WO2002030451A1 - Compositions and methods for reducing gnrh-induced bone loss - Google Patents

Abstract

Compositions, and methods for administering such compositions, are provided to reduce GnRH-induced bone loss. The compositions are a pharmaceutically acceptable formulation comprising a therapeutically effective amount of GnRH and a bone growth promoting agent. The GnRH and bone growth promoting agent are typically formulated with a pharmaceutically acceptable carrier and administered in an amount sufficient to treat, to prevent, or to reduce the symptoms of, a sex steroid hormone-responsive condition in a patient. The GnRH and bone growth promoting agent can be administered prophylactically or to treat existing sex steroid hormone-responsive conditions in patients by a variety of administration modes, including intramuscular, intravenous, intranasal, intrapulmonary, subcutaneous, parenteral, oral, transmucosal or transdermal delivery modes.

Description

COMPOSITIONS AND METHODS FOR REDUCING GπRH-INDUCED BONE LOSS

BACKGROUND OF THE INVENTION

Gonadotropin-releasing hormone ("GnRH"), also referred to as luteinizing hormone releasing hormone ("LHRH"), is a ten amino acid peptide that is produced and secreted by the hypothalamus. The amino acid sequence of the GnRH peptide is conserved in mammals and is believed to function in a similar fashion' in both males and females. The major pharmacologic role of GnRH is to control the release of gonadotropins. In particular, GnRH binds to specific receptors on pituitary cells and causes the release of gonadotropins. Subsequently, the gonadotropins exert their ^" "^" pharmacologic activity on the gonads by controlling gametogenesis and by regulating the production of the sex steroid hormones (e.g., estrogens, testosterones and progestins). Under normal physiological conditions, the hypothalamus produces GnRH in rhythmic pulses. If GnRH is present at a constant, high level for an extended period of time, however, the cells of the pituitary become desensitized and cease producing gonadotrophins. The cessation of gonadotrophin release causes the cessation of gametogenesis and the cessation of production of the sex steroid hormones. Thus, the constant presence of GnRH affects not only the reproductive potential, but other biological activities linked to the action of the sex steroid hormones.

The administration of GnRH is useful for treating a variety of sex steroid hormone-responsive conditions. Because GnRH administration causes the cessation of sex steroid production, continuous or prolonged administration of GnRH is useful for treatment of a sex steroid hormone-responsive condition, in which the condition requires, or is stimulated by, a sex steroid hormone. For example, in women a reduction of estrogen levels is beneficial in the treatment of endometriosis, uterine fibroids, dysfunctional uterine bleeding, and the like. In men, a reduction of testosterone levels is beneficial in the treatment of prostatic carcinoma, benign prostatic hypertrophy, and the like.

One of the more common sex steroid hormone-responsive conditions is endometriosis, which affects a significant number of women of childbearing age. Endometriosis is a condition of severe dysmenorrhea. In some patients, endometriosis is accompanied by severe pain, bleeding into the endometrial masses or the peritoneal cavity, and often leads to infertility. Endometriosis is commonly associated with ectopic growth of endometrial tissues. Endometrial growths are located in inappropriate tissues, which initiates local inflammatory-like responses causing macrophage infiltration and a cascade of events leading to initiation of a painful response.

Endometriosis and other sex steroid hormone-responsive conditions can be treated by the administration of GnRH. GnRH agonists, which mimic or enhance the effects of GnRH, can also be used. Such GnRH agonists include buserelin, leuprolide, histrelin, nafarelin, goserelin, triptorelin and their physiologically acceptable salts. Like GnRH, prolonged administration of these agonists induces decreased levels of sex steroid hormones. For example, sustained and prolonged administration of GnRH agonists induces hypoestrogenemia in women. (See, e.g., Dawood, Acta Obstet. Gvnecol. Scand. 159:22-34, 1994.)

Despite the potentially beneficial effects of GnRH and GnRH agonists, . their administration can cause severe, detrimental side effects, including osteoporosis. In particular, administration of GnRH agonists can cause trabecular bone loss, particularly in the vertebrae and hip bones. (See, e.g., Dawood, supra.) Such bone loss can be detected as early as three months after the start of treatment, and can take six to twelve months to recover after cessation of treatment. (See, e.g., Dawood, supra.) Such bone loss can result in increased bone fragility and/or a greater susceptibility to osteoporosis in later life.

Thus, there is an urgent need in the art for additional tools and methods to treat sex steroid hormone-responsive conditions, such as endometriosis, that prevent or alleviate the adverse side effects associated with sex steroid therapy, particularly bone loss associated with GnRH and GnRH agonist treatments.

SUMMARY OF THE INVENTION

The instant invention provides pharmaceutically acceptable compositions and methods for reducing bone loss associated with the administration of GnRH or a GnRH agonist (hereafter also referred to collectively as "GnRH"). The compositions typically include GnRH in combination with one or more agent(s) that promotes bone deposition (e.g., by promoting an increase in bone mass), or inhibits bone loss (e.g., by inhibiting bone resorption by osteoclasts), in a patient. These agents are collectively referred to herein as "bone growth promoting agents". Alternatively, the GnRH and bone growth promoting agent(s) are administered separately to the patient. The GnRH can be GnRH or a GnRH agonist such as, for example, buserelin, leuprolide, nafarelin, goserelin, histrelin or triptorelin, as well as analogs and pharmaceutically acceptable salts of any of these. The bone growth promoting agent can be a calcitonin, such as for example, human calcitonin, salmon calcitonin, hog calcitonin, chick calcitonin, cattle calcitonin, or eel calcitonin, or an androgen, such as for example, danazol or a structurally related androgen compound. In other embodiments, the bone growth promoting agent is a bisphosphonate that inhibits bone loss, such as for example, a nitrogen-containing bisphosphonate (e.g., risedronate, zoledronate, ibandronate, aldendronate, or pamidronate); a non-nitrogen- containing biphosphonate; an alpha v beta antagonist; an inhibitor of osteoclastic V-H(+) ATPase (e.g., SB 242784); an inhibitor of pp60 (src) (e.g., AP22408); a chemically modified tetracycline (e.g., doxycycline and CMT-3); the peptide amylin; and the like. The compositions are useful for treating a variety of sex steroid hormone-responsive conditions, such as endometriosis, uterine fibroids, dysfunctional uterine bleeding, uterine leiomyomas, mastalgia, prostatic carcinoma, benign hypertrophy, and the like. The bone growth promoting agent reduces boss loss caused by GnRH administration.

In one aspect, the GnRH and the bone growth promoting agent are administered in a pharmaceutically acceptable carrier in an effective amount to treat a sex steroid hormone-responsive condition (e.g., endometriosis), to alleviate one or more of the symptoms of the condition, or to inhibit proliferation of a sex steroid hormone- responsive cell or tissue (e.g., ectopic endometrial tissue) in a mammalian patient. Suitable pharmaceutically acceptable carriers include those useful for intramuscular, intravenous, intranasal, intrapulmonary, subcutaneous, parenteral, oral, transmucosal, and transdermal delivery. In preferred embodiments, compositions are formulated for administration to a mucosal surface of the patient (e.g., via intrapulmonary or intranasal delivery). In more detailed aspects according to the present invention, the compositions are suitable for intranasal delivery.

In another aspect, methods are provided for administering compositions comprising a therapeutically effective amount of a GnRH and a bone growth promoting agent to a patient suffering from a sex-steroid hormone-responsive condition, such as for example endometriosis, uterine fibroids, dysfunctional uterine bleeding, uterine leiomyomas, mastalgia, prostatic carcinoma, benign hypertrophy, and the like. Alternatively, separate compositions comprising GnRH and the bone growth promoting agent are administered to the patient. The compositions are administered to a patient by a variety of administration modes, including intramuscular, intravenous, intranasal, intrapulmonary, subcutaneous, parenteral, oral, transmucosal, or transdermal delivery methods. In preferred embodiments, the compositions are administered to a mucosal surface of the patient (e.g., via intrapulmonary or intranasal delivery).

In another aspect, methods are provided for prophylaxis by administering a composition comprising a therapeutically effective amount of GnRH and the bone growth promoting agent to a patient to prevent the onset, or the recurrence, of a sex steroid hormone-responsive condition, such as for example endometriosis, uterine fibroids, dysfunctional uterine bleeding, uterine leiomyomas, mastalgia, prostatic carcinoma, benign hypertrophy, and the like. In one embodiment, the patient has an increased risk for developing, or suffering a recurrence of, the sex steroid hormone-responsive condition. The composition is administered to a patient by a variety of administration modes, including intramuscular, intravenous, intranasal, intrapulmonary, subcutaneous, parenteral, oral, transmucosal, or transdermal delivery methods. In preferred embodiments, the composition comprising GnRH and a bone growth promoting agent, such as calcitonin, is administered to a mucosal surface of the patient (e.g., via intrapulmonary or intranasal delivery).

For prophylactic and treatment purposes, GnRH and the bone growth promoting agent can be administered to the patient in a single bolus injection delivery protocol, via continuous IV or transdermal delivery over an extended time period, or in a repeated administration protocol (e.g., on a daily or weekly basis). In preferred embodiments, GnRH and the bone growth promoting agent are self-administered by the patient daily as an intranasal spray or powder formulation. The various dosages and delivery protocols thus contemplated for administration of GnRH and the bone growth promoting agent are therapeutically effective to treat or prevent a sex steroid hormone- responsive condition (e.g., endometriosis), to alleviate one or more symptoms of such a condition, or to inhibit the proliferation of sex steroid hormone-responsive cells or tissue (e.g., ectopic endometrial tissue). Intranasal formulations are provided as aqueous solutions or suspensions or powders for intranasal delivery as an aerosol or propelled particulate bolus to facilitate administration and enhance delivery. Various excipients, stabilizers, absorption enhancers and other additives are optionally provided to optimize shelf-life, delivery and efficacy and to minimize irritation and other adverse side effects. In yet additional aspects of the invention, GnRH and the bone growth promoting agent are administered according to the foregoing methods in a coordinate treatment or prophylaxis protocol with another therapeutic agent. In one embodiment, a composition comprising GnRH and the bone growth promoting agent are administered cpordinately with a secondary therapeutic agent to prevent or treat the sex steroid hormone-responsive condition in a patient suffering from, or at risk of developing, the condition. The composition is administered as above, simultaneously or sequentially with administration of the therapeutic agent.

DEFINITIONS

Prior to setting forth the invention in more detail, it may be helpful to a further understanding thereof to set forth definitions of certain terms used herein.

The term "GnRH" refers to gonadotropin releasing hormone ("GnRH"), and its pharmaceutically acceptable salts. GnRH can be from a naturally-occurring source, including human cells, hogs, chickens, cattle, and the like, or it can be chemically synthesized. The term "GnRH" further includes GnRH agonists, such as derivatives of GnRH that stimulate the release of gonadotropins. Such derivatives of GnRH include those in which one or more of the amino acids of GnRH is altered by substitution, addition, deletion or chemical modification (e.g. , addition of acetyl, benzyl, naphthyl, or alkyl groups, conjugation to a carrier or a fusion protein, PEGylation, and the like) to provide a biologically active molecule. For example, in some embodiments, one or more amino acid residues within the sequence can be substituted by another amino acid, typically by another amino acid having a similar polarity, hydrophobicity or hydrophilicity, which acts as a functional equivalent of the substituted amino acid.

Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, following six groups each contain amino acids that can be considered functionally equivalent to one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins. W. H. Freeman and Company, 1984, incorporated herein by reference.)

The term "calcitonin," also known as thyrocalcitonin, refers to a peptide hormone that increases the deposition of calcium and phosphate in bone. Calcitonin can be produced by the parathyroid, thyroid and thymus glands. The term "calcitonin" further refers to derivatives of calcitonin, such as those having substitution, addition or deletion of one or more amino acids to provide a biologically active molecule. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, hydrophobicity or hydrophilicity, which acts as a functional equivalent (See supra). Calcitonin can also be chemically modified (e.g., addition of acetyl, benzyl, naphthyl, or alkyl, groups, conjugation to a carrier or a fusion protein, PEGylation, and the like). The term "bone growth promoting agent" refers to a compound that reduces bone loss, or promotes additional bone formation, through the deposition of calcium and/or phosphate. "Bone growth promoting agents" include calcitonin and androgens, such as danazol that promote bone formation, e.g., increased bone mass. Inhibitors of bone loss include, for example, bisphosphonates, and other agents that impair natural bone loss or bone loss stimulated by hormonal treatment and other factors, for example by inhibiting, destroying or impairing the function of osteoclasts that mediate bone resorption.

The terms "biologically active" and "functionally active," in the context of GnRH (including an agonist thereof), refer to the ability of GnRH to stimulate the release of gonadotropins. In this regard, biological activity of GnRH according to the methods of the invention may be reflected by an ability of a GnRH to bind to GnRH receptor and/or induce a response in a target cell or tissue, or to alleviate symptoms of a sex steroid- responsive disease or condition in a subject. The terms "biologically active" and "functionally active" in the context of a bone growth promoting agent generally refer to the ability of the agent to modulate bone formation, such as by promoting the deposition of calcium and/or phosphate, reducing bone loss or promoting bone gain.

The terms "sex steroid" and "sex steroid hormone" refer to estrogens, testosterones and progestins.

The terms "sex steroid hormone-responsive condition" or "hormone- responsive condition" refer to a disease or condition that is dependent on, or stimulated by, a sex steroid, and which is treatable or preventable by administration of GnRH.

The terms "hormone-responsive cell" or "hormone-responsive tissue" refer to a cell or tissue having a proliferative activity that is dependent on, or stimulated by, a sex steroid. The terms "proliferation" and "proliferative activities" refer to cellular activities such as the division (i.e., mitosis), growth and/or migration of hormone- responsive cells or tissue that are associated with a hormone-responsive condition.

The term "endometriosis" refers to a disease or condition typically characterized by the ectopic presence of endometrial tissue in a patient. Ectopic endometrial tissue can appear as adhesions in the peritoneum, in the retroperitoneum, as intra-ovarian lesions, and the like.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In a preferred embodiment according to the present invention, compositions are provided which comprise GnRH and a bone growth promoting agent for the therapeutic or prophylactic treatment of a sex steroid hormone-responsive condition, _. such as endometriosis, uterine fibroids, dysfunctional uterine bleeding, uterine leiomyomas, mastalgia, prostatic carcinoma, benign prostatic hypertrophy, and the like.

The general structure of GnRH is depicted in formula I, below, where the amino acids are in L-configurations.

5-oxo-prolyl-histidyl-tryptophyl-seryl-tyrosyl-glycyl-leucyl-arginyl-prolyl-glycyl-NH₂

Further included are pharmaceutically acceptable salts of GnRH, such as, for example, acid or base addition salts that are known to be non-toxic and are commonly used in the pharmaceutical literature. Commonly used acid addition salts are inorganic salts formed by the addition of sulfuric acid, nitric acid, hydrochloric acid, hydrobromic acid phosphoric acid, phosphorous acid, and the like; or organic salts formed by the addition of acetic acid, formic acid, benzoic acid, citric acid, methanesulfonic acid, and the like. Commonly used basic addition salts are the salts formed by alkali or alkaline earth hydroxides, ammonium hydroxide, alkyl or aromatic amines, and the like. In one embodiment, the salt is acetate.

According to the present invention, GnRH further includes GnRH agonists that are useful for treating hormone-responsive conditions. GnRH agonists are typically of the following formula II, below, where the amino acids are their natural L- configuration, except as noted. 5-oxo-prolyl-histidyl-tryptophyl-seryl-tyrosyl-X-leucyl-arginyl-prolyl-glycyl-NH₂-Y

These GnRH agonists include, but are not limited to: Leuprolide (LUPRON™), wherein X is D-leucine and Y is -CH₂CH₃, as well as the acetate salt of leuprolide (see, e.g., U.S. Patent. Nos. 4,005,063; 4,005,194; 4,652,441; 4,677,191; 4,728,721; and 4,849,228, each of which is incorporated by reference herein);

Histrelin (SUPPRELIN™), wherein X is D-(N-benzyl)-His and Y is - CH₂CH₃, as well as the acetate salt of histrelin (see, e.g., U.S. Patent No. 4,224,946, which is incorporated by reference herein);

Nafarelin (SYNAREL™), wherein X is D-3-(2-naphthyl)-Ala and Y is_.... hydrogen, as well as the acetate salt of nafarelin (see, e.g., U.S. Patent No. 4,244,946, which is incorporated by reference herein); Triptorelin, wherein X is D-Trp and Y is hydrogen;

Buserelin, wherein X is D-(O-t-butyl)Ser and Y is -CH₂CH₃; and Goserelin (ZOLADEX™), wherein X is O-(t-butyl)-serine and the tenth amino acid of formula II (glycine) is replaced with azgly-NH , as well as the acetate salt of goserelin. Other biologically active GnRH agonists are also within the scope of the invention. Such GnRH agonists can be synthetically prepared, such as, for example, by manual peptide synthesis (see, e.g., HunkapiUer et α/., Nature 310:105-111, 1984; Stewart and Young, Solid Phase Peptide Synthesis, 2^nd Ed., Pierce Chemical Co., Rockford, IL, 1984), or by automated peptide synthesis (e.g., using a synthesizer manufactured by Applied BioSystems (Foster City, CA)).

Compositions according to the present invention further comprises a bone growth promoting agent. In one embodiment, the bone growth promoting agent is a calcitonin. Examples of suitable calcitonins include, for example, human calcitonin, salmon calcitonin, hog calcitonin, chick calcitonin, cattle calcitonin, eel calcitonin, and the like. The calcitonin can comprise one or more of these calcitonins. Calcitonin can be isolated from a natural source, such as a human cells, salmon, hogs, chickens, cattle, and the like, or can be synthetically prepared, such as, for example, by manual peptide synthesis (see, e.g. , HunkapiUer, supra); Stewart and Young, supra), or automated peptide synthesis (e.g. , manufactured by Applied BioSystems (Foster City, CA)).

In another embodiment, the bone growth promoting agent is an androgen, such as danazol ((17α)-pregna-2,4-dien-20-ynol[2,3-d]isoxazol-17-ol), and structurally related androgen compounds.

In other embodiments, the bone growth promoting agent is a bisphosphonate, such as for example, a nitrogen-containing bisphosphonate such as risedronate, zoledronate, ibandronate, aldendronate, or pamidronate (see, e.g., Coxon et al, J. Bone Miner. Res.15:1467-1476, 2000); a non-nitrogen-containing biphosphonate (see, e.g., Rogers et al, Cancer 11:2961-2978, 2000); alpha v beta antagonists (see, e.g., Carron et al. , J. Endocrinol. 165:587-598, 2000); inhibitors of osteoclastic V-H(+) ATPase (e.g., SB 242784 (Visentin et al, J. Clin. Invest. 106:309-318, 2000)); inhibitors of pp60 (src) (e.g., AP22408); chemically modified tetracyclines (e.g., doxycycline and CMT-3 (Bettany et al. , Bone 27:75-80, 2000)); the peptide amylin (Horcajada et al , I . Endocrinol. 165:663-668, 2000); and the like (the disclosures of which are incorporated by reference herein.

The GnRH and bone growth promoting agent are administered in a therapeutically effective amount. As used herein, a "therapeutically effective amount" of GnRH is an amount of the compound which, depending on the selected mode, frequency and duration of administration, effectively treats a hormone-responsive condition (e.g. , endometriosis), inhibits the occurrence or recurrence of the hormone-responsive condition in the patient, or alleviates one or more symptoms of an existing hormone-responsive condition in the patient. Therapeutically effective dosages for patients presenting with existing hormone-responsive conditions can be determined in the patient or using in vitro or in vivo animal or human treatment models and measuring the amount (reflective of dose, administration mode and frequency) of GnRH required to significantly inhibit the proliferation of hormone-responsive cells or tissue (e.g., ectopic endometrial cell explants). The time to pharmacokinetic and clinical steady state following GnRH administration is readily determined depending on the mode, frequency and duration of administration (e.g., bolus dosage, repeat administration, or steady infusion). It is therefore a routine matter to determine an appropriate concentration and dose of GnRH to administer an effective amount (e.g., intranasally effective, transdermally effective, intravenously effective, or intramuscularly effective) to elicit a desired response. Effective amounts of the GnRH for prophylaxis (i.e., to inhibit the occurrence or recurrence of a hormone-responsive condition (e.g., endometriosis) in a patient) may be administered over a prolonged course of preventive therapy to a patient at risk of developing the hormone-responsive condition. Determination of effective dosages in this case is typically based on animal model studies followed up by human clinical trials and is approximated by determining effective dosages that significantly reduce the occurrence or incidence of the hormone-responsive condition in model patients and administration protocols.

The actual dosage of GnRH will vary according to factors such as the particular hormone-responsive condition, prognosis of that condition, age, the weight of the patient, and the ability of GnRH to elicit a desired response in the patient. Dosage regimens may be adjusted to provide an optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the GnRH are outweighed by the therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of GnRH is about 0.001 μg/kg to about 10 mg/kg, typically between about 10 μg to about 1500 μg per day, although greater and lesser amounts are within the scope of the invention. In a specific embodiment, a non-limiting range is about 50 to about 1000 μg per day for nasal administration.

A therapeutically effective amount of the bone growth promoting agent means an amount effective to reduce bone loss, or to increase the deposition of calcium and/or phosphorus as new bone growth, in a patient receiving the administration of GnRH. Therapeutically effective amounts of the bone growth promoting agent administered to a patient will depend on the condition being treated, the particular GnRH administered, and the formulation being used. In one embodiment, about 5 to about 500 IU daily dosage of the bone growth promoting agent calcitonin are administered, although greater and lesser amounts are within the scope of the invention. In another embodiment, from about 10 to about 1000 mg per day of an androgenic bone growth promoting agent (e.g., danazol) are administered, although greater and lesser amounts are within the scope of the invention. The bone growth promoting agent typically reduces GnRH-induced bone loss by at least 5 percent, more typically at least 15 percent, and preferably at least 25 percent, or more.

The administration of GnRH and bone growth promoting agent can be achieved by single or multiple dosages (e.g., multiple administrations per day, daily or weekly administrations). Per administration, it is desirable to administer at least about one quarter of the desired daily dosage. It is to be further noted that dosage values can vary with the severity of the condition to be alleviated. In addition, for any particular patient, specific dosage regimens should be adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of GnRH and the bone growth promoting agent. As will be appreciated by the skilled artisan, the dosage may also be adjusted based on the release rate of the administered formulation (e.g., nasal spray versus powder, sustained release oral or injected particles, transdermal formulations, and the like). The GnRH and bone growth promoting agent are typically formulated with a pharmaceutically acceptable carrier. For use according to the present invention, GnRH and/or the bone growth promoting agent are provided for administration by, for example, intranasal, intrapulmonary, intramuscular, intravenous, subcutaneous, parental, oral, transmucosal or transdermal modes. The pharmaceutically acceptable carrier can be a biologically suitable liquid or solid carrier. GnRH and the bone growth promoting agent are typically provided in substantially pure form and can be obtained from a variety of commercial providers in the United States and elsewhere.

Therapeutic compositions can be formulated as a solution, microemulsion, liposome, or other composition suitable to delivery of high concentration of the active compounds (i.e., GnRH and/or bone growth promoting agent). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g. , glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, poly alcohols (e.g., mannitol or sorbitol), or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. In various embodiments according to the present invention, the GnRH and the bone growth promoting agent are formulated with a pharmaceutically acceptable carrier. Such a pharmaceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, viscogens, preservatives, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and other excipients or additives. Pharmaceutically acceptable carriers further include those described in, for example, Remington's Pharmaceutical Sciences, by E.W. Martin, which is incorporated by reference herein. Suitable preservatives include, for example, sodium benzoate, quaternary ammonium salts, sodium azide, methyl paraben, propyl paraben, sorbic acid, ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, dehydroacetic acid, ethylenediamine, monothioglycerol, potassium benzoate, potassium metabisulfite, potassium sorbate, sodium bisulfite, sulfur dioxide, and organic mercurial salts. Suitable viscogens include, for example, carboxymethylcellulose, sorbitol, dextrose, and polyethylene glycols. In various embodiments, the carrier is suitable for intranasal, intrapulmonary, intravenous, intramuscular, subcutaneous, parenteral, oral, transmucosal or transdermal modes of administration. Depending on the route of administration, the GnRH and bone growth promoting agent can be coated in a material to protect the active compounds (i.e., GnRH and bone growth promoting agent) from the action of acids and other natural conditions which may inactivate those compounds.

In certain embodiments of the invention, the active compounds (i.e., GnRH and bone growth promoting agent) are administered in a time release formulation, for example in a composition which includes a slow release polymer, or by depot injection. The active compounds can be prepared with carriers that protect the active compounds against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, and polyglycolic copolymers ("PLG"). Many methods for preparing such formulations are generally known to those skilled in the art. (See, e.g. , Sustained and Controlled Release Drug Delivery Systems. J. R. Robinson (ed.), Marcel Dekker, Inc., New York, 1978, which is incorporated herein by reference). Such formulations include controlled-release compositions such as are known in the art for the administration of leuprolide (trade name: LUPRON^™), such as, for example, microcapsules (see, e.g., U.S. Patent Nos. 4,652,441 and 4,917,893, each incorporated herein by reference), injectable formulations (see, e.g., U.S. Patent No. 4,849,228, incorporated herein by reference), lactic acid- glycolic acid copolymers useful in making microcapsules or injectable formulations (see, e.g., U.S. Patent Nos. 4,677,191 and 4,728,721, each incorporated herein by reference), and sustained-release compositions for water-soluble peptides (see, e.g., U.S. Patent No. 4,675,189, incorporated herein by reference). A long-term sustained release implant also can be used. These can be readily constructed to deliver therapeutic levels of GnRH and the bone growth promoting agent for at least 30 days, preferably 60 days or longer. Long-term sustained release implants are well known to those of ordinary skill in the art and can incorporate some of the absorption delaying components described above. Such implants can be particularly useful in treating sex steroid hormone-responsive conditions by placing the implant near or directly within the site of the hormone-responsive cells or tissue or proximal to the site of pain experienced by the patient, thereby effecting localized, high-doses of the active compounds at one or more sites of interest.

In alternate embodiments, the active compounds (i.e., GnRH and the bone growth promoting agent) are orally or rectally administered for treatment or prophylaxis of the hormone-responsive condition. For example, the active compounds can be administered, for example, with an inert diluent or an assimilable carrier. The active compounds can thus be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the patient's diet (for oral compositions). For oral therapeutic administration, the active compounds can be administered with excipients and used in the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the active compounds in these compositions and preparations can be varied. The amounts of the active compounds in such therapeutic composition are such that a suitable dosage will be delivered to the patient.

For oral or rectal administration, the active compounds (i.e., GnRH and bone growth promoting agent) can be worked into tablets or dragee cores by being mixed with solid, pulverulent carrier substances, such as sodium citrate, calcium carbonate or dicalcium phosphate, and binders such as polyvinyl pyrrolidone, gelatin or cellulose derivatives, and can optionally include lubricants such as magnesium stearate, sodium lauryl sulfate, "Carbowax" or polyethylene glycol. Taste-improving substances can optionally be added in the case of oral administration forms. Solid delivery vehicles can contain the active compounds in mixture with fillers, such as lactose, saccharose, mannitol, starches, such as potato starch or amylopectin, cellulose derivatives or highly dispersed silicic acids. In soft-gelatin capsules, the active compounds are typically dissolved or suspended in suitable liquids, such as vegetable oils or liquid polyethylene glycols. As further orally-administrable forms, one can use plug capsules, for example, of hard gelatin, as well as dosed soft-gelatin capsules comprising a softener or plasticizer (e.g., glycerin).

Alternatively, liquid dosage forms for delivering the active compounds (i.e., GnRH and bone growth promoting agent) to mucosal surfaces include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms can contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms would also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

To administer the active compounds in a stable form according to the methods of the invention, it may be necessary to coat the active compounds with, or co- administer the compounds with, a material to prevent its inactivation. In this context, the active compounds can be administered to a patient in an appropriate carrier (e.g., liposomes or a diluent). Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Suitable liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (see, e.g. , Strejan et al. , J. Neuroimmunol. 7:27-41 , 1984, which is incorporated herein by reference). Suitable pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable or aerosol solutions or dispersible powder formulations. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compounds or yields unacceptable toxic or other adverse side effects, use thereof in the pharmaceutical compositions of the invention is contemplated. Secondary therapeutic compounds, including hormonal therapeutic and chemotherapeutic agents useful for the treatment or prevention of sex steroid hormone- responsive conditions, can also be incorporated into the compositions of the invention. (See infra.)

In more detailed aspects of the invention, the active compounds (i.e., GnRH and bone growth promoting agent) are stabilized to extend their effective half-life following delivery to the patient, particularly for extending metabolic persistence in an active state in the bloodstream, at a mucosal surface, or within a connective tissue compartment or fluid-filled body cavity. For this purpose, the active compounds can be chemically modified or chemical derivatization (e.g., by chemical conjugation, N- terminal capping, PEGylation, and the like), or by recombinant means (e.g., site-directed mutagenesis or construction of fusion proteins), or formulated with various stabilizing agents or carriers. Thus stabilized active compounds administered as above retain substantial activity (e.g., at least 20%, preferably 50%, 80%, or more activity) as compared to a comparable activity observed for an unmodified active compound (i.e., GnRH and/or bone growth promoting agent) for an extended period at the intended target site.

Numerous reports in the literature describe the potential advantages of PEGylated proteins, which include their increased resistance to proteolytic degradation, increased plasma half-life, increased solubility and decreased antigenicity and immunogenicity (see, e.g. , Nucci et al. , Advanced Drug Deliver Reviews 6:133-155, 1991 ; Lu et al. , Int. J. Peptide Protein Res. 43:127-138, 1994, each incorporated herein by reference). A number of proteins, including L-asparaginase, strepto-kinase, insulin, and interleukin-2 have been conjugated to a poly(ethyleneglycol) (PEG) and evaluated for their altered biochemical properties as therapeutics (see, e.g., Ho et al, Drug Metabolism and Disposition 14:349-352, 1986; Abuchowski et al, Prep. Biochem. 9:205-211, 1979; and Rajagopaian et al, J. Clin. Invest. 75:413-419, 1985, each incorporated herein by reference). Although the in vitro biological activities of PEGylated proteins can be decreased, this loss in activity is usually offset by the increased in vivo half-life in the bloodstream (see, e.g., Nucci et al. Advanced Drug Deliver Reviews 6:133-155. 1991, incorporated herein by reference). Several procedures have been reported for the attachment of PEG to proteins and peptides and their subsequent purification (see, e.g., Abuchowski et al, Biol. Chem. 252:3582-3586, 1977; Beauchamp et al. Anal. Biochem. 131:25-33. 1983, each incorporated herein by reference). Lu et al. (Int. J. Peptide Protein Res. 43:127-138, 1994) describe various technical considerations and compare PEGylation procedures for proteins versus peptides (see also Katre et al , Proc. Natl. Acad. Sci. USA 84:1487-1491, 1987; Becker et al. Makromol. Chem. Rapid Commun. 3:217-223, 1982; Mutter et al, Makromol. Chem. Rapid Commun. 13:151-157, 1992; Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1993: Lu et al. Peptide Res. 6:142-146. 1993: Lee et al. Bioconiugate Chem. 10:973-981. 1999: Nucci et al. Adv. Drug Deliv. Rev. 6:133-151. 1991; Francis et al. J. Drug Targeting 3:321-340, 1996: Zalipsky. Bioconiugate Chem. 6:150-165. 1995; Clark et al , J. Biol. Chem. 271 :21969-21977, 1996; Pettit et al. , J. Biol. Chem. 272:2312- 2318, 1997; Delgado et al. Br. J. Cancer 73:175-182. 1996; Benhar et al. Bioconiugate Chem. 5:321-326. 1994: Benhar et al. J. Biol. Chem. 269:13398-13404. 1994; Wang et al. Cancer Res. 53:4588-4594, 1993; Kinstler et al. , Pharm. Res. 13:996-1002. 1996; Filpula et al, EXP. Qpin. Ther. Patents 9:231-245, 1999; Pelegrin et al, Hum. Gene Ther. 9:2165-2175, 1998; each incorporated herein by reference). Following these and other teachings in the art, the conjugation of the active compounds with poly (ethylenegly col) polymers is readily undertaken with the expected result of prolonging circulating life and/or reducing immunogenicity while maintaining an acceptable level of activity of the PEGylated active compounds.

In addition to PEGylation, the active compounds can be modified to enhance circulating half-life by shielding the peptides via conjugation to other known protecting or stabilizing compounds, or by the creation of fusion proteins between the active compounds and proteins such as immunoglobulin chains. These modifications will decrease the degradation, sequestration or clearance of the active compounds and result in a longer half-life of the peptide (e.g., in the nasal sinus, lung, circulatory system, or synovium). The active compounds thus modified maintain activity for greater periods at . a target site compared to unmodified active compounds, and retain substantial biological activity for inhibiting the hormone-responsive condition and/or symptoms thereof, or for reducing bone loss, or increasing bone growth, when administered to a patient.

• The therapeutic compositions according to the invention typically are typically sterile and stable under the conditions of manufacture, storage and use. Sterile injectable solutions can be prepared by incorporating the active compounds (i.e., GnRH and bone growth promoting agent) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compounds into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolonged delivery of the active compounds in various compositions according to the present invention can be brought about by inclusion in the compositions of agents delaying absorption (e.g., aluminum mono stearate hydrogels and gelatin). In specific embodiments, when controlled release formulations of the active compounds (i.e., GnRH and bone growth promoting agent) are desired, controlled release binders suitable for use in accordance with the invention include any biocompatible controlled-release material which is inert to the active compounds and which is capable of incorporating the active compounds. Numerous such materials are known in the art. Preferred controlled-release binders are materials that are metabolized slowly under physiological conditions following their subcutaneous or intramuscular injection in mammals (e.g., in the presence of bodily fluids that exist there). Appropriate binders include but are not limited to biocompatible polymers and copolymers previously used in the art in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues (e.g., following subcutaneous or intramuscular injection), and do not trigger significant adverse effects such as immune response, inflammation, and the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body. For example, a polymeric matrix derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages can be used. A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity. Typically, such preferred polymers are polyglycolic acids ("PGA"), polylactic acids ("PLA"), poly(DL-lactic acid- co-glycolic acid)("DL PLGA"), poly(D-Iactic acid-coglycolic acid)("D PLGA") and poly(L-lactic acid-co-glycolic acid)("L PLGA"). The preferred ratio for lactic acid and glycolic acid polymers in poly(lactic acid-co-glycolic acid) is in the range of 100:0 (i. e. , pure polylactide) to 50:50. Other useful biodegradable or biodegradable polymers include but are not limited to such polymers as poly(epsilon-caprolactone), poly(epsilon- aprolactone-CO-lactic acid), poly(epsilon.-aprolactone-CO-glycolic acid), poly(β- hydroxy butyric acid), poly(alkyl-2-cyanoacrylate), hydrogels such as poly(hydroxyethyl methacrylate), poly amides, poly(amino acids) (e.g., L-leucine, glutamic acid, L-aspartic acid, and the like), poly (ester urea), poly (2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides and copolymers thereof. In certain embodiments of the invention, the active compounds are administered by topical delivery to a mucosal surface of the patient, preferably via intranasal delivery in the form of an aerosol spray or powder. According to this aspect of the invention, the active compounds are delivered in intranasally effective amounts, preferably in a selected volume of administered spray or powder, to achieve prophylaxis or treatment of a sex steroid hormone-responsive conditions. In related aspects of the invention, novel pharmaceutical compositions are provided for intranasal delivery that incorporate the active compounds in a powder or aqueous formulation for intranasal delivery. Intranasal administration of the active compounds is preferred for a variety of reasons. This method allows self-admimstration of treatment by patients, provided that sufficient safeguards are in place to control and monitor dosing and side effects. Nasal administration also overcomes certain drawbacks of other administration forms, such as injections that are painful and expose the patient to possible infections and may present drug bioavailability problems. Compositions according to the present invention may be administered in solution as a nasal spray and can be dispensed as a spray by a variety of methods known to those skilled in the art. Systems for intranasally dispensing liquids as a spray are well known (see, e.g., U.S. Patent No. 4,511,069, incorporated herein by reference). Preferred nasal spray solutions comprise the active compounds in a liquid carrier that optionally include a nonionic surfactant for enhancing absorption of the drug and one or more buffers or other additives to minimize nasal irritation. In addition, any of the enhancers and other excipients used to deliver peptides across absorptive mucosae can be included. (See, e.g., Sayani and Chien, Critical Reviews in Therapeutic Drug Carrier Systems 13:185-184, 1996, incorporated herein by reference). In some embodiments according to the present invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution is typically between pH 3.0 and 8.0.

For intranasal administration, compositions which improve the absorption of nasally administered active compounds (i.e., GnRH and bone growth promoting agent) and reduce nasal irritation, especially when used in a chronically administered treatment protocol, are desirable. In this context, the utilization of surface-active agents to enhance absorption of polypeptide therapeutics has been previously demonstrated. For example, Hirai and coworkers (Hirai et al, Int. J. Pharmaceutics 1:173-184, 1981; Great Britain Patent Publication 1 527 605, each incorporated herein by reference). However, nasal administration of drugs enhanced by surfactants can be accompanied by nasal irritation, including stinging, congestion and rhinorrhea. Thus, compositions which enhance absorption through the nasal mucosa with reduced irritation are desirable.

To achieve this goal, a combination of surfactants can be used. Nonionic surfactants such as nonoxynol-9, laureth-9, poloxamer-124, octoxynol-9 and lauramide DEA are particularly useful in the practice of the present invention. Nonoxynol-9 ("N- 9") is an ethoxylated alkyl phenol, the polyethyleneoxy condensate of nonylphenol with 9 moles of ethylene oxide. This surfactant has been used in detergent products and is sold under trade names, such as SURFONIC^® N-95 (Jefferson), NEUTRONYX^® 600 (Onyx) and IGEPAL^® CO-630 (GAF). N-9 is considered to be a hard detergent. N-9 has also been used as a spermatocide (See The Merck Index. 1 Oth Edition, Entry 6518). To minimize irritation attributed to employment of surfactants, one or more anti-irritant additives are included with the GnRH and bone growth promoting agent. In one example, polysorbate-80 has been shown to reduce the irritation caused by intranasally administered compounds where delivery was enhanced by use of a nonionic surfactant. (See, e.g. , U.S. Patent No. 5,902,789, incorporated herein by reference).

Thus, preferred nasal spray solutions according to the present invention comprise the active compounds in solution with a nonionic surfactant that enhances nasal absorption of the drug and polysorbate-80, together with one or more pharmaceutically acceptable carriers. Other secondary therapeutic ingredients, including secondary hormonal therapeutic agents, can also be included. Desirably, the formulation does not include oxidizing agents and other substances with which the active compounds, or secondary therapeutic agents to be administered, are known to be incompatible. Formulations according to the present invention suitable for nasal administration of the active compounds conveniently comprise sterile aqueous solutions containing a nonionic surfactant which enhances absorption of the active compounds and polysorbate-80, which solutions are typically in the range of pH 3.0 and 8.0. Such formulations can be conveniently prepared by dissolving compositions according to the present invention in water to produce an aqueous solution, and rendering the solution sterile. The formulations can be prepared in multi-dose containers, for example in the sealed dispensing system as disclosed in U.S. Patent No. 4,511 ,069, incorporated herein by reference.

In preferred embodiments, a nasal spray solution is provided containing approximately 0.001 and 50 milligrams per milliliter and preferably about 0.1 to 50 mL of liquid carrier or per gram of solid carrier. The solution is administered into the nose with a squeeze bottle or other intranasal delivery device held in the upright position while the patient is in a sitting position. One or two sprays of approximately 0.05 to 0.5 ml of a solution of the active compound per spray are administered into each nostril in a fine mist or aerosol. Approximately 0.1 mL is a preferred spray volume. The number and volume of sprays administered, as well as the concentration of the active compounds in the solution, can be adjusted according to well known pharmacokinetic principles. (See, e.g., Newton, Ann. N.Y. Acad. Sci. 652:481-483, 1992; Mena, Neuroendocrinology 61:722- 730, 1995; Gonser. Arch. Gvnecol. Obstet. 256:63-66. 1995; Orhue, Obstet. Gvnecol. 83:229-233, 1994; Satin et al, Am. J. Obstet. Gvnecol. 166:1260-1261, 1992; and Satin et al. , Obstet. Gvnecol. 83 :234-238, 1994, each incorporated herein by reference in its entirety) and further in accordance with the examples below to ensure that the amount of the active compounds administered to the patient corresponds to an intranasally effective amount to prevent or treat a hormone-responsive condition, and to reduce bone loss, or promote bone growth, in the patient. In other embodiments, gastric mucosa-protecting and/or tissue-repairing agents are administered with the active compounds (i.e., GnRH and bone growth promoting agent). Such protecting agents include gefarnate, aceglutamide aluminum, sucralfate, L-glutamine, sofalcone, teprenone, plaunotol (KELNAC^®), rebamipide (MUCOSTA^®), aldioxa, cetraxate, and troxipide. Such protecting agents can also be used in nasally administrable compositions by homogeneously dispersing the active compounds with these agents.

Alternative means of intranasal administration of the active compounds are provided by the use of ion exchange resins or adsorbent resin powders as carriers. Use of these materials is also adaptable for administration of the active compounds via oral, percutaneous, gasu-ointestinal, rectal, or subcutaneous routes. For intranasal use, these materials minimize irritation to the nasal mucosa and deliver the active compounds in a stable form and with efficient absorption. Thus, methods and compositions are provided wherein the active compounds are formulated with an ion exchange resin or an adsorbent resin powder as a carrier which effectively delivers the active compounds to, and supports their transfer across, the surface of the nasal mucosa for absorption into the general circulation. Other methods useful for compounding the active compounds with the carrier are described in U.S. Patent No. 5,942,242, which is incorporated herein by reference. These methods include: (1) preparing a suspension by adding an ion exchange resin or adsorbent resin powder to a solution or suspension; (2) mixing dried active compounds (e.g., GnRH and bone growth promoting agent) with an ion exchange resin or adsorbent resin powder by means of a mortar or ball mill while maintaining an appropriate relative humidity; (3) freeze drying a suspension obtained by method (1); and (4) increasing the homogeneity of a mixture of step (2) by adding an organic solvent such as ethanol.

Ion exchange resins suitable for use in the present invention include: polystyrenes, methacrylic resin, acrylic resins, phenol-formaldehyde resins, cellulose polymers, dextran polymers, and mixtures thereof. Examples of such polymers include, sodium polystyrene sulfonate prepared according to The Pharmacopeia of Japan, calcium polystyrene sulfonate prepared according to The Pharmacopeia of Japan. AMBERLITE^® 1RP64, AMBERLITE^® CU-SO, AMBERLITE^® DP-1, and DOWEX^® 2, and mixtures thereof, which bear as cation exchange functional groups, sulfonic acid or carboxylic acid, or a salt thereof (e.g., a sodium, potassium, or calcium salt). The anion exchange resins include, for example, quaternary ammonium resin derivatives or chloride salts thereof. Examples of such chloride salts include cholestyramine, AMBERLITE^® 1RP67, AMBERLITE^® IRA-68, DOWEX^® SOw, and mixtures thereof.

Examples of adsorbent resins for use within the invention include styrenedivinylbenzenes, such as: DIAION^® HP 10; DIAION^® HP2O; Sepabead 207; AMBERLITE^® XAD2; LEWATIT^® 0C103 1; DOLITE^® E586 1; methacrylic acid esters, such as DIAION^® HP2MG and AMBERLITE^® XAD-7; polyethylenes; vinyl chloride resins; amino acid sulfoxides; and mixtures thereof.

To provide an optimal intranasal powder for delivery of the active compounds, a mean particle size of the ion exchange resin or adsorbent resin is not typically larger than 200 μ, preferably 10 to 150 μ, and still more preferably 40 to 70 μ. The total amount of powdered medicament to be administered into the human nasal cavity as a single dose is typically approximately 10 μg to 50 mg, preferably 1 to 30 mg, and more preferably 10 to 25 mg. In this case, the total amount is the sum of the amount of the active compounds and carrier as well as any other active ingredients or additives. As for solid medicaments delivered in a liquid suspension (e.g., GnRH and bone growth promoting agent in a resin solid suspended in a liquid carrier), the amount for a single dose is typically 0.1 to 2.5 ml, preferably 0.2 to 2.0 ml, more preferably 0.3 to 1.5 ml.

To increase both adherence to the nasal mucosa and the stability of nasal powders and solid suspensions, the composition can include a water-soluble polymer powder, such as: polyacrylic acid or polymethacrylic acids or metal salts, such as sodium salt or potassium salts, thereof, with a mean particle size of 0.5 to 200 μ, preferably 20 to 100 μ; a water-soluble acrylate polymer such as polyacrylamide, having a molecular weight of 30,000 or greater, preferably 50,000 to 10,000,000; carboxyvinyl polymers, methylcelluloses, ethylcelluloses, hydroxymethylcelluloses, hydroxypropyl- methylcelluloses, carboxymethylcelluloses, carboxymethylchitin, polyvinylpyrrolidone, polyvinylalcohols, ester gums, polybutene, synthetic hydroxypropyl-starch, synthetic carboxymethyl-starch, synthetic polyvinylethers, and polyethylene oxide, having an average molecular weight of 20,000 to 9,000,000, and preferably 100,000 to 7,000,000; natural polymers such as hyaluronic acid, sodium alginate, gelatin, gluten, carboxymethyl-starch, hydroxypropyl-starch, gum arabic, mannan, dextran, tragacanth, amylopectin, xanthan gum, locust bean gum, casein, polyvinylethers, and pectin; and... mixtures thereof.

In yet additional aspects of the invention, the active compounds (i.e., GnRH and bone growth promoting agent) are administered according to the foregoing methods in a coordinate treatment or prophylaxis protocol with a secondary therapeutic agent. In one embodiment, preferred secondary therapeutic agents are conventional hormonal treatment agents. For example, the active compounds can be administered coordinately with an androgen (e.g., danazol) or a structurally related androgen compound. The active compounds are administered as above, simultaneously or sequentially with administration of the secondary therapeutic agent, the latter of which is administered in an amount sufficient to independently inhibit proliferation of hormone- responsive cells (e.g., ectopic endometrial tissue), and to reduce bone loss or promote bone growth, in the patient. Other secondary therapeutic agents include anti-fungal agents, such as ketoconazole, nystatin, griseofulvin, flucytosine, miconazole, amphotericin B, ricin, and β-lactam antibiotics (e.g., sulfazecin); vitamins such as cyanocobalamin neinoic acid, retinoids and derivatives such as retinol palmitate, and α-tocopherol; peptides, such as manganese super oxide dismutase; enzymes such as alkaline phosphatase; anti-allergic agents such as amelexanox; circulatory drugs such as propranolol; metabolic potentiators such as glutathione; antituberculars such as para-aminosalicylic acid, isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin sulfate; antivirals such as acyclovir, amantadine azidothymidine (AZT or ZIDOVUDINE™), ribavirin, amantadine, vidarabine, and vidarabine monohydrate (adenine arabinoside, ara-A); antianginals such as diltiazem, nifedipine, verapamil, erythrityl tetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritol tetranitrate; anticoagulants such as phenprocoumon, heparin; and antibiotics such as dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin and tetracycline. Other secondary therapeutic agents include drugs, such as antiinflammatories such as difunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac,_. .. tolmetin, aspirin and salicylates; antirheumatics such as penicillamine; narcotics such as paregoric; opiates such as codeine, heroin, methadone, morphine and opium; cardiac glycosides such as deslanoside, digitoxin, digoxin, digitalin and digitalis; neuromuscular blockers such as atracurium besylate, gallamine triethiodide, hexafluorenium bromide, metocurine iodide, pancuronium bromide, succinylcholine chloride (suxamethonium chloride), tubocurarine chloride and vecuronium bromide; sedatives (hypnotics) such as amobarbital, amobarbital sodium, aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride, methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium, secobarbital sodium, talbutal, temazepam and triazolam; local anesthetics such as bupivacaine hydrochloride, chloroprocaine hydrochloride, etidocaine hydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride, procaine hydrochloride and tetracaine hydrochloride; and general anesthetics such as droperidol, etomidate, fentanyl citrate with droperidol, ketamine hydrochloride, methohexital sodium and thiopental sodium.

The instant invention also includes kits, packages and multi-container units containing the above described compositions, active compounds, and/or means for administering the same for use in the prevention and treatment of hormone-responsive conditions, as discussed above. Typically, these kits include a pharmaceutical preparation of GnRH and a bone growth promoting agent in a biologically suitable carrier which is optionally contained in a bulk dispensing or unit or multi-unit dosage form. Optional dispensing means may be provided for administering these active compounds, preferably including an intranasal spray applicator. Preferred applicators include pressurized aerosol or hand-pump reservoirs equipped with a nozzle for placement in a nostril of the patient and functional to direct a liquid spray of the active compounds in solution therein. The following examples are offered by way of illustration, not by way of limitation.

EXAMPLE I GnRH -Calcitonin Formulations for Nasal Administration The following nasal formulations comprising GnRH and calcitonin exemplify useful formulations provided for therapeutic administration within the methods of the invention. The GnRH can comprise, for example, GnRH, leuprolide, histrelin, nafarelin, triptorelin, buserelin, goserelin, or a pharmaceutically acceptable salt thereof. The calcitonin can comprise, for example, human calcitonin, salmon calcitonin, hog calcitonin, chick calcitonin, cattle calcitonin, eel calcitonin, or a pharmaceutically acceptable salt thereof. Nasal formulations of GnRH and calcitonin for animal testing, human clinical trials and commerce are manufactured under current Good Manufacturing Practice (cGMP). The formulations are sterile when manufactured.

Formulation A: A 20 liter solution of GnRH and calcitonin for therapeutic use is formulated in a 25 liter glass vessel. Eighteen liters of Water for Injection USP are placed in the vessel and under stirring, the following are sequentially added and a solution created:

Sodium acetate 20 g

Sodium chloride 340 mg Chlorobutanol NF 100 mL

To the solution is added 80 g of GnRH and 20 million IU of a calcitonin with stirring until all of the active ingredients are dissolved. The pH is adjusted to between 3.7 and 4.3 and the batch is transferred to a balance and additional Water for Injection USP is added to complete the formulation. The batch of solution is terminally sterilized by aseptic filtration through a 0.2 μ membrane filter system and filled into 5.0 mL glass vials that are closed with a mist activator that discharges 0.1 mL liquid per spray. Between 3600 and 3950 vials are obtained from the batch. Formulation B: A 20 liter solution of GnRH and calcitonin for therapeutic use is formulated in a 25 liter glass vessel under an inert atmosphere of nitrogen. Eighteen liters of Water for Injection USP are placed in the vessel and under stirring, the following are sequentially added and a solution created: Chlorobutanol NF 10 mL

Glycerol 400 mL

Benzalkonium chloride 2 g

Sorbitol 1 kg

To the solution is added 160 g of GnRH and 40 million IU of calcitonin with stirring until all of the active compounds are dissolved. The pH is adjusted to between 3.7 and 4.3 with 50 mM citric acid and/or 50 mM sodium phosphate, monobasic and the batch is transferred to a balance and additional Water for Injection USP is added to complete the formulation. The batch of solution is terminally sterilized by aseptic filtration through a 0.2 μ membrane filter system and filled into 5.0 mL glass vials that are closed with a mist activator that discharges 0.1 mL liquid per spray.

Formulation C: The benzalkonium chloride of formulation B is substituted with methyl paraben and propyl paraben.

Formulations D. E. and F: Various formulations designated Formulation D can be obtained from Formulation A by altering the GnRH and/or calcitonin amounts from 2 grams per batch to 300 grams or more of GnRH, and from 1 million IU to 100 million IU or more of calcitonin per batch. Various formulations designated Formulation E can be obtained from Formulation B by altering the GnRH and/or calcitonin amounts from 2 grams per batch to 300 grams or more of GnRH, and from 1 million IU to 100 million IU or more calcitonin per batch. Various formulations designated Formulation F can be obtained from Formulation C by altering the GnRH and/or calcitonin amounts from 2 grams per batch to 300 grams or more of GnRH, and from 1 million IU to 100 million IU or more of calcitonin per batch.

EXAMPLE II Reduction of GnRH-Induced Bone Loss in the Mouse by Nasal Administration of a

GnRH-Calcitonin Composition To demonstrate the efficacy of the present invention in reducing the incidence of the growth of ectopic endometrial tissue in mammalian patients, mice are surgically implanted with autologous endometrial tissue, according to the method of Rossi et al (Arch. Gvnecol Obstet 263: 102-107, 2000). At 3 weeks the mice will show evidence of endometriosis. For one group, a solution of GnRH and calcitonin is administered intranasally at stepwise dosages and frequencies. The GnRH and calcitonin nasal spray solution will contain commercially available GnRH and calcitonin in 0.1 M NaH₂PO -H₂0 buffer, and varying concentrations of polysorbate-80 and nonoxynol-9. For this purpose, stock solutions containing 10:90, 50:50 and 25:75 (wt:wt) polysorbate , 80:nonoxynol-9 are prepared and added to the spray solution to a final concentration of 0.1% to 0.75% by weight. Nitrous oxide (Union Carbide, New York) is used as a propellant. Formulations A, B, or C ofExample I can also be used in these studies. The control group receives GnRH without the calcitonin. Exemplary dosages for test animals range between about 0.01 and 5 mg/kg of GnRH and about 1 to about 500 IU of calcitonin, exclusive of the selected carrier, administered one or more times daily.

Test and control animals are monitored for growth of endometrial tissue and for bone loss. The status of the endometrial tissue is monitored by histological examination. The mice are monitored during a period from about 30 to 90 days. Using standard methods, changes in bone mass (i.e., gain or loss) are monitored (e.g., by single- photon absorptiometry, dual-photon absorptiometry, duel-energy X-ray absorptiometry, quantitated computerized tomography, or by sacrifice and subsequent analysis of bone loss), for each test group and compared with the average bone loss per animal in the control group. In accordance with the teachings herein, animals treated with a composition comprising GnRH and a calcitonin will show significantly less bone loss as compared with the control group (i.e., treatment with GnRH alone).

The GnRH-calcitonin dosing schedule and formulations used in these animal studies, when used in human patients, will also show a significant reduction in bone loss compared to human patients treated with a GnRH alone.

All publications, patents and patent applications are herein incorporated by reference for all purposes, particularly those pertaining to practice, description and use of the invention. The invention now having been fully described, it will be apparent that many changes and modifications can be made in accordance with the description above by applying ordinary knowledge and skill in the art without departing from the spirit or scope of the appended claims which are offered by way of illustration and exemplification, not limitation.

Claims

WHAT IS CLAIMED IS:

1. A method for reducing GnRH-induced bone loss in a mammalian patient comprising administering to the patient having a sex steroid hormone-responsive condition a pharmaceutically acceptable formulation comprising a therapeutically effective amount of GnRH and a therapeutically effective amount of a bone growth promoting agent to reduce bone loss due to GnRH administration.

2. The method of claim 1 , wherein the hormone-responsive condition is endometriosis, uterine fibroids, dysfunctional uterine bleeding, uterine leiomyomas, mastalgia, prostatic carcinoma, or benign prostatic hypertrophy.

3. The method of claim 1 , wherein the GnRH is GnRH, leuprolide, histrelin, nafarelin, triptorelin, buserelin, goserelin, or a pharmaceutically acceptable salt thereof.

4. The method of claim 1 , wherein the bone growth promoting agent is calcitonin, danazol, a bisphosphonate, an alpha v beta antagonist, an inhibitor of osteoclastic V-H(+) ATPase, an inhibitor of pp60 (src), a chemically modified tetracyclines, or amylin.

5. The method of claim 4, wherein the calcitonin is human calcitonin, salmon calcitonin, hog calcitonin, chick calcitonin, cattle calcitonin, eel calcitonin, or a pharmaceutically acceptable salt thereof.

6. The method of claim 1 , wherein the GnRH and bone growth promoting agent are administered coordinately.

7. The method of claim 1 , wherein the GnRH and bone growth promoting agent are administered separately.

8. The method of claim 1 , wherein the GnRH and bone growth promoting agent are administered to the patient by a mode of administration selected from intramuscular, intravenous, intranasal, intrapulmonary, subcutaneous, parenteral, oral, transmucosal and transdermal delivery.

9. The method of claim 8, wherein the GnRH and bone growth promoting agent are administered to the patient intranasally.

10. The method of claim 9, wherein the GnRH and bone growth promoting agent are formulated in a powder or aqueous formulation for intranasal delivery.

11. The method of claim 8, wherein the GnRH and bone growth promoting agent are formulated for intranasal or intrapulmonary administration.

12. The method of claim 1 , further comprising a pharmaceutically acceptable carrier.

13. The method of claim 12, wherein the GnRH and bone growth promoting agent are combined in an aqueous formulation with an excipient selected from nonoxynol-9, laureth-9, poloxamer-124, octoxynol-9, lauramide DEA, chlorobutanol, glycerol, citric acid, sodium phosphate, methyl paraben, propyl paraben, sorbitol, sodium chloride, and sodium acetate for intranasal delivery.

14. The method of claim 1 , wherein the GnRH is administered in a dose of at least ten micrograms.

15. The method of claim 1 , wherein the calcitonin is administered in a dose of at least 50 IU.

16. The method of claim 1 , wherein the GnRH and bone growth promoting agent are administered daily in an intranasal formulation.

17. The method of claim 1 , further comprising administering a secondary therapeutic agent.

18. A pharmaceutical composition for prophylaxis or treatment of a sex steroid hormone-responsive condition in a mammalian patient comprising a therapeutically effective amount of a GnRH and a bone growth promoting agent in a pharmaceutically acceptable carrier, wherein GnRH-induced bone loss is reduced.

19. The pharmaceutical composition of claim 18, wherein the GnRH is GnRH, leuprolide, histrelin, nafarelin, triptorelin, buserelin, goserelin, or a pharmaceutically acceptable salt thereof.

20. The pharmaceutical composition of claim 18, wherein the bone growth promoting agent is a calcitonin selected from human calcitonin, salmon calcitonin, hog calcitonin, chick calcitonin, cattle calcitonin, eel calcitonin, or a pharmaceutically acceptable salt thereof.

21. The pharmaceutical composition of claim 20, prepared in a unit dosage form containing at least 50 IU of calcitonin.

22. The pharmaceutical composition of claim 18, wherein the bone" growth promoting agent is a bisphosphonate, an alpha v beta antagonist, an inhibitor of osteoclastic V-H(+) ATPase, an inhibitor of pp60 (src), a chemically modified tetracyclines, or amylin.

23. The pharmaceutical composition of claim 18, prepared in a unit dosage form containing at least one 10 micrograms of the GnRH.

24. The pharmaceutical composition of claim 18, wherein the composition is formulated in the carrier for intranasal or intrapulmonary administration.

25. The pharmaceutical composition of claim 18, wherein the composition is formulated in a powder or aqueous formulation for intranasal delivery.

26. The pharmaceutical composition of claim 18, wherein the composition is combined in an aqueous formulation with one or more excipients selected from the group consisting of nonoxynol-9, laureth-9, poloxamer-124, octoxynol-9, lauramide DEA, chlorobutanol, glycerol, citric acid, sodium phosphate, methyl paraben, propyl paraben, sorbitol, sodium chloride, and sodium acetate for intranasal delivery.

27. The pharmaceutical composition of claim 18, further comprising a secondary therapeutic agent.

28. A kit comprising one or more unit dosages of GnRH and a bone growth promoting agent admixed together or formulated separately.

29. The kit of claim 28, wherein the GnRH is GnRH, leuprolide, histrelin, nafarelin, triptorelin, buserelin, goserelin, or a pharmaceutically acceptable salt thereof.

30. The kit of claim 28, wherein the bone growth promoting agent is a calcitonin selected from human calcitonin, salmon calcitonin, hog calcitonin, chick calcitonin, cattle calcitonin, eel calcitonin, or a pharmaceutically acceptable salt thereof.

31. The kit of claim 28, wherein the kit further includes administration means for intramuscular, intravenous, intranasal, intrapulmonary, subcutaneous, parenteral, oral, transmucosal or transdermal delivery of the GnRH and bone growth promoting agent.

32. The kit of claim 31 , where the administration means is intranasal or intrapulmonary administration means.