MXPA99005351A - Pharmaceutical formulations for sustained drug delivery - Google Patents

Abstract

Sustained delivery formulations comprising a water-insoluble complex of a peptidic compound (e.g., a peptide, polypeptide, protein, peptidomimetic or the like) and a carrier macromolecule are disclosed. The formulations of the invention allow for loading of high concentrations of peptidic compound in a small volume and for delivery of a pharmaceutically active peptidic compound for prolonged periods, e.g., one month, after administration of the complex. The complexes of the invention can be milled or crushed to a fine powder. In powdered form, the complexes form stable aqueous suspensions and dispersions, suitable for injection. In a preferred embodiment, the peptidic compound of the complex is an LHRH analogue, preferably an LHRH antagonist, and the carrier macromolecule is an anionic polymer, preferably carboxymethylcellulose. Methods of making the complexes of the invention, and methods of using LHRH-analogue-containing complexes to treat conditions treatable with an LHRH analogue, are also disclosed.

Description

PHARMACEUTICAL FORMULATIONS THAT COMPRISE AN INSOLUBLE COMPLEX IN WATER FOR THE RELEASE OF DRUGS SUSTAINED BACKGROUND OF THE INVENTION A variety of clinical diseases and disorders are treated by administration of a pharmaceutically active peptide. An example is prostate cancer, which is a cancer that depends on the sex hormone and that can be treated by the administration of a luteinizing hormone-releasing hormone analog.

(LHRH) that alters the production of luteinizing hormone (LH), which regulates the synthesis of male hormones. In particular, peptide analogs of LHRH that act as superagonists of the luteinizing hormone-releasing hormone receptor, such as leuprolide and goserelin, have been used to decrease LH production. In many cases the therapeutic effectiveness of a pharmaceutically active peptide depends on its continued presence in vivo for prolonged periods of time. To achieve continuous delivery of the peptide in vivo, a sustained release or sustained delivery formulation is desired, to avoid the need for repeated administrations. A method for sustained release of the drug is by microencapsulation, in which the active ingredient is included within a polymeric membrane to produce microparticles. For example, LHRH superagonists, such as leuprolide and goserelin, are typically encapsulated within a microparticle comprising a poly-lactide / poly-glycolide copolymer to prepare formulations suitable for depot injection that provide sustained release of the superagonist for several weeks. up to months (see for example, US Patents Nos. 4,675,189; 4,677,191; 5,480,656 and 4,728,721). Further sustained release formulations are necessary for administration of pharmaceutically active peptides in vivo continuously for extended periods.

SUMMARY OF THE INVENTION The present invention provides pharmaceutical compositions containing a stable, water insoluble complex, consisting of a peptide compound (eg, a peptide, polypeptide, protein, peptidomimetic and the like), preferably a pharmaceutically active peptide compound and a Carrier acromolecule allowing sustained release of the peptide compound in vivo with administration of the complex. Accordingly, the complex of the invention can allow the continuous release of a pharmaceutically active peptide compound to an individual for prolonged periods, for example, a month. In addition, the association of the peptide compound and the carrier macromolecule in a compact, stable complex allows high concentrations of the peptide compound to be loaded into the formulation. The complex of the invention is formed by combining the peptide compound and the carrier macromolecule under conditions such that a complex substantially insoluble in water is formed, for example, the aqueous solutions of the peptide compound and the macromolecules of the carrier are mixed until the complex precipitates. The complex may be in the form of a solid (for example, a paste, granules, powder or lyophilisate) or the complex in powder form may be sufficiently finely pulverized to form stable liquid suspensions or semi-solid dispersions. In a preferred embodiment, the peptide compound of a water-insoluble complex is an analogue of LHRH, more preferably, an LHRH antagonist, and the carrier macromolecule is an anionic polymer, preferably carboxymethylcellulose. The complex of the invention is suitable for sterilization, such as by gamma radiation or electron beam radiation, before in vivo administration. The method for treating an individual in a treatable condition with an LHRH analog is also provided by administering to the individual a composition containing the LHRH analogue of the invention. In a preferred embodiment, the methods of treatment of the invention are used in the treatment of prostatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the graphs representing plasma testosterone levels (in ng / ml, light black squares) and levels of PP1-149 in plasma (in ng / ml, dark box) in rats (graphic left) and in dogs (right graph) for time following the intramuscular injection of a complex of PP1-149 and carboxymethylcellulose. Figure 2 is a graph depicting plasma testosterone levels (in ng / ml, clear frames) and levels of plasma PPl-149 (in ng / ml, dark squares) in rats for time after intramuscular injection of a complex of PP1-149 antagonist of LHRH and carboxymethyl cellulose on day 0 and injection of the Lupron ™ LHRH agonist on day 30, demonstrating the suppression of the testosterone surge induced by Lupron ™ by the pretreatment of PP1-149. Figures 3A-3C are a series of graphs representing plasma testosterone levels (in ng / ml) (in male Sprague-Dawley rats over time, followed by intramuscular injection of a PP1-149-CMC (Figure 3A ), PPI-258-CMC (Figure 3B) or Cetrorelix ™ -CMC (Figure 3C) Figure 4 is a graph depicting plasma testosterone levels (in ng / ml, light squares) and levels of PP1-149 in plasma (in ng / ml, dark squares) in dogs with time after a subcutaneous injection of PP1-149-CMC at the indicated doses at 28-day intervals, demonstrating prolonged suppression of plasma testosterone levels. Figure 5 is a graph depicting plasma testosterone levels (in ng / ml, clear squares) and plasma PP1-149 levels (in ng / ml, dark squares) in dogs over time after an intramuscular injection of PP1-149-CMC at the indicated doses at 28-day intervals, demonstrating the prolonged suppression of plasma testosterone levels.

DETAILED DESCRIPTION OF THE INVENTION This invention pertains to pharmaceutical compositions containing a stable, water-insoluble complex consisting of a peptide compound (eg, a peptide, polypeptide, protein, peptidomimetic and the like) and a carrier macromolecule, the methods of making such compositions and methods of using these compositions- The advantages of the pharmaceutical compositions of the invention include the ability to deliver a pharmaceutically active peptidic compound, systemically or locally, for prolonged periods (eg, a few weeks, a month or several months) -and the ability to carry high concentrations of the peptide compound in the complex. For the invention to be more quickly understood, certain terms are defined first. As used herein, the term "peptide compound" is intended to refer to compounds composed of, at least in part, amino acid residues linked by amide linkages (eg, peptide bonds) .The term "peptide compound" is proposed to include peptides, polypeptides and proteins. Typically, a peptide will be composed of less than about 100 amino acids, more commonly less than about 50 amino acid residues and even more regularly, less than about 25 amino acid residues. The term "peptide compound" is further proposed to include analog peptides, derived peptides and peptidomimetics that mimic the chemical structure of a peptide composed of naturally occurring amino acids Examples of peptide analogs include peptides comprising one or more non-natural amino acids Examples of peptide derivatives include peptides in which an amino acid side chain, the peptide backbone chain or the amino- or carboxy terminal chain have been derivatives (eg, peptide compounds with methylated amide bonds) Examples of peptidomimetics include peptide compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see, for example, James, GL et al. (1993) Science 260; 1937-1942). The "reverse" peptides in which all the L-amino acids are substituted with the corresponding D-amino acids, "reverse-reverse" peptides (see US Pat. No. 4,522,752 of Sisto) in which the amino acid sequence is inverted (' "retro") and all L-amino acids are replaced with D-amino acids) "inverse") and other isosteres, such as mimetic peptide backbones (ie, amide linkages), which include modifications of the amide nitrogen, the alpha carbon , carbonyl amide, complete replacement of the amide bond, extensions, deletions or crosslinks of the main chain. Some modifications of the peptide backbone are known, including? [CH2S],? [CH2NH],? [CSNH2],? [NHC0],? [C0CH2] and? [(E) or (Z) CH = CH]. In the nomenclature used before,? indicates the absence of amide bond. The structure that replaces the amide group is specified within "brackets- Other possible modifications include an N-alkyl (or aryl) substitution (? [CONR]), cross-linking of the main chain to build lactams and other cyclic structures and other derivatives that include C-terminal hydroxymethyl derivatives, O-modified derivatives and N-terminal modified derivatives including substituted amides, such as alkylamides and hydrazides As used herein, the term "pharmaceutically active peptide compound" is intended to refer to a peptide compound showing pharmacological activity, in its present form or on an in vivo processing (ie, the pharmaceutically active peptide compounds include peptide compounds with constitutive pharmacological activity and peptide compounds in a 'prodrug' form which has to be metabolized or processed from some way in vivo after the administration for most for pharmacological activity). As used herein, the terms "multivalent cationic peptide compound" and "multivalent anionic peptide compound" are intended to refer to peptide compounds that comprise a multiplicity of positive or negative charges respectively. A "bivalent cationic" or "bivalent anionic" peptide compound is proposed to refer to a peptide compound comprising two positive or negative charges respectively. A "trivalent cationic" or "trivalent anionic" peptide compound is proposed to refer to a peptide compound comprising three positive or negative charges respectively. As used herein, the term "LHRH analog" is intended to include peptide compounds that mimic the structure of the luteinizing hormone releasing hormone An LHRH analog can be an LHRH agonist or an LHRH antagonist. As used herein, an 'LHRH agonist' is proposed to refer to a compound that stimulates the hormone receptor (LHRH-R) releasing luteinizing hormone so that the release of luteinizing hormone is stimulated, or an "LHRH antagonist", which refers to a compound that inhibits LHRH-R in a manner that inhibits the release of luteinizing hormone Examples of LHRH agonists include leuprolide (registered name: Lupron®; Abbott / TAP), goserelin (registered name: Zoladex®, Zeneca), buserelin (Hoechst), triptorelin (also known as Decapeptyl, D-Trp-6-LHRH and Debiopharm®, Ipsen / Beaufour), nafarelin (registered name 'Synarel®; Syntex), lutrelin (Wyeth) , cistoreline (Hoehst), gonadorelin (Ayerst) and histrelin (Ortho). As used herein, the term "LHRH antagonist" is intended to refer to a compound that inhibits the releasing hormone receptor and luteinizing hormone, so that the release of luteinizing hormone is inhibited. Examples of LHRH antagonists include Antide Cetrorelix, compounds described in U.S. Patent No. 5,470,947 to Fol ers et al .; PCT Publication No. WO 89/01944 of Folkers et al.; U.S. Patent No. 5,413,990 to Haviv; U.S. Patent No. 5,300,492 to Haviv; U.S. Patent No. 5,371,070 to Koerber et al: U.S. Patent No. 5,296,468 to Hoeger et al; U.S. Patent No. 5,171,835 to Janaky et al .; U.S. Patent No. 5,003,011 to Coy et al .; U.S. Patent No. 4,431,635 to Coy; U.S. Patent No. 4,992,421 to De et al .; U.S. Patent No. 4,851,385 to Roeske; U.S. Patent No. 4,801,577 to Néstor, Jr. et al; and U.S. Patent No. 4,689,396 to Roeske et al and the compounds described in the patent application Serial No. 08 / 840,494, entitled "LHRH Antagonist Peptides", and a PCT application corresponding thereto (PCT Application No. PCT / US96 / 09852, also entitled 'LHRH Antagonist Peptides', the total contents of both are expressly incorporated herein by reference, an especially preferred LHRH antagonist comprises the structure: Ac-D-Nal1, 4-Cl-D-Phe2 , D-Pal3, N-Me-Tyr5, D-Asn6, Lys (iPr) 8, D-Ala10-LHRH, referred to herein as PP1-149.

As used herein, the term "carrier molecule" is intended to refer to a macromolecule that can be complexed with a peptide compound to form a water-insoluble complex.Prepared complexing with the peptide compound, the carrier macromolecule, so common, it is soluble in water Preferably, the macromolecule has a molecular weight of at least 5 kDa, more preferably 10 kDa .. By the term 'anionic carrier macromolecule' is meant to include high molecular weight macromolecules negatively charged, such as polymers anionic The term "cationic carrier macromolecule" is intended to include high molecular weight molecules positively charged, such as cationic polymers As used herein, the term "water insoluble complex" is intended to refer to a physically and chemically stable complex which is formed by appropriate combinations of a peptide compound and a carrier macromolecule according to methods described herein. This complex, in general, has the form of a precipitate which is produced by the combination of aqueous preparations of the peptide compound and the carrier macromolecule. Although not intended to "limit the mechanism, the formation of the preferred water insoluble complexes of the invention is thought to involve (e.g., that it is mediated at least in part by) ionic interactions in situations where the peptide compound is cationic and the carrier molecule is anionic or vice versa Additionally, or alternatively, the formation of a water insoluble complex of the invention may involve (eg, being mediated at least in part by) hydrophobic interactions.Furthermore, the formation of an insoluble complex Water of the invention may involve (eg, being mediated at least in part by) covalent interactions The description of the complex as being 'insoluble in water' is proposed to indicate that the complex is not rapidly or substantially dissolved in water, as it is indicated by its precipitation of aqueous solutions. However, it should be understood that a "water-insoluble" complex d < - d The invention may show limited solubility (ie, partial solubility) in water in vitro or in vivo in the aqueous physiological medium. present, the term 'sustained release' is proposed to refer to a continuous release of a pharmaceutical agent in vivo for a period after administration, preferably at least a few days, a week or several weeks. The sustained release of the agent can be demonstrated, for example, by the continuous therapeutic effect of the agent over time (for example, for an LHRH analogue, the sustained release of the analogue can be demonstrated by the continuous suppression of testosterone synthesis with time) . Otherwise, the sustained release of the agent can be demonstrated by detecting the presence of the agent in vivo over time. As used herein, the term "individual" is proposed to include is proposed to include [sic] warm-blooded animals, preferably mammals, more preferably primates and even more preferably humans. As used herein, the term "administration to an individual" is proposed to refer to the dosage, delivery or application of a composition (e.g., pharmaceutical formulation) to an individual by any suitable means for the release of the composition at the desired location in the individual, including the parenteral or oral administration, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colon, vaginal, intranasal or respiratory tract. As used herein, the term 'a condition treatable with an LHRH analogue' is intended to include diseases, disorders and other conditions in which the administration of an LHRH agonist or LHRH antagonist has a desired effect, for example, a beneficial therapeutic effect. Examples of conditions treatable with an LHRH analog include cancers that depend on hormones (including prostate cancer, breast cancer, ovarian cancer, uterine cancer and testicular cancer), benign prostatic hypertrophy, precocious puberty, endometriosis, uterine fibroids, infertility (a through in vitro fertilization) and fertility (that is, contraceptive uses). One aspect of the present invention corresponds to a pharmaceutical composition comprising a water-insoluble complex of a pharmaceutically active peptide compound and a carrier macromolecule. In a preferred embodiment, the formation of the water-insoluble complex is mediated, at least in part, by ionic interactions between the pharmaceutically active peptide [sic] and the carrier macromolecule. In these embodiments, the pharmaceutically active peptide component is cationic and the carrier macromolecule * is anionic or the pharmaceutically active peptide compound is anionic and the carrier macromolecule is cationic- In another embodiment, the formation of the water-insoluble complex is added, at least in part, by hydrophobic interactions between the pharmaceutically active peptidic compound and the carrier macromolecule. In a preferred embodiment, the peptide compound used in the complex is a multivalent cationic peptide compound, such as a bivalent or trivalent cationic peptide compound and the carrier macromolecule is an anionic macromolecule. The pharmaceutical compositions of the invention allow sustained release of the peptide compound to an individual in vivo after administering the composition to the individual, wherein the duration of sustained release may vary depending on the concentration of peptide compound and the carrier macromolecule used to form the the complex - For example, in one embodiment, a single dose of the water-insoluble complex provides sustained release of the peptide compound to an individual for at least one week after the pharmaceutical composition is administered to the individual. In another embodiment, a single dose of the water-insoluble complex provides sustained release of the peptide compound to an individual for at least one week after the pharmaceutical composition is administered to the individual. In yet another embodiment, a single dose of the water-insoluble complex provides sustained release of the peptide compound to an individual for at least three weeks after the pharmaceutical composition is administered to the individual. In yet another embodiment, a single dose of the water-insoluble complex provides sustained release of the peptide compound to an individual at least four weeks after the pharmaceutical composition is administered to the individual. Formulations that provide sustained release with longer or shorter durations are also included by the invention, such as formulations that provide continuous release for one day, 1-7 days, one month, two months, three months and the like. The continuous release of the peptide compound for a period of several months can be carried out, for example, by repeated monthly doses, each of which provides sustained release of the peptide compound for about one month (see Example 14). Any size of the peptide compound may be suitable for use in the complex provided that the peptide compound has the ability to form a water-insoluble non-covalent complex with the carrier macromolecule by combination of the peptide compound and carrier macromolecule. However, in certain preferred embodiments, the peptide compound is a peptide that is about 5 to about 20 amino acids in length, about 8 to about 15 amino acids in length or about 8 to about 12 amino acids in length. A variety of pharmaceutically active peptides can be used "in the formulations, non-limiting examples of which include analogs of LHRH (discussed below), bradykinin analogs, parathyroid hormone, adenocorticotrophic hormone [sic] (ACTH), calcitonin, and vasopressin analogues (e.g., 1-deamino-8- D-arginine vasopressin (DDAVP)). Although a variety of carrier macromolecules may be suitable for the formation of water-insoluble complexes of the invention, the preferred macromolecules are polymers, preferably water-soluble polymers. In a preferred embodiment, the carrier macromolecule is an anionic polymer such as an anionic polyalcohol derivative or fragment thereof, and salts thereof (e.g., sodium salts). The anionic fractions with which the polyalcohol can be derivatized include, for example, carboxylate, phosphate or sulfate groups. A particularly preferred anionic polymer is an anionic polysaccharide derivative, or fragment thereof, and salts thereof (e.g., sodium salts). The carrier macromolecule can comprise a single molecular species (e.g., a single type of polymer) or two or more different molecular species (e.g., a mixture of two types of polymers). Examples of the specific anionic polymers include carboxymethylcellulose, algin, alginate, anionic acetate polymers, anionic acrylic polymers, gums, xanthame, sodium glycolated starch, and fragments, derivatives and pharmaceutically acceptable salts thereof, as well as anionic carrageenan derivatives, derivatives anionics of polygalacturonic acid, and sulfated and sulphonated polystyrene derivatives. A preferred anionic polymer is the sodium salt of carboxymethylcellulose. Examples of cationic polymers include poly-L-lysine and other basic amino acid polymers. In a particularly preferred embodiment of the invention, the peptide compound of the water-insoluble complex is an LHRH analog, for example an LHRH agonist or, more preferably, an LHRH antagonist. Such LHRH analogues are usually 10 amino acids in length. Preferred LHRH antagonists include LHRH antagonists comprising a peptide compound, wherein a peptide compound residue corresponding to the amino acid at position 6 of the mammalian LHRH. , natural, comprises a structure D-asparagine (D-Asn). As used herein, the term "D-Asparagine structure" is understood to include D-Asn and the analogs, derivatives and mimetics thereof that retain the functional activity of D-Asn.Preferred other preferred LHRH antagonists include antagonists of LHRH comprising a peptide compound containing a structure: ABCDEFGHIJ, wherein: A is Pro-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal B is His or 4 -Cl-D-Phe C is Trp, D-Pal, D-Nal, L-Nal, D-Pal (NO), or D-Trp D is Ser E is N-Me-Ala, Tyr, N-Me- Tyr, Ser, Lys (iPr), 4-Cl-Phe, His, Asn, Met, Ala, Arg or lie; F is: X L, R O wherein R and X are, independently H or alkyl; and L consists of a small polar fraction G is Leu or Trp; H is Lys (iPr), Gln, Met or Arg I is Pro; and J is Gly-NH2 or D-Ala-NH2; or a pharmaceutically acceptable salt thereof. The term "small polar fraction" refers to a fraction that has a small steric volume and is relatively polar Polarity is measured as hydrophilicity by the P scale. The partition coefficient P, between 1-octanol and water has been used as a reference for measuring the hydrophilicity of a compound Hydrophilicity can be expressed as the logarithm of P, the logarithm of the partition coefficient (Hansch et al-, Nature 194: 178 (1962), Fujita et al-, J. 7? Chem. Soc. 86: 5175 (1964).) The standard tables of hydrophilicity for many molecules, and lipophilicity substituents (hydrophobicity) (designated by p) for many functional groups, have been compiled (see, for example, Hansch and Leo 'Substituent Constants for Correlation Analysis in Chemistry and Biology, "Wiley, New York, New York (1979)). The hydrophilicity of a wide range of candidate hydrophilicity fractions can be accurately predicted with the help of these tables. For example, the measured log P (octanol / water) of naphthalene is 3.45. The constant p substituent for -OH is -0.67. Therefore, the predicted log P for ß-naphthol is 3.45 + (-0.67) = 2.78. This value is a good match with the log P measured for ß-naphthol, which is 2.84. As used herein, the term "small polar fraction" refers to fractions having a log P between -1 and +2 and a steric charge that is less than the steric charge of Trp. In certain embodiments, L comprises a small polar fraction with the proviso that F is not D-Cit, D-Hci or a lower alkyl derivative of D-Cit or D-Hci Preferably, F is selected from the group consisting of D-Asn, D-Gln and D- Thr Most preferably F is D-Asn Preferably, E is tyrosine (Tyr) or N-methyl-tyrosine (N-Me-Tyr) In a particularly preferred embodiment, the LHRH antagonist has the following structure: Ac-D-Nal1, 4-Cl-D-Phe2, D-Pal3, N-Me-Tyr5, D-Asn6, Lys (iPr) 8, D-Ala10-LHRH (referred to herein as PP1-149). A particularly preferred complex of the invention consists of PP1-149 and carboxymethylcellulose In addition to the water insoluble complex, the pharmaceutical formulations of the invention may additionally contain the excipients and / or po Pharmaceutically acceptable ingredients. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic absorption and delaying agents, and the like that are physiologically compatible, Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous or parenteral administration (for example by injection) The excipients include pharmaceutically acceptable stabilizers and disintegrants In addition to the pharmaceutical formulations of the LHRH analogs complexed with a carrier macromolecule, the invention includes packaged formulations containing these complexes and syringes containing the complexes For example, the invention provides a packaged formulation for treating an individual of a condition treatable with an LHRH analog consisting of a water-insoluble complex of an LHRH analogue (preferably PPL- 1 49) and a carrier macromolecule (preferably carboxymethylcellulose), packaged with instructions for use of the water-insoluble complex to treat, to an individual, a condition treatable with an LHRH analogue. In another embodiment, the invention provides a syringe having a lumen, wherein a water-insoluble complex of an LHRH analogue (preferably PP1-149) and a carrier macromolecule (preferably, carboxymethylcellulose) is included in the lumen. The complex of the invention is prepared by combining the peptide compound and the carrier macromolecule under conditions such that a water-insoluble complex of the peptide compound and the carrier macromolecule is formed. Accordingly, another aspect of the invention pertains to the methods of preparing the pharmaceutical formulations. In one embodiment, the method consists of: providing a peptide compound and a carrier macromolecule; combining the peptide compound and the carrier macromolecule under conditions such that a water-insoluble complex of the peptide compound and the carrier macromolecule is formed; and preparing a pharmaceutical formulation containing the water-insoluble complex.

For example, a solution of the peptide compound and a solution of the carrier macromolecule are combined until the water-insoluble complex of the peptide compound and the carrier macromolecule precipitate from the solution. In certain embodiments, the solutions of the peptide compound and the carrier macromolecule are aqueous solutions. Otherwise, if the peptide compound or carrier macromolecule (or both) is not substantially soluble in water prior to the combination of the two, then the carrier peptide and / or macromolecule can be dissolved in a water-miscible solvent, such as an alcohol (eg, ethanol) before the combination of the two components of the complex. In another embodiment, of the method of preparing the water-insoluble complex, the solution of the peptide compound and the solution of the carrier macromolecule are combined and heated until the water-soluble complex of the peptide compound and the carrier macromolecule precipitate from the solution. The amounts of carrier peptide and macromolecule required to obtain the water-insoluble complex can vary depending in particular on the peptide compound and carrier macromolecule used, the particular solvent (s) used and / or the procedure used to obtain the complex. However, in general, the peptide compound will be in excess relative to the carrier macromolecule on a molar basis.

Often the peptide compound will also be in excess on a weight / weight basis, as demonstrated in the examples. In certain embodiments, the carrier macromolecule * preferably sodium carboxymethyl cellulose and the peptide compound, preferably PPI-149, is combined in a ratio of 0.2: 1 (w / w) carrier macromolecule: peptide compound. In other embodiments, the ratio of carrier macromolecule to peptide compound (w / w) can be, for example, 0.5: 1, 0.4: 1, 0.3: 1, 0.25: 1, 0.15: 1, 0.1: 1. Non-limiting examples of the conditions and methods for preparing a water-insoluble complex of the invention are further described in Examples 1-5 and 8-9. Once the peptide / macromolecule complex complex precipitates out of the solution, the precipitate can be separated from the solution by means known in the art, such as filtration (for example, through a 0.45 micron nylon membrane); centrifugation and the like. The recovered paste can then be dried (for example, in vacuum or in an oven at 70 ° C) and the solid can be milled or pulverized to a powder by means known in the art (for example, in a hammermill or knife, or crushed in mortar and pestle). After grinding or pulverizing, the powder can be sieved through a mesh (preferably a 90 micron mesh) to obtain a uniform particle distribution. In addition, the recovered paste can be frozen and lyophilized to dryness. The complex in powder form can be dispersed in a carrier solution to form a liquid suspension or semisolid dispersion suitable for injection. As a consequence, "in various embodiments, a pharmaceutical formulation of the invention is a dry solid, a liquid dispersion or a semi-solid dispersion." Examples of suitable liquid carriers for use in liquid suspensions include saline solutions, glycerin solutions and lecithin solutions. In another embodiment, the pharmaceutical formulation of the invention is a sterile formulation, for example, after the formation of the water-insoluble complex, the complex can be sterilized, optimally by gamma radiation or electron beam sterilization. of the invention for preparing a pharmaceutical formulation described above can further comprise sterilization of the water insoluble complex by gamma radiation or electron beam radiation.Preferably, the formulation is sterilized by gamma radiation using a gamma radiation dose of at least 15 KGy In other modalities, the formulation is sterilized by gamma radiation using an irradiation dose of at least 19 KG and at least 24KGy. As demonstrated in Example 11, the formulations of the invention remain acceptably stable with gamma radiation. Alternatively, to prepare a sterile pharmaceutical formulation, the water insoluble complex can be isolated using conventional sterilization techniques (e.g., using sterile starting materials and carrying out the production process aseptically). Accordingly, in another embodiment of the method for preparing a pharmaceutical formulation described above, the water-insoluble complex is formed using aseptic methods. The methods of formation of the water-insoluble complex of the invention are further described in Examples 1-5 and . 8-9. Pharmaceutical formulations include powders, liquid suspensions, semi-solid dispersions, dry solids (eg, lyophilized solids) and sterilized forms thereof (eg, by gamma radiation), prepared according to the methods of the invention, are also included in the invention. "Yet another aspect of the invention pertains to methods for using the pharmaceutical formulations of the invention to treat an individual suffering from a treatable condition - for the pharmaceutically active peptide compound included in the water-insoluble complex. Preferred embodiment, the invention provides a method for treating an individual with a LHRH analogue treatable condition comprising: administering to the individual a pharmaceutical formulation containing a water insoluble complex of an LHRH analogue and a carrier macromolecule. can be administered to the individual by any suitable route to achieve the desired therapeutic result (s), although preferred routes of administration are parenterally, in particular intramuscular (im) injection and subcutaneous / intradermal injection (sc / id.) Otherwise, the formulation can be administered to The individual orally Other suitable parenteral routes include intravenous injection, buccal administration, intradermal delivery and administration via the rectal, vaginal, intranasal or respiratory tract. It should be noted that when a formulation provides sustained release for weeks to months via i.m s.c./i.d. is administered by an alternate route, there may be no sustained release of the agent in an equivalent amount of time due to the removal of the agent by other physiological mechanisms (ie, the dosage form may be removed from the release site so that the effects prolonged therapies are not observed in periods of time as long as those observed with injection im or sc / id). The pharmaceutical formulation contains a therapeutically effective amount of the LHRH analog. A "therapeutically effective amount" refers to an effective amount, at the doses and for periods of time necessary to achieve the desired result A therapeutically effective amount of an LHRH analog may vary according to factors such as the state of the disease , age and weight of the individual, and the ability of the LHRH analogue (alone or in combination with one or more "of other medications) to produce a desired response in the individual- Dosage regimens can be adjusted to provide the optimal therapeutic response . A therapeutically effective amount is also that in which no toxic or detrimental effects of the antagonist are worth more than the beneficial, therapeutic effects. A non-limiting range for a therapeutically effective amount of an LHRH analogue is 0.01 to 10 mg / kg. A preferred dose of LHRH analog PPI-149 sustained reduction of plasma testosterone levels for 28 days is approximately 0.1-10 mg / kg, more preferably 0.3-1.2 mg / kg (expressed as free peptide) in a liquid suspension volume of about 1 ml or less. It should also be noted that the dosage values may vary with the severity of the condition to be alleviated. In addition, it should be understood that for any particular individual, the specific dosage regimens should be adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the compositions, and that the ranges of Dosages set forth herein are examples only and are not intended to limit the scope or practice of the claimed composition. The method of treatment of the invention can be applied for the treatment of various conditions, diseases and disorders in which the administration of LHRH has a desired clinical effect. Examples of diseases and disorders include hormone-dependent cancers, such as prostate cancer, breast cancer, ovarian cancer, uterine cancer and testicular cancer, benign prostatic hypertrophy, precocious puberty, endometriosis and uterine fibroma. Accordingly, the invention provides methods for treating these diseases and disorders by administering a pharmaceutical formulation of the invention. Additionally, LHRH analogs can be used to alter fertility. Accordingly, the methods of the invention can also be used for in vitro fertilization and contraceptive purposes.

In a particularly preferred embodiment, the method is used to treat prostate cancer, the LHRH analogue used in the formulation is an LHRH antagonist, more preferably PPI-149, and the method allows sustained release of the LHRH analogue in vivo by at least four weeks after administration by intramuscular or subcutaneous administration. An LHRH analogue, preferably PPI-149, formulated according to the invention can be used to inhibit the growth of prostate cancer cells by administration of the LHRH analog to an individual suffering from prostatic cancer. In addition, an LHRH antagonist, preferably PPI-149, formulated according to the invention, can be used to inhibit the surge of testosterone that accompanies the use of an LHRH agonist by pre-administration of the LHRH antagonist, preferably PPI-149, an individual suffering from prostatic cancer before initiating therapy with the LHRH agonist. Methods for inhibiting the surge of testosterone induced by the LHRH agonist, and other methods for treating prostate cancer using an LHRH antagonist, to which the formulations of the present invention can be applied, are further described in the US Patent Application. Series No. 08 / 573,109, entitled "Methods for the treatment of prostate using LHRH antagonists" filed on December 15, 1995 and a continuation in part of the patent application of the same Series No. 08 / 755,593, also entitled " Methods for -the treatment of borrowed using LHRH antagonists "presented on November 25, 1996, the contents of both are incorporated in the published PCT application WO 97/22357. The total contents of the published US applications and PCT application are expressly incorporated in present as reference The specific processes for complexing a pharmaceutically active peptide compound with a macromolecule to carrier are set forth in Examples 1-5 and 9-8 in the following. Also described are the results of tests demonstrating that a complex containing LHRH antagonists can facilitate the sustained release of the pharmaceutically active peptide in vivo (Example 6) and that it can inhibit the surge of testosterone induced by the LHRH agonist (Example 7) . The following examples, in addition to illustrating the invention, should not be considered as limiting. The contents of all references, patents and published patent applications cited by this application are incorporated herein by reference.

EXAMPLE 1: A 100 ml solution of the PPI-149 LHRH antagonist was prepared by dissolving 6.25 mg / ml of PPI-149 in water. An equal sample (100 ml minimum) of sodium carboxymethylcellulose USP (CMC) (low viscosity grade, Hercules Chemical Co.) was prepared at 0.125% w / v and mixed until dissolved. Equal portions of the PPI-149 and CMC solutions were mixed (giving a CMC: peptide ratio of 0.2: 1 (w / w)) and a solid material was obtained. The solid material was stirred overnight and then collected by filtration on a 0.45 micron nylon filter. HPLC evaluation of the filtered solution indicated that at least 95% of the PPI-149 compound was converted to- * a solid complex was removed from the solution. The recovered white paste was rinsed twice with water and then transferred to a vial and dried under vacuum. After drying for 72 hours, 633 mg of a white powder was obtained. The solid material was then pulverized in a mortar and pestle. Elemental analysis indicated 57% peptide in the complex.

EXAMPLE 2: 25 mg of PPI-149 was dissolved in 1 ml of water. To this was added 1 ml of a 0.5% solution of carboxymethylcellulose- The mixture formed a smooth white solid with mixing. The mixture was heated to reflux for 5 minutes and a white flocculent precipitate formed. This material was separated by centrifugation / decantation. The solid was resuspended in water and collected by repeated centrifugation. HPLC evaluation of the filtered solution indicated that when less than 90% of the PPI-149 compound was converted to the solid complex. The white precipitate was dried in vacuum and the solid material was ground in a mortar and pestle. Elemental analysis indicated 77% peptide in the complex.

EXAMPLE 3: 50 mg of PPI-149 was dissolved in 2 ml of 5% mannitol and mixed with 2 ml of 0.5% carboxymethylcellulose (low viscosity, USP, Spectrum Quality Chemicals). The mixture was stirred and immediately produced a white precipitate. The suspension was frozen and lyophilized to dryness to produce a sustained release complex of PPI-149.

EXAMPLE 4: 25 mg of PPI-149 was dissolved in 1 ml of water. To this was added 1 mL of 0.5% sodium alginate. USP (Spectrum). The mixture immediately formed a white precipitate with mixing. This material was separated by centrifugation / decantation. The solid was resuspended in water and collected by repeated centrifugation. The white precipitate was dried in vacuum. Elemental analysis was performed to obtain a peptide content of 66%.

EXAMPLE 5: 25 mg of PPI-149 was dissolved in 1 ml of water. Ammonia was added to adjust the pH to 11.0. To this was added 1 ml of 0.5% alginic acid, USP (Spectrum). The mixture immediately formed a white precipitate with mixing. This material was separated by centrifugation / decantation. The solid was resuspended in water and collected by repeated centrifugation. The white precipitate was dried under vacuum. The elemental analysis was performed to obtain a peptide content of 79%.

EXAMPLE 6: A water insoluble complex of PPI-149, LHRH antagonist, and carboxymethylcellulose was prepared according to the previous examples. A suspension of the PPI-149 / CMC complex was prepared and a single dose was injected intramuscularly in rats and dogs. The dose for the rats was 50 μg / kg / day X 60 days the dose for the dogs was 40 μg / kg / day X 28 days. Plasma testosterone levels (in ng / ml) were determined at different points in time as a measure of the activity of the LHRH antagonist in the animal. Representative results, shown in the graph of Figure 1, demonstrate that intramuscular injection of the PPI-149 / CMC complex results in a sustained suppression of plasma testosterone levels for at least 42 days in rats and at least 28 days in the dogs (indicated by the clear boxes in Figure 1), demonstrating the sustained release of the LHRH antagonist. The levels of PPI-149 (in ng / ml) in plasma were also monitored in the animals (indicated by the dark boxes in Figure 1). An initial peak of PPI-149 was observed for approximately the first 8 days, after which time PPI-149 was virtually undetectable in plasma. Despite the inability to detect PPI-149 in the plasma after approximately day 8, the results of the testosterone levels showed that PPI-149 was still therapeutically active in vivo during the course of the experiment.

EXAMPLE 7: A water soluble complex of PPI-149 LHRH antagonist and carboxymethylcellulose was prepared according to the previous examples. A suspension of the PPI-149 / CMC complex was prepared and a single dose was injected intramuscularly in rats on day 0. On day 30, it was injected to the rats Lupron ™ (leuprolide), an LHRH agonist. The plasma testosterone levels (in ng / ml, indicated by the light boxes in Figure 2) were determined at various points in time as a measure of the activity of the LHRH antagonist in plasma. Plasma PPI-149 levels (in ng / ml) were also monitored in animals (indicated by the dark boxes in Figure 2). The representative results, shown in the graph of Figure 2, show that previous treatment with the PPI-149 / CMC complex rapidly reduces plasma testosterone to castration levels and, in addition, blocks the testosterone surge induced by the testosterone agonist. LHRH. Despite the inability to detect PPI-149 in the plasma after approximately day 8, the results of the testosterone levels showed that the PPI-149 was still therapeutically active in vivo during the course of the experiment - EXAMPLE 8: In this example, an insoluble complex was formed between the PPI-258 analog of LHRH and carboxymethylcellulose (CMC). PPI-258 has a structure: acetyl-D-naphthylalanyl-D-4-Cl-phenylalanyl-D-pyridylalanyl-L-seryl-L-tyrosyl-D-asparaginyl-L-leucyl-L-Ne-isopropyl-lysyl-L -propyl-D-alanyl-amide. To prepare a deposit of PPI-258 / CMC, 174.8 mg (148.6 mg net) of PPI-258 were added to 29.72 ml of water and the material was stirred until the peptide was suspended and dissolved. 1.85 ml of a 2% sodium CMC solution (Hercules) was added to this stirring solution. A solid precipitate was immediately observed- With heating to reflux, the suspension became translucent and then a white precipitate appeared. After 5 minutes of reflux, the reaction was cooled and the solid was separated by centrifugation. The solid was rinsed with water, and dried under vacuum overnight. The dry powder was ground in a mortar and pestle and sieved through a 90 micron stainless steel mesh. The sieved powder (90 micron sieve) was collected and characterized. The total yields were 198.4 mg of a dry solid that produced 110.8 mg of fixed size powder after the milling step. The characterization provided the following composition of the complex: peptide PPI-258-80%, CMC-18.8%, water- 6.6%.

EXAMPLE 9: In this example, an insoluble complex was formed between Cetrorelix® analogue LHRH (also known as SB-75) and carboxymethylcellulose (CMC). Cetrorelix® has the structure: acetyl-D-naphthylalanyl-D-4-Cl-phenylalanyl-D-pyridylalanyl-L-seryl-L-tyrosyl-D-citrulyl-L-leucyl-L-arginyl-L-prolyl-D -alanil-amide. To prepare a Cetrorelix / CMC deposit. 102.8 mg (87 mg net) of Cetrorelix® were added to 17.4 ml of water and the material was shaken to suspend and dissolve the peptide. To this stirred solution were added 1.1 ml of 2% sodium CMC solution (Hercules). A lumpy white precipitate was immediately observed. The suspension was heated to reflux for 5 minutes and cooled to produce a solid white precipitate. The solid was separated by centrifugation, rinsed with water and dried under vacuum overnight. The dry powder was ground in mortar and pestle and sieved through a 90 micron stainless steel mesh. The dust was collected and characterized. The total yields were 95 mg of dry solid, which produced 60 mg of powder of the same size after the milling step. The characterization provided the following composition of the complex: peptide Cetrorelix® 75%, CMC-20.7%, water-6.5% .

EXAMPLE 10 In this example, the sustained release of three different analogues of LHRH, PPI-149, PPl-258 and Cetrorelix®, prepared as CMC depot formulations as described in the three previous examples was examined in vivo. Three different formulation vehicles were tested: saline solution, glycerin (15% glycerone / 4% dextrose) and lecithin. Sprague-Dawley rats (25 males, weight range 300-325 g) were used and the efficacy of the LHRH analogue was determined based on the reduction of plasma testosterone levels. Dosage and administration routes were as follows: The actual dose of the peptide was 300 μg / kg / day for 30 days, which was 2.7 mg / rat given as a single injection of 200 μl intramuscularly (IM) or subcutaneously (SC). The total volume required to inject 5 rats / group was 1.3 ml at a concentration of 13.5 mg / ml of active peptide. The volume of the injection was kept constant and the weight of the powder was adjusted to a total peptide content as follows: A single 200 μl injection, intramuscular or subcutaneous, of the test article was made from the upper side of the left hind limb or under the skin between the scapulae, respectively, on day 0 under anesthesia. To test plasma testosterone levels, approximately 0. ml of blood was removed from the retroorbital sinus on day 1 after the dose and on days 3, 7, 14, 21, 28 and 35. The blood was processed to plasma and frozen in dry ice for the determination of plasma testosterone levels by standard methods. The representative results, shown in Figures 3A-3C, demonstrate that plasma testosterone levels in male Sprague-Dawley rats were reduced and maintained at low levels for at least 28 days and as much as 50 days in "response to sustained release. of the LHRH analogs PPI-149, PPI-258 and Centrolix® [sic] prepared as CMC deposit formulations (shown in Figures 3A, 3B and 3C, respectively) These results indicate that the three formulations are effective in reducing , in vivo, testosterone levels in plasma and keep plasma testosterone levels reduced over time.

EXAMPLE 11: In this example the PPI-149-CMC formulations were exposed to gamma radiation for sterilization purposes, followed by evaluation of the physical and chemical properties of the irradiated formulations. The data described below indicate that radiation? they are a viable means for sterilizing the PPI-149-CMC deposit.

Peptide Stability Approximately 40 mg of each of two separate batches of PPI-149-CMC were packaged separately (under an air gap) for a number of type 1 glass vials, sealed with rubber stoppers and aluminum seals. The vials were then subjected to a variety of nominal doses of gamma radiation. Two vials were analyzed for the purity of the peptide (expressed as%) at each level of radiation? exposed for each of the two lots. The results indicated that radiation doses? up to and including 24 KGy, the PPI-149-CMC consistently showed less than a 2% reduction in peptide purity (determined by the impurity profile by HPLC). A second study, using higher exposure doses?, Was performed in an additional laboratory lot of PPI-149-CMC. The PPI-149-CMC showed, in a remarkable way, good chemical stability when exposed to high doses of radiation? A subsequent preformulation study was carried out to compare the degradation profile obtained after the gamma radiation of the PPI-149-CMC with that obtained after autoclaving an injectable solution of PPI-149 (1 mg / ml). Two samples were prepared: a) PPI-149-CMC exposed to 19 KGy of radiation? b) a solution of PPI-1 9 (1 mg / ml) subjected to sterilization by autoclaving (121 ° C / 20 minutes). The HPLC chromatograms of the data show that the degradation profile of the two samples appears to be qualitatively similar (giving relatively similar retention times of the major peaks).

Stability in forced storage followed by gamma radiation Stress studies were also carried out on forced storage in preformulation in vials after gamma radiation. The sealed vials of two laboratory batches of PPI-149-CMC were exposed to 19 KG and gamma radiation, stored at 25 ° C, 37 ° C and 50 ° C for up to one month. The chemical stability data in these preformulation studies indicated that radiation? at a dose of 19 KGy followed by storage-forced stability did not cause greater chemical instability even under high storage stress conditions (for example, one week at 50 ° C). The data indicated that a dose of radiation? on and including 19 KGy, storage of PPI-149-CMC for up to 28 days to or below 50 ° C, consistently exhibited less than 2% reduction in peptide purity (as determined by the impurity profile by HPLC ). Despite a clear difference in the initial moisture content between the two batches studied, no significant difference in the purity of the peptide was determined in the stability samples of the initial preformulation or in those stored for up to one month.

Particle size analysis of PPI-149-CMC A particle size-using laser light scattering method was developed, which is applicable to classification studies according to the size of PPI-149-CMC. To illustrate the usefulness of the method, a preformulation experiment was presented, which was carried out to investigate the effect of gamma radiation on the particle size of PPI-149-CMC. This experiment was conceived with the prior understanding that amorphous solid materials may be predisposed to the consolidation of particles with storage. Two samples of a laboratory lot of PPI-149-CMC were packaged in type 1 glass vials, closed with gray butyl rubber stoppers and sealed with aluminum seals. The evaluation of the particles was performed before and after exposure to a dose of 15.5 KGy of gamma radiation. The evaluation of the particles was performed by laser light scattering (using a Malvern Mastersizer S ™ equipped with an inverted Fourier lens). Samples of 20 mg for the analysis of particle size by scattering of laser light were dispersed in approximately 0.5 ml of deionized water with vigorous agitation, then voiced in a bath at room temperature for 5 minutes. After running a fund account, a method qualification experiment was conducted. The dispersion of the sample was added in the form of drops to the continuous feed tank (approximately 60 ml nominal volume) until approximately 20% darkening was obtained. The speed of rotation of the mixer was maintained at 2700 rpm throughout the experiment (more background verification). No vortex-induced bubbles were generated at this velocity, but an adequately stable dispersion was maintained. 8 records were made, the analysis of the acquired data indicated a standard deviation of < 0.03% as the end of any data point taken. When the dispersion of the samples was maintained in the tank for 15 minutes and then it was run again, no significant change was found, indicating the absence of particle dissolution during the course of the experiment. The samples were analyzed using the experimental parameters already provided. Eight records were made and the average particle diameter was determined. Two different size distributions were observed, and all had a clean cut in the high end particle size, indicating the absence of particle aggregation. A batch of PPI-149-CMC had seemingly lower volume diameter before gamma radiation compared to the sample after radiation. This preformulation study would seem to indicate that some consolidation of particles occurs during the sterilization process.

EXAMPLE 12: In this example several preformulation experiments were conducted to investigate the effect of gamma radiations and temperature / humidity changes in the solid state of PPI-149-CMC.

Diffraction of X-rays in the powder In the initial experiment, two samples of 60 mg of PPI-149-CMC were packed (under an air space) in type 1 glass vials, closed with butyl rubber stoppers and sealed with seals. aluminum. One sample was then exposed to gamma radiation dose of 19.0 KGy. The solid state form of the two 60 mg samples was studied by X-ray powder diffraction. The diffractograms were compared before and after exposure to a dose of 19.0 KGy of gamma radiation. In a subsequent study, a 60 ml sample of PPI-149-CMC (after gamma radiation) was placed in a type 1 glass vial, placed in a pre-equilibrated constant humidity incubator at 50 ° C / 75 % relative humidity for 5 days. Immediately after removing it from the incubator, the container with the sample was closed with a plug of gray butyl rubber and sealed with an aluminum seal. The X-ray powder diffractogram of this stressed sample was then compared to another sample of the same batch that was kept at room temperature in a closed container. The samples were analyzed using a Siemens D500 automated powder diffractometer equipped with a graphite mohochromator "and a Cu X-ray source (? = 1.54 Á) operated at 50 kV, 40 mA.The exploration range 2- teat was 4- 40 ° C using a gradual log window of 0.05 ° / l.2 second step The beam slots were set to No. (1) 1 °, (2) 1 °, (3) 1 °, (4) 0.15 And (5) 0.15 ° of amplitudes.Two-theta calibration was performed using a standard NBS mica (SRM 675) .The samples were analyzed using a zero-background sample plate.The data indicated that before gamma irradiation PPI-149-CMC had no apparent crystalline or pseudocrystalline structure- In fact, it gave a powder X-ray diffraction pattern characteristic of an amorphous solid (broad hillock between 2-20 ° 20 [sic], without significant peaks in the difratogram). The post-radiation PPI-149-CMC sample generated a diffraction pattern very similar to that of the non-irradiated sample, indicating that gamma radiation processing (at doses up to and including 19 KGy) apparently does not induce a polymorphic transition in the solid state within the material. In a similar way, the PPI-149-CMC sample stressed in temperature and humidity generated a very similar diffraction pattern for the non-irradiated sample and the irradiated sample, which strongly suggests that the PPI-149-CMC is not unduly prone to the induction of polymorphic transitions in the solid state within the material.

Hygroscopicity Preformulation studies in PPI-149-CMC (after radiation) were performed to determine the moisture uptake at equilibrium (measured by weight gain) at constant temperature (25 ° C) under various relative humidity conditions . Analysis of equilibrium moisture (% water) as a function of relative humidity (% RH) indicated that the moisture content is gradually increased to approximately 80% relative humidity. At high relative humidity (95% RH) the PPI-149-CMC was able to absorb significant moisture. At humidities relative to or less than 80% RH, significant precautions in terms of moisture protection are considered not necessary; in this way, certain manufacturing steps can be taken under ambient humidity conditions (provided extreme humidity is avoided).

EXAMPLE 13: In this example, dissolution studies were carried out in PPI-149-CMC. The experiments were carried out using immersion and non-immersion conditions. The PPI-149-CMC had a solubility of approximately 100 μg / ml (measured and expressed as free peptide) at 25 ° C in 0.1 M phosphate buffered saline at pH 7.3. Under immersion conditions (defined as <10% of the ~~ saturated solubility in the system at a given temperature), even in the absence of agitation, the PPI-149-CMC was rapidly dissolved (measured and expressed as free peptide). In a similar experiment, the equilibrium solubility of PPI-149-CMC was determined (measured and expressed as free peptide) at 25 ° C and in saline buffered with 0.1 M phosphate at pH 7.3, using three samples; PPI-149-CMC only, PPI-149-CMC in the presence of an additional 10% (by weight) of PPI-149 (expressed as free peptide, but produced as PPI-149 with associated acetate) and PPI-149-CMC in the presence of an additional 50% (by weight) sodium carboxymethylcellulose USP. All three samples ostensibly gave a similar solubility of the peptide at equilibrium. As the selected mortar system approximates physiological conditions, the presence of additional free carboxymethyl cellulose or peptide species present in PPI-149-CMC seems unlikely to affect solubility.

EXAMPLE 14: In this example, the pharmacokinetics, pharmacodynamics and safety of repeated subcutaneous (SC) and intramuscular (IM) doses of PPI-CMC were characterized in dogs. In a first study, conducted over three months, 40 male beagle dogs were evaluated using monthly IM or SC injections of PPI-149-CMC at 1.2 mg / kg (day 1). 0.3 or 0.6 mg / kg (day 29) and 1.2 mg / kg (day 57) in a variety of reconstitution vehicles- Eight groups of five dogs were assigned for the study as shown below: to. the reconstitution vehicles are used to reconstitute the PPI-149-CMC as a suspension, in particular those containing the following (in water): 1. glycerin = '15% glycerin / 5% dextrose 2. PEG = 4% polyethylene glycol-3350/4% mannitol 3. lecithin = 0.5% lecithin / 5% mannitol b. Note: reconstitution vehicles used in clinical studies are 0.9% sodium chloride USP c. All doses are expressed in terms of the peptide content (PPI-149). d. Three animals were sacrificed on day 85 to perform anatomical and microscopic histology. This study was designed so that the efficacy of PPI-149-CMC at an initial dose in different vehicles was assessed during the first month of treatment. During the second month of the study, dogs received a lower dose of PPI-149-CMC in an attempt to determine an effective "maintenance" dose, and the third month was scheduled to evaluate the long-term efficacy and safety characteristics of PPI- 149-CMC The IM or SC dose of PPI-149-CMC formulated in one of the reconstitution vehicles, or IM dose of the control article were administered in each dosage day in upper side of the right hind limb (IM) or in the mid scapular region (SC). The material was extracted with an Ice tuberculin syringe with a 23 g short bevel needle. The injection site was rubbed with a swab and alcohol immediately before dosing. The volume injected was based on a specific dose of peptide / kg body weight. It should be noted that all doses refer to the amount of peptide PPI-149 administered. Each animal was observed at least twice a day throughout the study for obvious signs of toxicity or pharmacological effects and changes in general behavior and appearance. All abnormal clinical observations were recorded.

Blood was collected before administration of the first dose and at different times after dosing, for whole blood counts (CBC), chemical analysis of the jsuero and determination of PPI-149 and testosterone concentrations, twice weekly by radioimmunoassays. After three months under study, nine animals were sacrificed and their tissues were collected for macroscopic, pathological and histopathological analysis. Animals for slaughter were selected from the control vehicle group, one from the IM dosing group and one from the SC dosing group. The tissues collected for pathology and macroscopic histopathology sacrificed at three months were: administration site (SC or IM), adrenal glands, aorta, bone, bone marrow, brain, diaphragm, epididymis, esophagus, eyes with optic nerve, heart, kidneys, small intestine (cecum, colon), liver with gallbladder, lungs -with bronchi, lymph nodes, pancreas, pituitary gland, prostate gland with urethra, salivary glands, sciatic nerve, skeletal muscle, skin, small intestine (duodenum, yoyo , ileo), spinal cord, spleen, stomach, testes, thymus, thyroid gland with parathyroid, tongue, trachea, urinary bladder and lesions with the naked eye.

There were no significant changes in hematology or blood chemistry from the baseline during the study for any of the treated or control animals. The histological evaluation with the naked eye of those sacrificed at the third month showed evident differences between dogs treated with PPI-149-CMC and control animals (treated with vehicle), with the exception of changes in the testes and prostate, as expected with this LHRH antagonist. With respect to the pharmacokinetics of PPI-149-CMC, all dogs treated with 1.2 mg / kg of PPI-149-CMC, resuspended in a variety of reconstitution vehicles and administered IM or SC, showed similar pharmacokinetic profiles of PPI-149 * in plasma, with a peak plasma concentration within the first two days and then slowly decreasing exponentially during * the following month. PPI-149-CMC gave a plasma distribution similar to PPI-149 when suspended in any of the three reconstitution vehicles used in the study. With respect to the endocrine efficacy of PPI-149-CMC, castrate levels of testosterone (<0.6 ng / ml) were observed within 24 hours of the initiation of PPI-149-CMC dosing in all dogs, and Levels generally remained in the castration range throughout the first month without considering the route of administration or choice of reconstitution vehicle.

Twenty-six (26) of 35 dogs (75%) had testosterone castration levels in a blood sample obtained immediately prior to the administration of the second dose of PPI-149-CMC on day 29. These results indicated that an initial dose of 1.2 mg / kg in dogs successively induces a rapid, prolonged suppression (> 28 days) of plasma testosterone- In the second month of dosing, when the efficacy of a dose of 'maintenance' (a dose lower than the initial dose ) was investigated, the results indicated that administration of 0.3 or 0.6 mg / kg of PPI-149-CMC maintained castrate levels of testosterone for more than 20 days in 30 out of 35 dogs at the end of the second month of treatment ( day 57), 21 of 35 dogs (60%) remained castrated, while 14 animals had testosterone in the normal range (> 0.6% ng / ml) .A dose of 1.2 mg / kg was administered beginning the third month. of PPI-149 in plasma on sustained during the next 28-day period while plasma testosterone levels were again 'castrating'. At the end of the third month (day 85), plasma testosterone levels were in the castrating range in 30 of 35 dogs treated with PPI-149-CMC. In sum, thirty-five (35) dogs received 1.2 mg / kg PPI-149-CMC on day 1, 0.3 or 0.6 ml / kg of PPI-149-CMC on day 29 and 1.2 mg / kg PPI-149-CMC in the day 57, using IM or SC dosing with a variety of reconstitution vehicles. Of these 35 dogs, 19 animals (54%) had plasma testosterone levels that remained in the castration range during the course of therapy. In this way, the administration of PPI-149-CMC at 28-day intervals was able to cause a complete suppression of testosterone in plasma, which is rapid (all animals had castration levels within 24 hours) durable for a long time (maintained through the course of administration). A study similar to that debed above was conducted for six months in dogs to evaluate the long-term efficacy and safety characteristics of PPI-149-CMC. The animals received an initial dose of 1.2 mg / kg of PPI-149-CMC, IM or SC, and five subsequent doses (at a concentration of 0.3 mg / kg, 0.6 mg / kg or 1.2 mg / kg) at intervals of 28 days. PPI-149 levels of plasma testosterone were evaluated by radio immunoassay at regular intervals. Representative results are shown in Figure 4 (for SC treatment) and Figure 5 (for IM treatment), which 'illustrate plasma testosterone levels (clear squares) and PPI-149 levels (dark squares). The specific dosages provided in each administration of PPI-149-CMC are shown in the graphs. The results illustrated in Figures 4 and 5 further demonstrate that the administration of PPI-149-CMC at 28-day intervals was capable of causing a complete suppression of plasma testosterone which is rapid and long-lasting, with reduced levels of testosterone in plasma being maintained for six months.

EQUIVALENTS Those skilled in the art will recognize, or may find out, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are proposed to be included by the following claims.

Claims (52)

1. CLAIMS 1. A pharmaceutical composition comprising a solid ionic complex of a pharmaceutically active peptide and a carrier macromolecule, wherein the content of the peptide of said complex is at least 57% by weight.
2. 2. A pharmaceutical composition comprising a solid ionic complex of a pharmaceutically active peptide and a carrier macromolecule, wherein the peptide content of said complex is 57% to 79% by weight.
3. 3. A pharmaceutical composition consisting essentially of a solid ionic complex of a pharmaceutically active peptide and a carrier macromolecule, wherein the peptide content of said complex is at least 57% by weight.
4. 4. A pharmaceutical composition consisting essentially of a solid ionic complex of a pharmaceutically active peptide and a carrier macromolecule, wherein the peptide content of said complex is 57% to 79% by weight. 5- A pharmaceutical composition comprising a solid ion complex of an LHRH analog and a carrier macromolecule, wherein the carrier and analog used to form the complex are combined in a carrier weight ratio: analog of 0.5: 1 to 0.1: 1 and wherein said complex is not a microcapsule. 6. A pharmaceutical composition consisting essentially of a solid ionic complex of a pharmaceutically active peptide and a carrier macromolecule, wherein the carrier and the peptide used to form the complex are combined in a carrier weight: analog ratio of 0.5: 1 up to 0.1: 1 and wherein the complex is not a microcapsule- 7. A pharmaceutical composition comprising a solid, sterile, ionic complex of a pharmaceutically active peptide and a carrier macromolecule, wherein the complex is capable of remaining stable after administration. radiation sterilization? 8. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7 wherein the pharmaceutically active peptide is an analogue of LHRH. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutically active peptide is an LHRH antagonist. _10. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7, wherein the pharmaceutically active peptide is selected from the group "consists of bradykinin analogs, parathyroid hormone, adenocorticotropic hormone, calcitonin, vasopressin analogues. 11. The pharmaceutical composition of any one of claims 1, 2, 3, 4, 6 or 7, wherein the pharmaceutically active peptide is cationic and the carrier macromolecule is anionic. The pharmaceutical composition of any one of claims 1, 2, 3, 4, 6 or 7, wherein the complex provides sustained release of the pharmaceutically active peptide to an individual for at least one week after the pharmaceutical composition is administered to the individual . The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7, wherein the complex provides sustained release of the pharmaceutically active peptide to an individual for at least two weeks after the pharmaceutical composition is administered to the individual . The pharmaceutical composition of any one of claims 1, 2, 3, 4, 6 or 7, wherein the complex provides sustained release of the pharmaceutically active peptide to an individual for at least three weeks after the pharmaceutical composition is administered. to the individual - 15. The pharmaceutical composition of any of claims 1, 2, 3, 7, wherein the complex provides sustained release of the pharmaceutically active peptide to an individual for at least four weeks after the pharmaceutical composition is administered to the individual. 16. The pharmaceutical composition of any one of claims 1, 2, 3, 4, 6 or 7, wherein the pharmaceutically active peptide is a multivalent cationic or anionic peptide. 17. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7, wherein the peptide has from 5 to 20 amino acids in length. 18. The pharmaceutical composition of any one of claims 1, 2, 3, 4, 6 or 7, wherein the peptide has from 5 to 15 amino acids in length. 19. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7, wherein the peptide is from 8 to 12 amino acids in length. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7, wherein the carrier macromolecule is an anionic polymer. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7, wherein the carrier macromolecule is an anionic polyalcohol derivative or a fragment thereof. 22. The pharmaceutical composition of any one of claims 1, 2, 3, 4, 6 or 7, wherein the carrier macromolecule is an anionic polysaccharide derivative, or fragment thereof. 23. The pharmaceutical composition of any one of claims 1, 2, 3, 4, 6 or 7, wherein the carrier macromolecule is carboxymethylcellulose, or a fragment or derivative thereof. 24. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6, or 7, wherein the carrier macromolecule is selected from the group consisting of algin, alginate, anionic acetate polymers, anionic acrylic polymers, xanthan gums , anionic carrageenan derivatives, anionic derivatives of polygalacturonic acid, sodium starch glycolate and fragments, derivatives and pharmaceutically acceptable salts thereof. 25. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7, which is a lyophilized solid. 26. The pharmaceutical composition of any of claims 1, 2, 3, 4, 6 or 7, wherein the solid ionic complex is suspended as a liquid suspension or dispersed as a semi-solid dispersion. The pharmaceutical composition of claim 8 wherein the LHRH analogue is an LHRH antagonist comprising a peptide compound, wherein a residue of the peptide compound corresponding to the amino acid at position 6 of the mammalian natural LHRH comprises a structure D- asparagine. The pharmaceutical composition of claim 8, wherein the LHRH analogue is an LHRH antagonist comprising a peptide compound containing a structure: ABCDEFGHIJ wherein: A is pyro-Glu, Ac-D-Nal, Ac.D -Qal, Ac-Sar or Ac-D-Pal B is His or 4-Cl-D-Phe C is Trp, D-Pal, D-Nal, L-Nal, D-Pal (NO) or D-Trp D is Ser E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys (iPr), 4-Cl-Phe, His, Asn, Met, Ala, Arg or Lie; F is D-Asn, D-Gln or D-Thr; G is Leu or Trp; H is Lys (iPr), Gln, Met or Arg I is- Pro; and J is Gly-NH2 or D-Ala-NH2; or a pharmaceutically acceptable salt thereof. 29. The pharmaceutical composition of claim 8, wherein the LHRH analogue is an LHRH antagonist having the following structure: Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-N-Me -Tyr-D-Asn-Leu-Lys (iPr) -Pro-D-Ala. 30. A packaged formulation for treating an individual for a condition treatable with an LHRH analog containing: a solid ion complex of an LHRH analog and a carrier macromolecule packaged with instructions for use of the complex, to treat an individual having a condition treatable, with an LHRH analogue, wherein the content of the peptide of said complex is at least 57% by weight. 31. The packaged formulation of claim 30, wherein the LHRH analogue has the following structure: Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-N-Me-Tyr-D-Asn -Leu-Lys (iPr) -Pro-D-Ala, and the carrier macromolecule is carboxymethylcellulose. 32. In a syringe having a lumen, the improvement comprises the inclusion in the lumen of a liquid suspension of a solid ion complex of an LHRH analog and a carrier macromolecule, wherein the peptide content of said complex is at least 57 % in weigh. 33. The syringe of claim 32, wherein the LHRH analogue has the following. structure: Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-N-Me-Tyr-D-Asn-Leu-Lys (iPr) -Pro-D-Ala, and the carrier macromolecule is carboxymethylcellulose. 34. The use of the pharmaceutical composition of any of claims 1, 2, 3, 4, 5, 6 or 7 in the manufacture of a medicament for the treatment of a condition treatable with an LHRH analogue. 35. The use of claim 34, wherein the complex provides sustained release of the LHRH analog to an individual for at least one week after the pharmaceutical composition is administered to the individual. 36. The use of claim 34, wherein the complex provides sustained release of the LHRH analogue to an individual for at least two weeks after the pharmaceutical composition is administered to the individual. 37. The use of claim 34, wherein the complex provides sustained release of the LHRH analogue to an individual for at least three weeks after the pharmaceutical composition is administered to the individual. 38. The use of claim 34, wherein the complex provides sustained release of the LHRH analogue to an individual for at least four weeks after the pharmaceutical composition is administered to the individual. 39. The use of claim 34, wherein the LHRH analogue is an LHRH antagonist. 40. The use of claim 39, wherein the LHRH antagonist has the following structure: Ac-D-Nal-4-Cl-D-Phe-D-Pal-Ser-N-Me-Tyr-D-Asn- Leu-Lys (iPr) -Pro-D-Ala. 41. The use of claim 34, wherein the carrier macromolecule is an anionic polymer. 42. The use of claim 34, wherein the carrier macromolecule is an anionic polyalcohol derivative, or fragment thereof. 43. The use of claim 34, wherein the carrier macromolecule is an anionic polysaccharide derivative, or fragment thereof. 44. The use of claim 34, wherein the carrier macromolecule is carboxymethylcellulose, or a fragment or derivative thereof. 45. The use of claim 34, wherein the carrier macromolecule is selected from the group consisting of algin, alginate, anionic acetate polymers, anionic acrylic polymers, xanthan gums, anionic derivatives of polygalacturonic acids, sodium starch glycollate and fragments , derivatives and pharmaceutically acceptable salts thereof. 46. The use of claim 34, wherein the pharmaceutical composition is administered to the individual parenterally - 47. The use of claim 34, wherein the pharmaceutical composition is administered to the individual orally. 48. The use of claim 34, wherein the pharmaceutical composition is administered by intramuscular injection or subcutaneous / intradermal injection. 49. The use of claim 34, wherein the condition treatable with an LHRH analogue is a hormone-dependent cancer. 50. The use of claim 43, wherein the hormone-dependent cancer is prostate cancer. 51. The use of claim 34, wherein the condition treatable with an LHRH analogue is selected from the group consisting of: benign prostatic hypertrophy, precocious puberty, endometriosis and uterine fibroma. 52. The use of claim 34, wherein the LHRH analog is administered for contraceptive or in vitro fertilization purposes.