phenylalanine, tryptophan, tyrosine, norleucine and valine. This represents another preferred feature of a stabilizer of the invention--the tendency to protect or mask the pharmaceutical agent, e.g., 4-.alpha. helical protein, during spray drying. In this particular embodiment, the pharmaceutical agent is shielded from the harsh conditions of spray drying by the presence of stabilizer molecules on the droplet surface. In this instance, the stabilizer is not an encapsulating agent and does not form a discrete coating, but rather simply accumulates at greater concentrations on the surface of the droplet during spray drying, resulting in particles having an enhanced surface concentration of stabilizer.

Also preferred for use as stabilizers are di- and tripeptides containing two or more leucyl residues, as described in Inhale Therapeutic System's International patent publication, WO 01/32144, incorporated herein by reference in its entirety. Representative of this class of stabilizer are dileucine and trileucine. A di-leucyl containing trimer may contain the two leucyl residues adjacent to each other (at the 1 and 2 positions), or they can form the ends of the trimer (occupying positions 1 and 3). The remaining amino acid contained in the trimer can be any amino acid as defined in section I above. Suitable are amino acids such as glycine (gly), alanine (ala), valine (val), leucine (leu), isoleucine (ile), methionine (met), proline (pro), phenylalanine (phe), trytophan (trp), serine (ser), threonine (thr), cysteine (cys), tyrosine (tyr), asparagine (asp), glutamic acid (glu), lysine (lys), arginine (arg), histidine (his), norleucine (nor), and modified forms thereof. Preferably, for di-leucyl containing trimers, the third amino acid component of the trimer is one of the following: leucine (leu), valine (val), isoleucine (ile), tryptophan (trp) alanine (ala), methionine (met), phenylalanine (phe), tyrosine (tyr), histidine (his), and proline (pro). Exemplary trimers for use in the invention include but are not limited to the following: leu-leu-gly, leu-leu-ala, leu-leu-val, leu-leu-leu, leu-leu-ile, leu-leu-met, leu-leu-pro, leu-leu-phe, leu-leu-trp, leu-leu-ser, leu-leu-thr, leu-leu-cys, leu-leu-tyr, leu-leu-asp, leu-leu-glu, leu-leu-lys, leu-leu-arg, leu-leu-his, leu-leu-nor, leu-gly-leu, leu-ala-lea, leu-val-leu, leu-ile-leu, leu-met-leu, leu-pro-lea, leu-phe-leu, leu-trp-leu, leu-ser-leu, leu-thr-leu, leu-cys-leu, leu-tyr-leu, leu-asp-leu, leu-glu-leu, leu-lys-leu, leu-arg-leu, leu-his-leu, and leu-nor-leu.

Dimers and trimers composed of any combination of the above described amino acids are also suitable for use in the invention. Most preferred are dimers and trimers containing at least two of the following amino acids: leucine, isoleucine, valine, norleucine, phenylalanine, and tryptophan.

Although less preferred due to their limited solubility in water, additional stability and aerosol performance-enhancing peptides for use in the invention are 4-mers and 5-mers containing any combination of amino acids as described above. More preferably, the 4-mer or 5-mer will comprise two or more leucine residues. The leucine residues may occupy any position within the peptide, while the remaining (i.e., non-leucyl) amino acids positions are occupied by any amino acid as described above, provided that the resulting 4-mer or 5-mer has a solubility in water of at least about 1 mg/ml. Preferably, the non-leucyl amino acids in a 4-mer or 5-mer are hydrophilic amino acids such as lysine, to thereby increase the solubility of the peptide in water.

Also preferred are di- and tripeptides having a glass transition temperature greater than about 40^oC., more preferably greater than 50^oC., even more preferably greater than 60^oC., and most preferably greater than 70^oC.

Polyamino acids, and in particular, those comprising any of the herein described amino acids, are also suitable for use as stabilizers. Preferred are polyamino acids such as poly-lysine, poly-glutamic acid, and poly(lys, ala).

Also suitable for use in protecting the protein during spray drying are carbohydrate excipients carbohydrates (e.g., sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and sugar polymers), and in particular, sugars. Carbohydrate 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. Preferred are non-reducing sugars, sugars that can form an amorphous or glassy phase with protein in a spray-dried solid, and sugars possessing relatively high Tgs, e.g., Tgs greater than 40^oC., preferably greater than 50^oC., more preferably greater than 60^oC., and even more preferably greater than 70^oC., and most preferably having Tgs of 80^oC. and above. Particularly preferred as stabilizing excipients are sucrose, mannitol and trehalose, as can be seen from the Examples.

Also beneficial in spray drying 4-.alpha. helical proteins are electrolytes, preferably strong electrolytes. In preliminary investigations carried out by the Applicants, it has been discovered that the incorporation of electrolytes (e.g., any of a number of pharmaceutically acceptable inorganic salts such as sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, and the like) into a liquid formulation containing a 4-.alpha. helical protein, to adjust the ionic strength of the solution, is also effective to "mask" the protein during spray drying. Without being bound by any theory, it is postulated that due to an increased concentration of electrolytes in solution, the ions tend to concentrate on the droplet surface during spray drying, thereby protecting or shielding the active protein in the droplet core during the spray drying process.

Additional excipients and additives useful in the present compositions and methods are provided in Inhale Therapeutic Systems' International Publication No. WO 96/32096 and in the "Handbook of Pharmaceutical Excipients" Third Ed., Kibbe, A. H. Editor (2000). Preferred are excipients having glass transition temperatures (Tg), above about 35^oC., preferably above about 40^oC., more preferably above 45^oC., most preferably above about 55^oC.

Exemplary protein excipients include albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like. The compositions of the invention may also include a buffer or a pH adjusting agent, typically but not necessarily 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. Other suitable buffers include Tris, tromethamine hydrochloride, borate, glycerol phosphate and phosphate. Amino acids such as glycine are also suitable.

The spray-dried compositions of the invention are typically considered to be rapid-acting, i.e., they do not exhibit controlled or sustained release characteristics when administered by inhalation. In general, the compositions of the invention are non-microspherical and non-liposomal, and the particles tend to be non-hollow. Moreover, the spray-dried human growth hormone particles of the invention are absent surfactant, which was shown in the model protein, lactate dehydrogenase, to have a deleterious effect on storage stability of the spray-dried product (Adler, M.; Lee, G., J. Pharm Sci: February 88(2): 199-208 (1999)). Surprisingly, the Applicants have produced non-aggregated spray dried powders of human growth hormone in the absence of surfactant, which in a previous study, was shown to be essential for the prevention of protein aggregation during spray (Maa, Y-F., et al., J. Pharm Sci, 87(2):152-159 (1998)).

The compositions may further include flavoring agents, taste-masking agents, inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80", and pluronics such as F68 and F88, available from BASE, although at levels less than about 0.1% by weight), sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, although preferably not in liposomal form), fatty acids and fatty esters, steroids (e.g., cholesterol), and chelating agents (e.g., EDTA, zinc and other such suitable cations). When employed in a 4-.alpha. helical protein powder, pluronics are typically present in percentages below about 1% by weight and more preferably at percentages below about 0.1% by weight. One preferred excipient combination of the invention comprises a pluronic such as F68 and trileucine. Preferred cations for use in the compositions and methods of the invention include aluminum, manganese, calcium, zinc, and magnesium. Such cations, when present, are typically present in relative molar amounts ranging from about 50:1 cation (mol)/protein (mol) to about 1:1, and are more preferably between about 20:1 and 2:1. Particularly preferred are compositions containing molar ratios of cation to protein of about 2:1, 7:1, 10:1, 15:1, and 20:1. Especially preferred at these ratios is the cation, zinc, although spray-dried formulations having insignificant levels of soluble aggregates were achieved even in the absence of zinc or other divalent cations. Preferably, the composition is absent permeation enhancers, or if present, such enhancers are typically present in small quantitities, e.g., less than about 10% or so by weight, and more preferably less than about 5% by weight in the dried solid. 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", 19^th ed., Williams & Williams, (1995), and in the "Physician's Desk Reference", 52^nd ed., Medical Economics, Montvale, N.J. (1998).

Generally, the pharmaceutical compositions of the invention will contain from about 1% to about 99% by weight stabilizer, preferably from about 5%-98% by weight stabilizer, more preferably from about 15-95% by weight stabilizer. Even more preferably, the spray dried composition will contain from about 0-40% by weight stabilizer, more preferably from 0-30% by weight stabilizer, e.g., preferred compositions will contain any of the following amounts; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30% by weight stabilizer. In general, a high protein concentration is desired in the spray drying particles, and in particular for spray dried human growth hormone. Thus, the stabilizers of the invention are typically very effective in protecting the protein during spray drying, since they are ideally present at concentrations of less than about 30% by weight (solids). Typically, the optimal amount of stabilizer is determined experimentally, i.e., by preparing compositions containing varying amounts of stabilizer (ranging from low to high), examining the protein aggregation, MIvIADs and dispersibilities of the resulting spray-dried compositions, and then further exploring the range at which optimal aerosol performance is attained with no significant adverse effect upon protein aggregation or storage stability.

III. Preparing Dry Powders

The dry powder formulations of the invention are prepared by spray drying under conditions which minimize the extent of protein aggregation. 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 Platz, R., et al., International Patent Publication Nos. WO 97/41833 (1997) and WO 96/32149 (1996), the contents of which are incorporated herein by reference.

The pharmaceutical agent, preferably a 4 .alpha.-helical protein such as a growth hormone, is typically spray dried from an aqueous solution or suspension, depending upon the solubility of the active protein at the pH range employed. Utilizing this approach, the 4-.alpha. helix bundle protein is first dissolved or suspended in water, optionally containing a physiologically acceptable buffer. In a preferred embodiment, the protein (e.g. hGH) is dissolved in an aqueous solution. The pH range of active agent-containing solutions is generally between about 3 and 11, more typically between about 3.5-9, with nearer neutral pHs being preferred in some instances (5.5-7.8), since such pHs may aid in maintaining the physiological compatibility of the powder after dissolution of powder within the lung. The solution is thus prepared at pHs that are at, above or below the pI of the protein. That is to say, the liquid formulation is spray dried at a pH ranging from about 3-4, 4-5, 5-6, 6-7, 7-8, or 8-9. As shown in by the Examples herein, surprisingly superior aerosol properties were obtained for hGH powders spray dried at low pH conditions, i.e., at pHs below about 4 (from 3.5-4). However, suitable powders were also obtained when spray dried at near neutral pHs (7-8). In one embodiment of the invention, a suspension of a 4-.alpha. helical protein such as hGH is spray dried at a pH that is about equal to its pI (isoelectric point), which in the case of hGH, is 5.3. In one particularly preferred embodiment of the invention, a suspension comprising trileucine in solution and the 4-.alpha. helical protein, hGH, in suspension at pH 5.3, is spray dried.

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, isopropanol, and the like. Such mixed solvent systems will typically contain from about 0-80% of the water miscible solvent, more preferably from about 20-40%, and most preferably from about 10-30% of the water miscible solvent. 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 0.05% to 10% (weight/volume, e.g., mg/ml), and preferably from about 0.1 to 2% (weight/volume, i.e., 1-20 mg/ml). Specifically, the pre-spray dried formulation will typically possess one of the following solids concentrations: 0.1 mg/ml or greater, 0.5 mg/ml or greater, 1 mg/ml or greater, 1.5 mg/ml or greater, 2 mg/ml or greater, 3 mg/ml or greater, 4 mg/ml or greater, or 5 mg/ml or greater. Preferred are solids concentrations from about 10-15 mg/ml. In one embodiment of the invention, the protein is spray dried at a solids concentration of 0.1 mg/ml, which is effective to provide a spray dried solid in which the native protein conformation is retained. Preferred embodiments of the invention will thus employ optimal solids concentrations during spray drying, optionally in concert with one or more stabilizers to provide respirable powders having minimized protein aggregation and superior aerosol properties.

Also, while working within solids concentrations which maintain alpha helicity of the protein (as described above), it is preferable to spray dry the protein at the higher ends (i.e., higher solids content) of the preferred concentration ranges, since higher protein concentrations correspond to high amounts of protein in a droplet, which will decrease the relative percentage of protein that can be denatured on the droplet surface due to contact with an air-water interface. Thus, if one assumes that only a certain number of protein molecules fit on a droplet surface, and that only those molecules are susceptible (or at least are most susceptible) to denaturation, then the remaining non-surface positioned protein molecules represent "protected payload".

The solutions are then spray dried in a conventional 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 50^oC. to about 200^oC., while the outlet temperature will range from about 30^oC. to about 150^oC. Preferred parameters include atomization pressures ranging from about 20-150 psi, and preferably from about 30-40 to 100 psi. Typically the atomization pressure employed will be one of the following (psi): 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or above. Spray dried powders are physically distinct from powders prepared by other evaporative drying methods, and typically exhibit morphologies and thermal histories (including glass transition temperatures, glass transition widths, and enthalpic relaxation profiles) that differ from those of powders prepared by other drying methods such as lyophilization.

Once formed, the 4-.alpha. helix bundle protein dry powder compositions are preferably maintained under dry (i.e., relatively low humidity) conditions during manufacture. processing, and storage. Irrespective of the particular drying parameters employed, the spray drying process will preferably result in inhaleable, non-protein aggregated, highly dispersible particles comprising an active 4-.alpha. helical bundle protein, preferably human growth hormone.

V. Features of Dry Powder Formulations

Powders of the invention are further characterized by several features, most notably, (i) consistently high dispersibilities, which are maintained, even upon storage (ii) small aerodynamic particles sizes (MMADs), (iii) improved fine particle dose values, i.e., powders having a higher percentage of particles sized less than 3.3 microns MMAD, all of which contribute to the improved ability of the powder to penetrate to the tissues of the lower respiratory tract (i.e., the alveoli) for systemic treatment by a protein such as hGH. These physical characteristics of the spray dried powders of the invention, to be described more fully below, are important in maximizing the efficiency of aerosolized delivery of such powders to the deep lung.

Dry powders of the invention are composed of aerosolizable particles effective to penetrate into the lungs. The particles of the invention have a mass median diameter (MMD) of less than about 20 .mu.m, preferably less than about 10 .mu.m, more preferably less than about 7.5 .mu.m, and most preferably less than about 4 .mu.m, and even more preferably less than about 3.5 .mu.m, and usually are in the range of 0.1 .mu.m to 5 .mu.m in diameter. Preferred powders are composed of particles having an MMD from about 0.2 to 4.0 .mu.m. In some cases, the powder will also contain non-respirable carrier particles such as lactose, where the non-respirable particles are typically greater than about 40 microns in size.

The powders of the invention are further characterized by an aerosol particle size distribution less than about 10 .mu.m mass median aerodynamic diameter (MMAD), preferably having MMADs less than about 5 .mu.m, more preferably less than 4.0 .mu.m, even more preferably less than 3.5 .mu.m, and most preferably less than 3 .mu.m. The mass median aerodynamic diameters of the powders will characteristically range from about 0.1-10 .mu.m, preferably from about 0.2-5.0 .mu.m MMAD, more preferably from about 1.0-4.0 .mu.m MMAD, and even more preferably from about 1.5 to 3.0 .mu.m. Illustrative MMAD values for exemplary spray dried hGH powders are provided in the Examples. Several of these examples demonstrate an improvement in aerosol particle size distribution achieved by a combination of optimized spray drying conditions and choice and concentration of stabilizer.

The powders of the invention may further be characterized by their densities. The powder will generally possess a bulk density from about 0.1 to 10 g/cubic centimeter, preferably from about 0.1-2 g/cubic centimeter, and more preferably from about 0.15-1.5 g/cubic centimeter.

The powders will generally have a moisture content below about 20% by weight, usually below about 10% by weight, and preferably below about 6% by weight. More preferably, the spray dried powders will typically possess a residual moisture content below about 3%, more preferably below about 2%, and most preferably between about 0.5 and 2% by weight. Such low moisture-containing solids tend to exhibit a greater stability upon packaging and storage. Generally, the powders of the invention are hygroscopic, i.e., they demonstrate a tendency to absorb moisture from the atmosphere if not stored in sealed containers such as blister packages.

One of the most striking features of the compositions of the invention is their high dispersibilities, as indicated by the ED value. The spray drying methods and stabilizers described herein are effective to provide formulations having significantly improved dispersibilities over their non-optimized counterparts. Generally, the emitted dose (ED) of these powders is greater than 30%, and usually greater than 40%. More preferably, the ED of the powders of the invention is greater than 50%, and is often greater than 55%. In fact, in looking at the Examples, the spray dried powders of the invention typically possess optimized ED values exceeding 65%, and often ranging from about 70-90% or greater.

An additional measure for characterizing the overall aerosol performance of a dry powder is the fine particle fraction (FPF), which describes the percentage of powder having an aerodynamic diameter less than 3.3 microns. The powders of the invention are particularly well suited for pulmonary delivery, and possess FPF values ranging from about 30%-64% or more. Preferred powders contain at least about 30 percent of aerosol particle sizes below 3.3 .mu.m to about 0.5 .mu.m and are thus extremely effective when delivered in aerosolized form, in reaching the regions of the lung, including the alveoli.

The compositions described herein also possess good stability with respect to both chemical stability and physical stability, i.e., aerosol performance over time. Generally, with respect to chemical stability, the 4-.alpha. helix bundle protein contained in the formulation will degrade by no more than about 10% upon spray drying. That is to say, the powder will possess at least about 90% intact active protein, preferably at least about. 95% intact active agent, and even more preferably will contain at least about 97% or greater intact active agent. Preferably, the spray drying process will result in powders having less than about 10% total protein aggregates, preferably less than about 7% total aggregates, and most preferably, less than 5-6% total aggregates. More specifically, the spray dried powder will typically possess less than about 10% insoluble aggregates, preferably less than about 7% insoluble aggregates, and more preferably will contain less than about 5% insoluble aggregates. In considering soluble aggregates, the spray dried powder will typically contain less than 7% soluble aggregates, preferably less than 6% soluble aggregates, more preferably less than 5% soluble aggregates, and most preferably less than 4%, 3%, 2% or 1% soluble aggregates. The total amount of monomer in the spray dried particles is typically greater than 90%, and is more preferably greater than one of the following: 91%, 92%, 93%, 94%, or 95%. Moreover, the compositions of the invention further demonstrate good stability upon storage, as characterized by a total protein aggregate content of less than about 10% after storage for one month at 40^oC. and ambient relative humidity. That is to say, the total aggregate content of many of the exemplary growth hormone compositions of the invention remained substantially unchanged upon storage for one month at 40^oC., thus illustrating the attainment of an optimized balance of both chemical and physical stability upon both spray drying and storage of the inhaleable growth hormone compositions of the invention.

With respect to aerosol performance, compositions of the invention are generally characterized by a drop in emitted dose of no more than about 20%, preferably no more than about 15%, and more preferably by no more than about 10%, when stored under ambient conditions for a period of three months.

The improvement in aerosol properties noted for the compositions herein, along with minimized protein aggregation, can result in several related advantages, such as: (i) reducing costly drug loses to the inhalation device, since more powder is aerosolized and is therefore available for inhalation by a subject; (ii) reducing the amount of dry powder required per unit dose, due to the high efficiency of aerosolization of powder, and (iii) reducing the number of inhalations per day by increasing the amount of aerosolized drug reaching the lungs of a subject.

VI. Administration of the Composition

The formulations 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. Preferred are Inhale Therapeutic Systems' dry powder inhalation devices as described in Patton, J. S., et al., U.S. Pat. No. 5,458,135 (1995) Smith, A. E., et al., U.S. Pat. No. 5,740,794 (1998); and in Smith, A. E., et. al., U.S. Pat. No. 5,785,049 (1998), Schuler, C., et al., International Patent Publication No. WO 01/00263, herein incorporated by reference.

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 receptable 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 Parks, D. J., et al., WO 97/41031 (1997) incorporated herein by reference.

Also suitable for delivering the powders described herein are dry powder inhalers of the type described, for example, in Cocozza, S., et al., U.S. Pat. No. 3,906,950 (1974), and in Cocozza, S., et al., U.S. Pat. No. 4,013,075 (1997), incorporated herein by reference, wherein a premeasured dose of dry powder for delivery to a subject is contained within a hard gelatin capsule.

Other dry powder dispersion devices for pulmonarily administering dry powders include those described, for example, in Newell, R. E., et al, European Patent No. EP 129985 (1988); in Hodson, P. D., et al., European Patent No. EP 472598 (1996); in Cocozza, S., et al., European Patent No. EP 467172 (1994), and in Lloyd, L. J. et al., U.S. Pat. No. 5,522,385 (1996), incorporated herein by reference. 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 Virtanen, R., U.S. Pat. No. 4,668,281 (1987); in Wetterlin, K., et al U.S. Pat. No. 4,667,668 (1987); and in Wetterlin, K., et al. U.S. Pat. No. 4,805,811 (1989), all of which are incorporated herein by reference. Other suitable devices include dry powder inhalers such as the Rotahaler.RTM. (Glaxo), Discus.RTM. (Glaxo), Spiros.TM. inhaler (Dura Pharmaceuticals), and the Spinhaler.RTM. (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 Mulhauser, P., et al, U.S. Pat. No. 5,388,572 (1997), incorporated herein by reference.

Dry powders may also be delivered using a pressurized, metered dose inhaler (MDI), e.g., the Ventolin.RTM. 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 Laube, et al., U.S. Pat. No. 5,320,094 (1994), and in Rubsamen, R. M., et al, U.S. Pat. No. 5,672,581 (1994), both incorporated herein by reference. Alternatively, the powders described herein may be dissolved or suspended in a solvent, e.g., water, ethanol, or saline, and administered by nebulization. Nebulizers for delivering an aerosolized solution include the AERx.TM. (Aradigm), the Ultravent.RTM. (Mallinkrodt), and the Acorn II.RTM. (Marquest Medical Products).

Prior to use, dry powders are generally stored under ambient conditions, and preferably are stored at temperatures at or below about 25^oC., 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.

VII. Utility

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 a 4-.alpha. helix bundle protein as described in section II.A above.

In particular, the exemplary 4-.alpha. helix bundle protein, hGH, when administered in therapeutically effective amounts, is useful in the treatment of conditions such as pediatric growth hormone deficiency, adult growth hormone deficiency, chronic renal insufficiency, and Turner syndrome.

Claim 1 of 27 Claims

What is claimed is:

1. A method for treating a mammalian subject having a condition that is responsive to the administration of a growth hormone comprising pulmonarily, administering a therapeutically effective amount of the growth hormone, wherein (a) the growth hormone is present in a spray-dried, inhaleable powder having aerosolizability and protein stability, (b) the spray-dried, inhaleable powder is free from surfactant and characterized by (i) an emitted close of at least about 65%, and (ii) a total protein aggregate content of less than 10% and, (c) the condition is selected from the group consisting of pediatric growth hormone deficiency adult growth hormone deficiency, chronic renal insufficiency, Turner Syndrome.

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