Provided are stabilized follicle stimulating protein (FSP) dry powder compositions for aerosolized delivery to the deep lung, methods of preparing and administering such compositions, and methods for treating infertility involving administering the dry powders by pulmonary delivery to the deep lung.

DETAILED DESCRIPTION OF THE INVENTION

FSP Dry Powders

The present invention provides stable, dispersible dry powder compositions for pulmonary delivery of FSP. The compositions developed by the applicants overcome many of the problems often encountered heretofore in formulating proteins, particularly gonadotropin hormones, for delivery to the deep lung. The FSP dry powder compositions described herein are readily dispersed (i.e., demonstrate good aerosol performance), are stable against both physical and chemical degradation (i) in solution, before powder manufacture, (ii) during powder manufacture and processing, and (iii) upon storage, and exhibit good bioavailabilities when delivered by inhalation for deposition in and absorption from the lung. The dry powder compositions according to the present invention generally include FSP and a pharmaceutically acceptable excipient, although dry powders composed of neat FSP (i.e., respirable powders composed of FSP and essentially lacking any additional excipients or additives) are also envisioned. Components of FSP dry powders suitable for delivery to the deep lung will now be described.

A. Follicle Stimulating Proteins

1. Naturally occurring FSP. The FSP contained in the solid preparations may be at least partially purified from natural sources (i.e., may be highly purified and may optionally include LH or other co-purified proteins), such as human urinary FSH (uFSH). Urinary-derived FSH may be obtained from a commercial source (e.g., Vitro Diagnostics, Boulder, Colo.) or purified by immunopurification as described in Arpaia, G., et al., 1992. A number of naturally occurring FSP heterodimers are known and are suitable for use in the dry powder of the invention. Such exemplary FSP heterodimers, i.e., comprising one alpha subunit and one beta subunit, include but are not limited to SEQ ID NOS: 1 and 2 (bovine FSH); 3 and 4 (equine FSH); 5 and 6 (hFSH); 7 and 8 (porcine FSH); 9 and 10 (ovine FSH).

2. Synthetic or Recombinant FSP. Alternatively, FSP may be produced by chemical synthesis or may be biosynthesized in host cells appropriately engineered using conventional techniques of molecular biology (i.e., recombinant technology). Recombinant FSP, a preferred form of FSP for use in the dry powder compositions of the invention, may be prepared using conventional techniques such as those described in Keene, et al., 1989; Boime, et al., Seminars in Reproductive Endocrinology, 10:45 50 (1992); Chappel, et al., 1992; or Reddy, et al., 1992; Shome, B., et al., J. Prot. Chem., 7:325 339 (1988); Saxena, B. B. and Rathnam, P., J. Biol. Chem., 251:993 1005 (1976); Watkins, et al., DNA, 6:205 212 (1987); Shome, B. and Parlow, A. F., J. Clin. Endocrizol. Metab., 39(1):203 205 (1974); Beck, et al., DNA, 4:76 (1985); Boime, et al., U.S. Pat. No. 5,405,945 (1995); and Reddy, V. B., et al., U.S. Pat. No. 5,639,640 (1997); Sambrook, et al. Molecular Cloning: A Laboratory manual, 2.sup.nd Edition, (1989); Ausubel, et al., Eds., Current Protocols in Molecular Biology, (1987 1998), Chapters 10, 12, 13, 16, 18 and 20, the contents of which are incorporated herein by reference. Exemplary preparations of FSH variants are provided in Examples 13,14,18 and 19.

More specifically, FSP products may be prepared by recombinant techniques in a prokaryotic or eucaryotic host, including, for example, bacterial, yeast, [Sherma, F., et al., Methods in Yeast Genetics, (1992)] higher plant, insect [Schneider, J. Embryol. Exp. Morphol., 27:353 365 (1987)] and mammalian cells such as CHO, COS and Bowes melanoma cells. Depending upon the host employed in a recombinant production procedure, the rFSP molecules thus produced may be glycosylated or can be non-glycosylated. In addition, a rFSP subunit may also include an initiating methionine residue. Such methods are described in many standard laboratory manuals.

FSP may be expressed in modified forms, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to an FSP molecule to facilitate purification. Such regions can be removed prior to final preparation of a polypeptide. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29 17.42 and 18.1 18.74; Ausubel, supra, Chapters 16, 17 and 18.

Briefly, the expression of isolated nucleic acids encoding a FSP used in the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eucaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.

Expression of a FSP in yeast cells is carried out by well-known methods [Sherma, et al., (1982)]. Two widely utilized yeast for production of eucaryotic proteins are Saccharomyces cerevisiae and Pichia. Vectors, strains, and protocols for expression in Saccharomzyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen, Inc.). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like.

Polynucleotide sequences encoding FSP can also be ligated to various expression vectors for use in transfecting mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing FSP include the HEK293, BHK21, AV12 and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., CMV promoter, EF1 alpha promoter, or a HSV tk promoter or phosphoglycerate kinase promoter), an enhancer, and processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. Other animal cells useful for production of FSP are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992).

Appropriate vectors for expressing FSP in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line [Schneider, supra, 1987].

Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 1 4 and 16 18; Ausubel, supra, Chapters 1, 9, 13, 15, 16, entirely incorporated herein by reference.

Alternatively, polynucleic acids encoding FSP can be expressed in host cells by introducing, by homologous recombination into the cellular genome at a preselected site, DNA which includes at least a regulatory sequence, an exon and a splice donor site. These components are introduced into the chromosomal (genomic) DNA in such a manner that this, in effect, results in production of a new transcription unit (in which the regulatory sequence, the exon and the splice donor site present in the DNA construct are operatively linked to the endogenous gene). As a result of introduction of these components into the chromosomal DNA, the expression of the desired endogenous gene is altered. Such methods are well known in the art, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.

Recombinant FSP can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, reverse phase chromatography, dye chromatography and lectin chromatography. Monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay or other standard immunoassay techniques.

3. hFSH Variants. The dry powder composition of the invention may contain a hFSH variant. hFSH variants can be produced by a number of techniques well known to those skilled in the art such as by chemical synthesis, recombinant biosynthesis, and in vivo or in vitro processing of naturally occurring or recombinant FSH by amino- and/or carboxy-peptidases to expose one or more internal amino acids.

B. Nucleic Acid Molecules Encoding FSP

Polynucleic acid molecules encoding FSP described herein are illustrative, not limiting, of sequences useful for preparing FSP by recombinant expression and they may be incorporated into the dry powder compositions of the invention, for expression of FSP in vivo, as described in greater detail below. Other polynucleic acids may be used to produce FSP without affecting the present invention. The polynucleic acid molecules can be in the form of RNA, such as mRNA, hnRNA, or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.

Isolated polynucleic acid molecules for use in expressing a FSP comprise an open reading frame (ORF) shown in at least one of SEQ ID NOS: 32, 37, 54, 55, or 56, or a nucleic acid molecule having a sequence complementary thereto, for expressing an alpha subunit, or comprise an ORF in at least one of SEQ ID NO:33, 34, 35, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53, or a nucleic acid molecule having a sequence complementary thereto, for expressing a beta subunit.

The polynucleic acid sequences can be made using (a) standard recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well known in the art. The polynucleic acids may conveniently comprise sequences in addition to those exemplary sequences provided herein. For example, a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of a translated FSH polynucleotide. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of an FSP polynucleotide, or to improve the introduction of the polynucleotide into a cell.

Polynucleic acids encoding FSP can be prepared by direct chemical synthesis methods such as the phosphotriester method of Narang, et al., Meth. Enzymol., 68:90 99 (1979); the phosphodiester method of Brown et al., Meth. Enzymol., 68:109 151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Letts., 22:1859 1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts., 22(20):1859 1862 (1981), e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter, et al., Nucleic Acids Res., 12:6159 6168 (1984); and the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single-stranded oligonucleotide, which may be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. Although chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

Representative polynucleotide sequences described in sequences 32 56 herein encode either an alpha or a beta subunit. The DNA of SEQ ID NO: 37 and 38 is designed and constructed from ligated oligonucleotides. The differences between SEQ ID NO: 32 and SEQ ID NO: 37 do not change the encoded amino acid sequence of the alpha subunit protein. Likewise, the differences between SEQ ID NO: 38 and SEQ ID NO: 33 do not change the encoded amino acid sequence of the beta variant subunit protein. The skilled person will know a multitude of sequences equivalent to the sequences described in SEQ ID NO: 32 56 exist because of the degeneracy of the genetic code.

The dry powder of the invention may contain a polynucleotide encoding FSP (e.g., genes encoding both the .alpha.- and .beta.-subunits of FSP), a fragment thereof (e.g., the .beta.-subunit, which confers biological specificity), and/or a variant as described herein, where the polynucleotide or FSP-coding region is operably linked to suitable transcriptional regulatory or control sequences (e.g., promoters, enhancers, and the like) to permit FSP transgene expression in target host cells of a subject, e.g., mammalian cells from the pulmonary regions of the lung (e.g., lung epithelial cells, alveolar type cells), or other suitable subject host cells. In addition to the sequences provided herein, representative gene sequences encoding FSP, its subunits, or precursor proteins, are found, for example, in the Entrez Nucleotide Database (as described above). Such exemplary coding sequences have the following Accession Nos.: J00152 V00487 (.alpha. subunit, bases 1 397), M54912 M38644 M21219 M18536 (.beta.-subunit gene, exon 1), M54913 M21220 M18536 (.beta.-subunit gene, exon 2), M54914 M38646 M21221 M18536 (.beta.-subunit gene, exon 3).

In a preferred embodiment of this aspect of the invention, regulatory sequences operably linked to a polynucleotide coding sequence will function to express FSP in a pulsatile fashion. Polynucleotides encoding FSP may optionally be contained in a viral or other conventional gene therapy vector, or may comprise a lipid-FSP transgene complex, as described in Eljamal, M., et al., International Patent Publication WO 96/32116, Oct. 17, 1996; in McDonald, R. J., et al., Pharm. Res., 15(5):671 9 (1998), and in Eastman, et. al., Hum. Gene Ther., 8(6):765 73 (1997), the contents of which are expressly incorporated herein by reference. Generally, FSP expression levels in the target tissue will roughly correspond to the quantities of FSP described herein for direct incorporation into the dry powders of the invention; such expression levels will characteristically correspond to a therapeutically effective amount of FSP. Representative quantities of nucleic acid constructs for incorporation in the dry powders of the invention for achieving the desired expression levels are described in Eljamal, M., et al., ibid.

C. Excipients and Other Additives

In the powders of the invention, FSP is generally combined with one or more pharmaceutical excipients that are suitable for respiratory and pulmonary administration. Such excipients may serve simply as bulking agents when it is desired to reduce the active agent concentration in the powder that is being delivered to a patient. An excipient may also serve to improve the dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the active agent and to improve the handling characteristics of the active agent (e.g., flowability and consistency) to facilitate manufacturing and powder filling into unit dosage forms. In particular, the excipient materials can often function to improve the physical and chemical stability of FSP, to minimize the residual moisture content, hinder moisture uptake, and to regulate particle size, degree of aggregation, particle surface properties (i.e., rugosity), ease of inhalation, and targeting of the resultant particles to the deep lung.

Alternatively, FSP may be formulated in an essentially neat form, wherein the composition contains FSP particles within the requisite size range and substantially free from other biologically active components, pharmaceutical excipients, and the like.

Pharmaceutical excipients and additives useful in the present composition include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers such as Ficolls), which may be present singly or in combination. Preferred are excipients that are soluble in either water (e.g., sugars, peptides, amino acids, and salts), alcohol (such as pectin, lecithin, povidone) or acetone (e.g., citric acid, PLGA). Also preferred are excipients having a glass transition temperature (Tg), above about 35.degree. C., preferably above about 45.degree. C., more preferably above about 55.degree. C. Illustrative excipients suitable for use in the FSP dry powders of the invention include those disclosed in Inhale Therapeutic Systems' International Patent Application No. WO98/16207.

Exemplary protein excipients include but are not limited to serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like. Polypeptides and proteins suitable for use in the dry powder composition of the invention are provided in Inhale Therapeutic Systems' International Patent Publication No. WO96/32096. HSA is a preferred proteinaceous excipient, and has been shown to increase the dispensability of dry powders for aerosolized delivery to the lungs (WO 96/32096, ibid).

Representative amino acid/polypeptide components, which may optionally function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, threonine, tyrosine, tryptophan and the like. Preferred are amino acids and peptides that function as dispersing agents. Amino acids falling into this category include hydrophobic amino acids such as leucine (leu), valine (val), isoleucine (isoleu), norleucine (nie), tryptophan (try), alanine (ala), methionine (met), phenylalanine (phe), tyrosine (tyr), histidine (his), and proline (pro). Examplary FSP formulations containing a variety of amino acid excipients are provided in Example 23. Leucine is one particularly preferred amino acid excipient for the FSP compositions described herein. Leucine, when used in the formulations described herein includes D-leucine, L-leucine, and racemic leucine. Exemplary FSP dry powders containing leucine are described in Examples 11, 21, 22 and 23. Dispersibility enhancing peptides for use in the invention include dimers, trimers, tetramers, and pentamers composed of hydrophobic amino acid components such as those described above, e.g., dileucine, trileucine, and the like.

Carbohydrate excipients suitable for use in the invention exclude melezitose and 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, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose. Preferred powder compositions in accordance with the invention are those that are stable in the absence of sucrose, and particularly those that are stable in the absence of combinations of sucrose and glycine.

The dry powder compositions may also include a buffer or a pH-adjusting agent. Typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid and Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers for use in the present compositions are organic acid salts such as citrate.

Additionally, the FSP dry powders of the invention may include polymeric excipients such as polyvinylpyrrolidones, Ficolls (a polymeric sugar), hydroxyethyl starch, dextrates, polyamino acids (e.g., polyleucine, polyglutamic acid), and/or polyethylene glycols, where such polymers are present as additives rather than as encapsulating agents. The dry powder may also optionally contain flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, steroids (e.g., cholesterol), and chelating agents (e.g., EDTA). Other pharmaceutical excipients and/or additives suitable for use in the FSP compositions according to the invention are listed in Remington, The Science & Practice of Pharmacy, 19.sup.th ed., (1995), and in the Physician's Desk Reference, 52.sup.nd ed., (1998), the disclosures of which are herein incorporated by reference.

In contrast to previously reported dry powders that are poorly absorbed into the pulmonary vasculature and into the systemic circulation when lacking one or more enhancer compounds [Backstrom, et al., U.S. Pat. No. 5,128,453, Jul. 7, 1992], the dry powder of the invention is surprisingly well-absorbed through the lung, and does not require the addition of absorption enhancers. This is evidenced by the bioavailability data provided herein, e.g., in Examples 10 and 17. Thus, the dry powders of the invention preferably lack typical enhancer compounds, such as surfactants (e.g., salts of a fatty acids), bile salts, alkyl glycosides, cyclodextrins, and phospholipids, or, if such compounds are present, they are typically present in very low concentrations--less than about 20% by weight relative to FSP, and more preferably less than about 15% to 10% by weight relative to FSP--such that they do not function to noticeably enhance absorption.

Preferred carrier or excipient materials are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or polymeric agents, optionally in combination with an amino acid or di- or tripeptide as described above.

In accordance with the invention, the solid state matrix formed by the excipient and the protein imparts a stabilizing environment to the FSP, and may optionally aid in dispersivity of the composition. The stabilizing matrix may be crystalline, an amorphous glass, or a mixture of both forms. Preferred are compositions that, irrespective of their percent crystallinity, are stable with respect to this percentage over time. Most suitable are dry powder formulations which are substantially amorphous (glasses) or substantially crystalline (i.e., are crystalline to the greatest extent possible, after adjusting for the amount of FSP contained in the powder, since FSH does not crystallize). More generally, preferred powders are substantially crystalline, such as FSP powder L2017 (Example 6.C.), i.e., 51 to 99% crystalline, 60% to 95% crystalline, 65% to 90% crystalline, or any range or value therein, or are substantially amorphous glasses, such as powders L2010 and L2018 (Example 6.C.), i.e., at least about 70% of the solid is an amorphous glass, preferably at least about 75% is an amorphous glass, and more preferably at least about 85% is an amorphous glass.

For FSP dry powder formulations which are substantially amorphous, preferred are those formulations exhibiting glass transition temperatures (T.sub.g) above about 30.degree. C., preferably above about 40.degree. C., and more preferably above about 60.degree. C. Glass transition temperatures for representative FSP dry powder compositions are presented in Table 6 (Example 6.B.) As can be seen from the values presented therein, for those compositions for which Tg values were determined, 22 formulations/lot numbers exhibited Tg values above 30.degree. C., while 19 formulations/lot numbers possessed Tg values about 60.degree. C. Formulations having Tg values at or above 60.degree. C. included the following: L2001, L2004, L2006, L2008, L2009, L2010, L2012, L2013, L2014, L2016, L2020 and L2018. Preferred storage temperatures for substantially amorphous powders are at least about 10.degree. C. lower than the T.sub.g of the composition, as set forth in Inhale Therapeutic Systems' International Patent Publication WO 98/16205.

FSP contained in the dry powder formulations is present in a quantity sufficient to form a pharmacologically effective amount when administered by inhalation to the lung. The dry powders of the invention will generally contain from about 0.1% by weight to about 99.9% by weight FSP, more typically from about 0.5 to 80% by weight FSP, and preferably from about 1 to 75% by weight FSP. Preferred compositions contain from about 1.5 to 70% FSP, and more preferably from about 12 to 60% by weight FSP, depending upon the specific activity of FSP contained in the formulation and the type of treatment (i.e., ovulation induction versus super ovulation therapy). In one preferred embodiment of the invention, the dry powder contains about 5% by weight FSP. In an alternate embodiment, the dry powder contains about 15% by weight FSP. Correspondingly, the amount of excipient material(s) will range from about 99.9 to 0.1% by weight, more typically from about 99.50% to 20% by weight, and preferably from about 99% to 25% by weight. Preferred compositions contain from about 98.5 to 30% by weight excipient material, and more preferably contain from about 88 to 40% by weight excipient material. Leucine-containing FSP powders will typically contain from about 15 85% by weight leucine, preferably from about 20 80% by weight leucine, and more preferably from about 40 60% by weight leucine.

The composition of representative FSP dry powders for pulmonary delivery is provided at least in Examples 2 and 11. As can be seen from the data provided in Example 2, Table 1, compositions containing various combinations and relative amounts of FSP and excipient(s) resulted in powders having good particle size characteristics (all powders possessed MMDs less than 3.5 microns, while 80 percent of powders possessed MMDs less than 1.5 microns) and which exhibited a minimal change in higher order aggregate formation of FSP upon powder manufacture (Example 3). The findings detailed in Example 3 provide evidence of another surprising feature of the FSP compositions of the invention, i.e., their ability to stabilize monomeric FSP against higher order aggregate formation during powder manufacture and processing. This is important, since the formation of higher order aggregates of FSP can adversely affect its bioactivity, due to impaired receptor binding, which will in turn reduce the potency of the resultant powders.

III. Preparing FSP Dry Powders

Dry powder FSP formulations are preferably prepared by spray drying under conditions that result in a substantially amorphous glassy or a substantially crystalline bioactive powder as described above. Spray drying of the FSP-solution formulations is carried out, for example, as described generally in the Spray Drying Handbook, 5.sup.th ed., (1991), and in Platz, R., et al., International Patent Publication No. WO 97/41833, Nov. 13, 1997.

To prepare an FSP solution for spray drying, FSP is generally dissolved in a physiologically acceptable aqueous buffer, e.g., a citrate buffer or a citrate glycine buffer, typically having a pH range from about 2 to 9. The pH range of pre-dried solutions is generally maintained between about 4 and 10, with near neutral pH being preferred, since such pHs may aid in maintaining the bioactivity or physiological compatibility of the powder after dissolution of powder within the lung. The aqueous formulation may optionally contain additional water-miscible solvents, such as alcohols and the like. Representative alcohols include methanol, ethanol, propanol, isopropanol, and the like. FSP solutions will generally contain FSP dissolved at a concentration from 0.01% (weight/volume) to about 10% (weight/volume), usually from 0.1% to 1% (weight/volume).

In turning now to the results provided in Example 1, it can be seen that representative solution formulations of FSP prior to spray drying showed no appreciable decline in bioactivity upon storage for several days, and after repeated freeze-thaw cycles, indicating the relatively high stability of such solution formulations.

The FSP-containing 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 stable, FSP dry powder. Optimal conditions for spray drying the FSP 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 deactivation of FSP in the sprayed material. Such temperatures are typically determined experimentally, although generally, the inlet temperature will range from about 50.degree. C. to about 200.degree. C. while the outlet temperature will range from about 30.degree. C. to about 150.degree. C.

Alternatively, FSP dry powders may be prepared by first drying a solution containing FSP and exciptient(s) by lyophilization, vacuum drying, spray freeze drying, super critical fluid processing, or other forms of evaporative drying, and then further processing the dry material to obtain a FSP dry powder having aerosol properties suitable for administration into the deep lung and an acceptable ED, by blending, grinding or jet milling the dried formulation. In some instances, it is desirable to make FSP dry powder formulations possessing 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 FSP dry powder particles, where the aggregates are readily broken back down to the fine powder components for pulmonary delivery, as described, e.g., Johnson, et al., U.S. Pat. No. 5,654,007, Aug. 5, 1997, incorporated herein by reference. Alternatively, the FSP powders may be prepared by agglomerating the powder components, sieving the materials to obtain the agglomerates, spheronizing to provide a more spherical agglomerate, and sizing to obtain a uniformly-sized product, as described, e.g., and in Ahlneck, C.; et al., International PCT Publication No. WO95/09616, Apr. 13, 1995, incorporated herein by reference. The FSP dry powders of the invention may also be prepared by blending, grinding or jet milling formulation components directly in dry powder form. The FSP dry powders 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 particles composed of FSP in non-aggregated form, or having an extent of higher order aggregate formation that is substantially unchanged from that observed in the pre-dried material, as illustrated in Example 3. Moreover, processes suitable for preparing the FSP formulations of the invention are those in which the bioactivity of the FSP is not adversely affected, i.e., the bioactivity of the resulting powder is reduced by no more than about 40 50%, preferably by no more than about 30 40%, and more preferably by no more than about 15% 30% in comparison to the bioactivity of the FSP formulation prior to drying/sizing. Illustrative FSP dry powders which exhibited no significant loss in bioactivity upon drying are described in Example 8.

Provided in Example 2, and more specifically in Table 1, are exemplary FSP compositions in accordance with the invention. As can be seen, FSP dry powders were obtained in high yields, typically between about 70 and 80%, illustrating the ability to reproducibly prepare large quantities of FSP dry powders suitable for pulmonary delivery. Moreover, the extent of FSP sialylation was shown to be unaffected by spray drying (Example 5), illustrating the chemical stability of FSP under conventional spray drying conditions. This is important since the prolonged bioactive half-life of FSP is a result of its oligosaccharide content; FSP asialoglycoproteins (i.e., proteins stripped of sialic acid) are cleared rapidly by the liver.

IV. Features of FSP Dry Powders

The FSP powders of the invention are further characterized by several features, most notably, the ability of the powder to penetrate to the tissues of the lower respiratory tract (i.e., the alveoli) for subsequent entry into the bloodstream (see, e.g., Examples 10 and 18). It has been found that certain physical characteristics of the FSP dry powders, to be described more fully below, are important in maximizing the efficiency of aerosolized delivery of such powders to the deep lung. The FSP dry powders are composed of particles effective to penetrate into the alveoli of the lungs, that is, having a mass median diameter (MMD) from about 0.1 to 20 .mu.m. Typically, the MMD of the particles is less than about 10 .mu.m (e.g., ranging from about 0.1 to 10 .mu.m), preferably less than 7.5 .mu.m (e.g., ranging from about 0.5 to 7 microns), and most preferably less than 5 .mu.m, and usually being 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 1 to 3.5 .mu.m. Numerous examples of respirable FSP powders of varying concentrations of active agent and excipients and composed of particles within this preferred size range have been prepared (e.g., Table 1, column 10; Example 2). In some cases, the FSP 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 FSP powders of the invention are further characterized by an aerosol particle size distribution less than about 10 .mu.m mass median aerodynamic diameter (MMAD), and preferably less than 5 .mu.m, and more preferably less than about 3.5 .mu.m. The mass median aerodynamic diameters of the powders will characteristically range from about 0.5 5.0 .mu.m, preferably from about 1.0 4.0 .mu.m MMAD, more preferably from about 1.5 3.5 .mu.m MMAD, and even more preferably from about 1.5 to 3.0 .mu.M. To further illustrate the ability to prepare FSP powders having an aerosol particle size distribution within a range suitable for pulmonary administration, exemplary FSP dry powders composed of particles having an aerosol particle size distribution less than about 5 .mu.m MMAD, and more specifically, characterized by MMAD values less than 3.5 .mu.m, are illustrated in Table 8 (Example 7), in Table 16 (Example 11), in Table 30 (Example 21) and in Table 31 (Example 22).

FSP dry powders of the invention are characterized, in another respect, by a relative pulmonary bioavailability between about 1% to 60%. The relative pulmonary bioavailability of a powder of the invention will typically fall between about: 1 60%, 1 30%, 5 30%, 10 30%, 15 30%, 15 25%, 20 25%, 1 20%, 2 20%, 3 20%, 4 20%, 1 15%, 1 10%, 2 10%, 3 10% and 4 10%. Preferably, an FSP powder will exhibit a relative pulmonary bioavailability between about 1 30% and more preferably between about 1 20%, as supported by the data provided herein.

The FSP dry powders generally have moisture content below about 10% by weight, usually below about 5% by weight, and preferably below about 3% by weight. Such low moisture-containing solids tend to exhibit a reduced tendency towards degradation of protein. The moisture content of representative FSP dry powders prepared as described herein is provided in Example 6.B., Table 6.

The delivered dose efficiency (DDE) of these powders is greater than 30% and usually greater than 40%. More preferably, the DDE of the FSP powders of the invention is greater than 50%, and is often greater than 55%. Even more preferably, the DDE of an FSP powder is greater than 60%. Highly preferred are powders having DDE values greater than 70%, and even more preferred are powders having DDE values greater than about 75%. In looking at Example 7, Table 7, and Example 11, Tables 14 and 15, it can be seen that the applicants have successfully prepared a large number of representative FSP dry powders with DDE values greater than or equal to 50%. Formulations which exhibited and maintained particularly good powder dispersibilities, as indicated by their DDE values, included L2001, L2005, L2006, L2008, L2010, L2012, L2014, L2016, L2017, L2018, and L2020, the compositions of which are provided in Table 6, and 99348, 99349, 99423, 99425, 99426, 99455, 99454, and 99457 (Tables 14, 15).

The FSP powders of the invention will typically possess a bulk density value ranging from about 0.10 to 10 gram/cubic centimeter, preferably from about 0.15 to 5 gram/cubic centimeter, more preferably from about 0.15 to 4.0 grams/cubic centimeter, even more preferably from about 0.17 to 1 gram/cubic centimeter, even more preferably from about 0.17 0.75 gram/cubic centimeter, and most preferably from about 0.2 to 0.75 gram/cubic centimeter.

Powders of the invention will possess a wide range of specific bioactivity values, depending upon the type of FSP (e.g., impure FSP mixtures containing FHP and other proteins versus highly purified forms of FSP) contained in the powder. Powders of the invention will generally possess a specific bioactivity greater than 100 IU per gram of powder. In most instances, the specific bioactivity of the FSP powder is: greater than 250 IU per gram of powder, greater than 500 IU per gram of powder, greater than 1,000 IU per gram of powder, greater than 2,000 IU per gram of powder, greater than 5,000 IU per gram of powder, greater than 10,000 IU per gram of powder, greater than 25,000 IU per gram of powder, greater than 50,000 IU per gram of powder, greater than 100,000 IU per gram of powder, greater than 250,000 IU per gram of powder, greater than 500,000 IU per gram of powder, greater than 1,000,000 per gram of powder, greater than 2,500,000 IU per gram of powder, greater than 5,000,000 IU per gram of powder, or greater than 10,000,000 IU per gram of powder.

An additional measure for characterizing the overall aerosol performance of a dry powder is the aerosol performance coefficient (APC). The APC value for FSP powders as described herein is greater than 0.10, typically greater than 0.15, and more preferably greater than 0.25. As can be seen from the APC values provided in Tables 8 and 14, FSP dry powders have been prepared which are particularly well suited for pulmonary delivery, as evidenced by APC values greater than 0.25. Such powders contain a large proportion of small aerosol particle sizes and are thus extremely effective when delivered aerosolized form, in (i) reaching the alveolar region of the lung, followed by (ii) diffusion to the interstitium and (iii) subsequent passage into the bloodstream through the endothelium.

V. Pulmonary Administration of FSP Compositions

The FSP dry powder 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, Oct. 17, 1995; Smith, A. E., et al., U.S. Pat. No. 5,740,794, Apr. 21, 1998; and in Smith, A. E., et. al., U.S. Pat. No. 5,785,049, Jul. 28, 1998, herein incorporated by reference. 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 (i.e., unit dose packages) with metered doses of dry powder medicament are described, e.g., in Parks, D. J., et al., International Patent Publication WO 97/41031, Nov. 6, 1997, incorporated herein by reference.

Also suitable for delivering the FSP 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, Sep. 23, 1974, and in Cocozza, S., et al., U.S. Pat. No. 4,013,075, Mar. 22, 1977, incorporated herein by reference, wherein a pre-measured dose of FSP dry powder for delivery to a subject is contained within a hard gelatin capsule.

Other dry powder dispersion devices for pulmonary administration of FSP dry powders include those described, for example, in Newell, R. E., et al. European Patent No. EP 129985, Sep. 7, 1988); in Hodson, P. D., et al., European Patent No. EP472598, Jul. 3, 1996; in Cocozza, S., et al., European Patent No. EP 467172, Apr. 6, 1994, and in Lloyd, L. J. et al., U.S. Pat. No. 5,522,385, Jun. 4, 1996, incorporated herein by reference. Also suitable for delivering the FSP 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. 4,668,218, May 26, 1987; in Wetterlin, K., et al. U.S. Pat. No. 4,667,668, May 26, 1987; and in Wetterlin, K., et al., U.S. Pat. No. 4,805,811, Feb. 21, 1989, all of which are incorporated herein by reference. Other suitable devices include dry powder inhalers such as 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, Sep. 30, 1997, incorporated herein by reference.

The FSP 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, Jun. 14, 1994, and in Rubsamen, R. M., et al, U.S. Pat. No. 5,672,581 (1994), both incorporated herein by reference. When administered by a metered dose inhaler, the FSP composition is preferably absent a surfactant (e.g., fatty acids, bile salts, phospholipids, or alkyl saccharides) for enhancing the systemic absorption of FSP, since, when administering the compositions of the invention, such compounds are unnecessary for achieving therapeutic levels of FSP in the bloodstream.

Alternatively, the powders described herein may be dissolved or suspended in a solvent, e.g., water 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, the FSP dry powders are generally stored under ambient conditions, and preferably are stored at a temperature at or below about 25.degree. C., and relative humidity (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 desiccating agent in the secondary packaging of the dosage form. The FSP powders of the invention demonstrate no significant loss of bioactivity upon storage; moreover, accelerated stability studies carried out on three representative dry powder formulations (Example 7.C., Table 9, and FIG. 1) illustrate the ability to prepare respirable FSP dry powders characterized not only by good aerosol performance, but having good stability, as well. Moreover, both preliminary in vitro results for intratracheally administered FSP solutions in rats (Example 9) and pulmonary administration of FSP powders to monkeys (Examples 10 and 17) revealed reasonably high bioavailability values, i.e., from about 5% to 20% and from 18% to 26%, relative to administered subcutaneously FSP, further indicating the operability of treating infertility by pulmonary administration of dry powders of the present invention.

VI. Therapeutic Applications

The FSP dry powder of the invention is useful, when administered by inhalation for deposition in and absorption from the lung in a therapeutically effective amount, for treating infertility in both male and female subjects. More specifically, the methods of the present invention are particularly useful in therapeutic applications for the treatment of patients who are deficient in, or could otherwise benefit from, levels of FSP that are augmented over those produced endogenously.

When inhaled into the deep lung in dry powder form, FSP is effective to stimulate ovarian follicular growth in women who are not suffering from primary ovarian failure. Following pulmonary delivery of FSP, measurement of plasma inhibin levels can be used to provide a pharmacodynamic marker of FSP activity. Levels of follicular growth (an increase in the number of follicles greater than about 10 mm in diameter, (e.g., determined by ultrasound) and estradiol secretion (i.e., serum estradiol levels) can also be used to assess the effects of FSP; in males, Serotoli cell production levels can also provide an additional indicator of the efficacy of treatment. Preferably, pulmonary delivery of an FSP dry powder formulation as described herein is effective to result in a level of one or more of the above markers (follicular growth, serum estradiol, inhibin, Serotoli cell production) that is increased relative to its baseline level measured prior to FSP treatment.

The FSP powders of the invention are also useful, when administered to the deep lung, for Assisted Reproductive Technologies (ART). In such instances, FSP is administered to ovulatory infertile females undergoing stimulation of multiple follicular development for In Vitro Fertilization (IVF), Embryo Transfer (ET), and other assisted reproductive technologies. Generally, for use in female infertility-related conditions, FSP is administered to the deep lung for one or more treatment cycles of a period of from about 7 to 21 days or more to stimulate follicular growth. In the case of infertile females, in order to effect final maturation of the follicle and ovulation in the absence of a LH (luteinizing hormone) surge, human chorionic gonadotropin (hCG) is administered after sufficient follicle development has occurred (as described above). When used for treating male infertility, the FSP dry powder is typically administered for at least about six months, and typically for at least about a year.

Typically, treatment of the above-described conditions is effected by administering therapeutically effective doses of FSP dry powder that, on average, range from at least about 0.5 to 35 micrograms FSP/kilogram of patient daily, and preferably at least about 1 to 20 micrograms FSP/kilogram of patient daily, depending upon the specific activity of FSP contained in the composition. Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. For example, the amount of FSP administered per unit dosage form will generally range from about 5 IU to about 12,000 IU FSP, preferably from 5 IU to about 1000 IU FSP more preferably from about 37 IU to 500 IU, and even more preferably from about 50 IU to 300 IU FSP. Preferably, a therapeutically effective amount will range, on average, from about: 10 to 1000 IU FSP per day, 50 to 3,000 IU FSP per day, 75 IU to about 600 IU of FSP per day, or from about 200 IU to 12,000 IU FSP per day, depending upon the dosing regimen followed by the subject (i.e., the number of aerosolized doses delivered over a period of 24-hours or greater) and the type of treatment. In some instances, to achieve the desired therapeutic amount, it may be necessary to provide for daily repeated administration, i.e., repeated individual inhalations of a particular metered dose per day, where the individual administrations are repeated until the desired daily dose is achieved.
 

Claim 1 of 68 Claims

1. A stabilized dry powder composition for delivery to the deep lung of a mammalian subject, comprising: (i) a pharmacologically effective amount of follicle-stimulating hormone (FSH), FSH glycoform, or mixture thereof, wherein the FSH, FSH glycoform, or mixture thereof is selected from the group consisting of mammalian urinary-derived FSH, FSH glycoform, or mixture thereof and recombinantly derived FSH, FSH glycoform, or mixture thereof; and (ii) a pharmaceutically acceptable excipient, wherein the composition comprises particles having a bulk density from 0.1 to 10 grams per cubic centimeter, wherein the composition possesses a specific activity of at least 50 IU/mg FSH, wherein the composition comprises a solid state matrix that imparts a stabilizing environment for the FSH, FSH glycoform, or mixture thereof, wherein the solid state matrix is crystalline, an amorphous glass, or a mixture thereof, and wherein the composition maintains at least about 70% of its initial bioactivity when stored for one month at room temperature under ambient conditions.

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