KR20070021331A - A pharmaceutical composition for the treatment of a condition treatable with a pharmaceutical active peptide and a packaged formulation - Google Patents

Abstract

The present invention relates to sustained release formulations containing insoluble complexes consisting of peptide compounds (eg, peptides, polypeptides, proteins, peptide analogs, etc.) and carrier polymers. The formulations of the present invention are capable of loading peptide compounds in small volumes and allow for the transport of pharmaceutically active peptide compounds for a prolonged period of time, eg 1 month after administration of the complex. The composite of the present invention can be ground or crushed into fine powder. In powder form, the complex can be formed into stable suspensions and dispersions suitable for injection. In a preferred embodiment, the peptide compound of the complex is an LHRH analog, preferably an LHRH antagonist, and the carrier polymer is an anionic polymer, preferably carboxymethylcellulose. The present invention also relates to the use of LHRH-analog-containing complexes for the treatment of symptoms treatable with LHRH analogs and to the preparation of the complexes of the invention.

LHRH analogues (LHRH antagonists), ionic interactions, carboxymethylcellulose, insoluble complexes, carrier polymers

Description

Pharmaceutical compositions and packaging formulations for the treatment of symptoms that can be treated with pharmaceutically active peptides {A PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF A CONDITION TREATABLE WITH A PHARMACEUTICAL ACTIVE PEPTIDE AND A PACKAGED FORMULATION}

1 shows plasma testosterone concentrations (ng / ml; □) and plasma PPI-149 concentrations (ng / ml; □) in rats (left graph) and dogs (right graph) during the period after intramuscular injection of PPI-149 and carboxymethylcellulose complex. / Ml; ■) is shown.

Figure 2 shows the plasma testosterone concentrations (ng / ml; □) of rats during the period following intramuscular injection of LHRH antagonist PPI-149 and carboxymethylcellulose complex on day 0 and injection of LHRH agonist Lupron ^™ on day 30. It is a graph which shows plasma PPI-149 concentration (ng / ml; ■) and demonstrates suppression of Lupron ^™ -induced testosterone spike by PPI-149 line treatment.

Figures 3A-3C show plasma of male Sprague-Dolay rats during the period following intramuscular injection of PPI-149-CMC (Figure 3A), PPI-258-CMC (Figure 3B) or Cetrorelix ^™ -CMC (Figure 3C). A series of graphs showing testosterone concentrations (ng / ml).

4 shows plasma plasma testosterone concentrations (ng / ml; □) and plasma PPI-149 concentrations (ng / ml; ■) for the period following subcutaneous injection of PPI-149-CMC at doses indicated at 28 day intervals. It is a graph demonstrating long term inhibition of plasma testosterone concentrations.

FIG. 5 shows plasma plasma testosterone concentrations (ng / ml; □) and plasma PPI-149 concentrations (ng / ml; ■) during the period following intramuscular injection of PPI-149-CMC at doses indicated at 28-day intervals. , A graph demonstrating long-term inhibition of plasma testosterone concentrations.

Various diseases and clinical diseases are treated by administering pharmaceutically active peptides. One such example is prostate cancer, which is a sex hormone-dependent cancer that can be treated by administering a luteinizing hormone releasing hormone (LHRH) analog that inhibits the production of luteinizing hormone (LH), which regulates the synthesis of testosterone. have. In particular, to reduce LH production, peptide analogs of LHRH that act as superagonists of luteinizing hormone releasing hormone receptors, such as leuprolide and goserelin, are used.

In many cases, the therapeutic effect of a pharmaceutically active peptide is due to its sustained in vivo for a long time. Sustained release formulations are preferred which do not require repeated administration to continuously release the peptide in vivo. One method for sustained release drugs is by microencapsulation, which encapsulates the active ingredient with a polymer membrane to make microparticles. For example, LHRH superagonists, such as leuprolide and goserelin, are typically encapsulated with microparticles containing poly-lactide / poly-glycolide copolymers to provide Formulations suitable for depot injection that provide sustained release (see, eg, US Pat. Nos. 4,675,189; 4,677,191; 5,480,656 and 4,728,721).

Additional sustained release formulations are needed to continuously administer the pharmaceutically active peptides in vivo for long periods of time.

The present invention provides a stable composition consisting of a peptide compound (e.g., peptide, polypeptide, protein, peptide analog, etc.), preferably a pharmaceutically active peptide compound, and a carrier polymer which makes the peptide compound sustained release in vivo after administration of the complex. Provided are pharmaceutical compositions containing water-insoluble complexes. Thus, the complex of the present invention can continuously release a pharmaceutically active peptide compound to a subject for a long time, for example 1 month. Moreover, when the peptide compound and the carrier polymer are compactly and stably combined, the peptide compound is contained at a high concentration in the formulation.

The complex of the present invention is formed by combining a peptide compound and a carrier polymer under conditions that form a practically water-insoluble complex. For example, an aqueous solution of the peptide compound and the carrier polymer is mixed until the complex precipitates. The complex may be in the form of a solid (eg, paste, granules, powder or lyophilized body) or the powder form of the complex may be finely ground to form a stable suspension or semi-solid dispersion.

In a preferred embodiment, the peptide compound of the water-insoluble complex is an LHRH analog, more preferably an LHRH antagonist, and the carrier polymer is an anionic polymer, preferably carboxymethylcellulose. The composite of the present invention is suitable for sterilization by gamma irradiation or electron beam irradiation before administration in vivo.

Also provided is a method of treating a subject having symptoms that can be treated with an LHRH analog by administering the LHRH-analog-containing composition of the present invention to the subject. In a preferred embodiment, the treatment method of the present invention is used to treat prostate cancer.

The present invention relates to pharmaceutical compositions containing a stable insoluble complex consisting of a peptide compound (e.g., peptide, polypeptide, protein, peptide analog, etc.) and a carrier polymer, a method for preparing such a composition and a method of using such a composition. . An advantage of the pharmaceutical compositions of the present invention is the ability to release pharmaceutically active peptide compounds systemically or locally and for long periods of time (e.g., weeks, months or months) and to allow the complex to contain high concentrations of peptide compounds. Is in ability.

In order to make the present invention easier to understand, terms are first defined.

As used herein, the term "peptide compound" refers to a compound consisting of at least a portion of amino acid residues linked by amide bonds (ie peptide bonds). "Peptide compounds" include peptides, polypeptides and proteins. Generally, a peptide consists of up to about 100 amino acids, more generally up to about 50 amino acid residues, most commonly up to about 25 amino acid residues. "Peptide compounds" also include peptide analogs that mimic the chemical structure of peptides consisting of peptide analogs, peptide derivatives and naturally-occurring amino acids. Examples of peptide analogs are peptides containing one or more non-natural amino acids. Examples of peptide derivatives are peptides from which amino acid side chains, peptide backbones or amino- or carboxy-terminus are derived (eg peptide compounds with methylated amide linkages). Examples of peptide analogs include peptide compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see, eg, James, G. L. et al. (1993), Science 260 : 1937-1942), all L-amino acids corresponding. "Inverso" peptides substituted with D-amino acids, amino acid sequences are reversed ("retro"), "retro-inverso" peptides and peptide backbones in which all L-amino acids are replaced ("inverso") with D-amino acids That is, there are other isotopes that include amide nitrogen, α-carbons, amidecarbonyls, complete substitution, expansion, deletion of amide bonds, or modification of skeletal cross linkages, such as amide bonds). Several peptide backbone modifications are known and include Ψ [CH ₂ S], Ψ [CH ₂ NH], Ψ [CSNH ₂ ], Ψ [NHCO], Ψ [COCH ₂ ] and Ψ [(E) or (Z) CH = CH]. In the predicates used above, Ψ indicates no amide bond. The structure which substituted the amide group is specified in parentheses. Other possible modifications include N-alkyl (or aryl) substitutions (Ψ [CONR]), skeletal crosslinks and other ring structures that make up lactams, while other derivatives include C-terminal hydroxy methyl derivatives, O-modified derivatives and alkyls. There are N-terminal modified derivatives comprising substituted amides such as amides and hydrazides.

As used herein, the term “pharmaceutically active peptide compound” refers to a peptide compound that is present in vivo or exhibits pharmacological activity upon treatment (ie, a pharmacologically active peptide compound is characterized by structural pharmacological activity). And peptide compounds in the form of "drug precursors" that are administered to exhibit pharmacological activity and then metabolized or treated in vivo in several ways).

As used herein, the terms "polyvalent cation peptide compound" and "polyvalent anion peptide compound" refer to peptide compounds having a variety of positive or negative charges, respectively. "Divalent cation" or "divalent anion" peptide compound refers to a peptide compound having two positive or negative charges, respectively. By "trivalent cation" or "trianion" peptide compound is meant a peptide compound having three positive or negative charges, respectively.

As used herein, the term "LHRH analogue" refers to a peptide compound that mimics the structure of luteinizing hormone releasing hormone. LHRH analogs include LHRH agonists or LHRH antagonists.

; 아보트/TAP), 고세레린(상품명 : Zoladex

; 제네카), 부세레린(획스트), 트리프토레린(데카펩틸로 알려진 D-Trp-6-LHRH 및 Debiopharm

; 입센/비유포어), 나파레린(상품명 Synarel

; 신텍스), 루트레린(위에트), 시스토레린(획스트), 고나도레린(아이엘스트) 및 히스트레린(오르토)이 있다.As used herein, "LHRH agonist" refers to a compound that stimulates the release of luteinizing hormone releasing hormone receptors (LHRH-R) to stimulate the release of luteinizing hormones, "LHRH antagonist" inhibits LHRH-R to corpus luteum Means a compound that inhibits the release of forming hormones. An example of an LHRH agonist is leuprolide (trade name: Lupron

; Abbott / TAP), Goserelin (Product Name: Zoladex

; Geneca), Buserelin (Chult), Tripphrine (D-Trp-6-LHRH and Debiopharm, also known as decapeptyl)

; Ibsen / Beyond Pore), Naparerine (trade name Synarel)

; Syntex), leutrerin (Wyatt), cystorerin (Chulst), gonadorerin (Ielst) and hystrerin (Ortho).

The term "LHRH antagonist" as used herein refers to a compound that inhibits the release of luteinizing hormone by inhibiting the luteinizing hormone releasing hormone receptor. Examples of LHRH antagonists include US Pat. No. 5,470,947 to Folkers et al .; PCT Publication No. WO 89/01944 to Folkers et al .; Haviv, US Pat. No. 5,413,990; Haviv, US Pat. No. 5,300,492; US Patent No. 5,371,070 to Koerber et al .; US Patent No. 5,296,468 to Hoeger et al .; US Patent No. 5,171,835 to Janaky et al .; US Patent No. 5,003,011 to Coy et al .; US Patent No. 4,431,635 to Coy; US Patent No. 4,992,421 to De et al .; US Patent No. 4,851,385 to Roeske; Nestor Jr. Antide, Cetrorelix, a compound described in U.S. Pat. No. PCT / US96 / 09852, name “LHRH antagonist peptide”), in particular their entire contents are hereby incorporated by reference. Particularly preferred LHRH antagonists have the structure: Ac-D-Nal ¹ , 4-Cl-D-Phe ² , D-Pal ³ , N-Me-Tyr ⁵ , D-Asn ⁶ , Lys (iPr) ⁸ , D-Ala ¹⁰ -LHRH, referred to herein as PPI-149.

As used herein, the term "carrier polymer" refers to a polymer capable of forming an insoluble complex with a peptide compound. Prior to complexing with the peptide compound, the carrier polymer is generally water soluble. Preferably, the polymer has a molecular weight of at least 5 KDa, more preferably 10 KDa. The term "anionic carrier polymer" refers to a negatively charged high molecular weight molecule, such as an anionic polymer. The term "cationic carrier polymer" refers to a positively charged high molecular weight molecule, such as a cationic polymer.

As used herein, the term "water-insoluble complex" refers to a physically and chemically stable complex formed by suitably combining a peptide compound and a carrier polymer according to the processes described herein. This complex generally takes the form of a precipitate prepared by combining an aqueous formulation of a peptide compound and a carrier polymer. Formation of the preferred water-insoluble complexes of the present invention is not limited by the mechanism, but includes ionic interactions in which the peptide compound is cationic and the carrier molecule is anionic or vice versa. (Ie at least partially regulated). Additionally or alternatively, the formation of the water-insoluble complex of the present invention may comprise hydrophobic interactions (ie at least partially controlled). In addition, the formation of the water-insoluble complex of the present invention may include (ie at least partially regulated) covalent interactions. Description of a complex that is “insoluble” indicates that the complex is substantially or insoluble in water, such as exhibiting precipitation in aqueous solution. However, it should be understood that the "insoluble" complex of the present invention exhibits limited solubility (ie partial solubility) in water in an aqueous physiological environment in vitro or in vivo.

As used herein, the term "sustained release" means that the drug is released in vivo continuously for a period of time following administration, preferably for at least several days, one week, or several weeks. Sustainability of the drug may be demonstrated, for example, by the continuous therapeutic effect of the drug for a period of time (e.g., for LHRH analogs, the sustained release of the analog may be demonstrated by continued inhibition of testosterone synthesis for a period of time). Can be). Alternatively, the sustained release of the agent can be demonstrated by detecting the presence of the agent in vivo for a period of time.

As used herein, the term "subject" means warm blooded animals, preferably mammals, more preferably primates, most preferably humans.

As used herein, the term “administered to a subject” refers to parenteral or oral methods, intramuscular injection, subcutaneous / endothelial injection, intravenous injection, oral administration, release by transdermal release and rectal, colon, vaginal, intranasal or respiratory route. Administration by means of dispensing, releasing or using the composition (e.g., pharmaceutical formulation) to the subject by a suitable method of releasing the composition in the desired position.

As used herein, the term "symptoms that can be treated with LHRH analogs" refer to diseases, diseases and other conditions that have a desired effect, eg, a therapeutically beneficial effect, by administration of an LHRH agonist or LHRH antagonist. Examples of symptoms that can be treated with LHRH analogs include hormone-dependent cancers (prostate cancer, breast cancer, ovarian cancer, uterine cancer and testicular cancer), benign prostatic hyperplasia, precocious adolescence, endometriosis, uterine fibroids, infertility (through in vitro fertilization) and fertilization Ability (ie use of contraceptives).

One aspect of the invention relates to a pharmaceutical composition containing an insoluble complex of a pharmaceutically active peptide compound and a carrier polymer. In a preferred embodiment, the formation of the water insoluble complex is at least partially regulated by the ionic interaction between the pharmaceutically active peptide and the carrier polymer. In such embodiments, the pharmaceutically active peptide compound is cationic and the carrier polymer is anionic or the pharmaceutically active peptide compound is anionic and the carrier polymer is cationic. In another embodiment, the formation of the water insoluble complex is at least partially regulated by hydrophobic interactions between the pharmaceutically active peptide compound and the carrier polymer. In a preferred embodiment, the peptide compound used in the complex is a polyvalent cationic peptide compound, such as a divalent or trivalent cationic peptide compound, and the carrier polymer is an anionic polymer.

The pharmaceutical composition of the present invention allows the peptide compound to be released to the subject in vivo after administration of the composition to the subject, wherein the sustained period may be changed depending on the concentration of the peptide compound and the carrier polymer used to form the complex. have. For example, in one embodiment, the single dose of insoluble complex provides sustained release of the peptide compound to the subject for at least one week after administering the pharmaceutical composition to the subject. In another embodiment, the single dose of insoluble complex provides the sustained release of the peptide compound to the subject for at least two weeks after administering the pharmaceutical composition to the subject. In another embodiment, the single dose of insoluble complex provides sustained release of the peptide compound to the subject for at least 3 weeks after administering the pharmaceutical composition to the subject. In another embodiment, the single dose of insoluble complex provides sustained release of the peptide compound to the subject for at least 4 weeks after administering the pharmaceutical composition to the subject. Also included in the invention are formulations that provide sustained release for longer or shorter periods of time, such as formulations that provide continuous release for periods of 1 day, 1-7 days, 1 month, 2 months, 3 months, and the like. Continuous release of the peptide compound over a period of months can be achieved, for example, by repeating monthly administration, each administration providing sustained release of the peptide compound for about one month (see, eg, Example 14). ).

Peptide compounds of any size are suitable for use in the complex as long as the peptide compound has the ability to form an insoluble non-covalent complex with the carrier polymer, depending on the combination of the peptide compound and the carrier polymer. However, in a preferred embodiment, the peptide compound is a peptide having 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. Various pharmaceutically active peptides can be used in the formulation and non-limiting examples thereof include LHRH analogs (described further below), bradykinin analogs, parathyroid hormones, corticosteroids (ACTH), calcitonin and vasopressin analogs (eg For example, 1-diamino-8-D-arginine vasopressin (DDAVP)).

Although various carrier polymers are suitable for forming the water-insoluble composite of the present invention, preferred polymers are polymers, preferably water-soluble polymers. In a preferred embodiment, the carrier polymer is an anionic polymer such as an anionic polyhydric alcohol derivative, or a fragment thereof and a salt thereof (eg sodium salt). Anionic moieties capable of inducing polyhydric alcohols are, for example, carboxylate, phosphate or sulfate groups. Particularly preferred anionic polymers are anionic polysaccharide derivatives, or fragments thereof and salts thereof (eg sodium salts). The carrier polymer may comprise a single molecular species (eg a single type of polymer) or two or more other molecular species (eg a mixture of two types of polymers). Examples of special anionic polymers include carboxymethylcellulose, algin, alginate, anionic acetate polymers, anionic acrylic polymers, as well as anionic carrageenan derivatives, anionic polygalacturonic acid derivatives and sulfated and sulfonated polystyrene derivatives. Xantham gum, sodium starch glycolate and fragments, derivatives and pharmaceutically acceptable salts thereof. The anionic polymer is preferably a carboxymethylcellulose sodium salt. Examples of cationic polymers are polymers of poly-L-lysine and other basic amino acids.

이고, 여기에서, R 과 X 는 독립적으로 H 또는 알킬이고 ; L 은 비교적 극성인 부분을 포함하며 ; G 는 Leu 또는 Trp 이고, H 는 Lys(iPr), Gln, Met 또는 Arg 이고, I 는 Pro 이고, J 는 Gly-NH₂ 또는 D-Ala-NH₂ 이다.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 analogs are generally 10 amino acids long. Preferred LHRH antagonists include LHRH antagonists containing peptide compounds wherein the residues of the peptide compounds corresponding to amino acids at position 6 of the natural mammalian LHRH have a D-asparagine (D-Asn) structure. The term -asparagine structure "means analogs, derivatives and mimetics thereof that retain the functional activity of D-Asn and D-Asn. Other preferred antagonists include LHRH antagonists or pharmaceutically acceptable salts thereof containing peptide compounds having the 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 Ile, F Is

Wherein R and X are independently H or alkyl; L comprises a relatively polar moiety; G is Leu or Trp, H is Lys (iPr), Gln, Met or Arg, I is Pro and J is Gly-NH ₂ or D-Ala-NH ₂ .

The term "small polar moiety" refers to a portion that has a small steric bulk and is relatively polar. Polarity is measured hydrophilically by P scale. The partition coefficient between 1-octanol and water, P, is used as a reference to determine the hydrophilicity of the compound. Hydrophilicity can be expressed as log P, logarithm of the partition coefficient [Hansch et al. Nature 194: 178 (1962); J. Am. Chem. Soc. 86: 5175 (1964). Prepare a hydrophilic baseline of several molecules and a lipophilic (hydrophobic) substituent constant (π) of several functional groups (see, for example, "Substituent Constants for Correlation Analysis in Chemistry and Biology" by Hansch and Leo, New York, NY). Willie (1979)]. The hydrophilicity of a wide range of candidate hydrophilic moieties can be very accurately predicted with the help of these tables. For example, the measured log P (octanol / water) of naphthalene is 3.45. The substituent constant p of -OH is -0.67. Therefore, the predicted log P for β-naphthol is 3.45 + (-0.67) = 2.78. This value closely matches the log P measured at 2.84 for β-naphthol. The term "relatively polar moiety" as used herein means a moiety having a log P between -1 and +2 and a steric bulk smaller than the steric bulk of Trp.

In some embodiments, L comprises a relatively polar moiety unless F is D-Cit, D-Hci or a lower alkyl derivative of D-Cit or D-Hci. Preferably, F is selected from D-Asn, D-Gln and D-Thr. More preferably, F is D-Asn. Preference is given to when E is tyrosine (Tyr) or N-methyl-tyrosine (N-Me-Tyr). In a particularly preferred embodiment, the LHRH antagonist has the structure: Ac-D-Nal ¹ , 4-Cl-D-Phe ² , D-Pal ³ , N-Me-Tyr ⁵ , D-Asn ⁶ , Lys (iPr) ⁸ , D-Ala ¹⁰ -LHRH (herein referred to as PPI-149). Particularly preferred composites of the present invention contain PPI-149 and carboxymethylcellulose.

In addition to the water-insoluble complex, the pharmaceutical formulation of the present invention may additionally contain a pharmaceutically acceptable carrier and / or excipient. As used herein, “pharmaceutically acceptable carrier” includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antibacterial agents, isotonic and absorption delaying agents, and the like. The carrier is preferably suitable for intravenous, intramuscular, subcutaneous or parenteral administration (eg by injection). Excipients include pharmaceutically acceptable stabilizers and disintegrating agents.

In addition to pharmaceutical formulations of LHRH analogs complexed with carrier polymers, the present invention includes packaging formulations containing such complexes and syringes containing such complexes. For example, the present invention provides an LHRH analogue (preferably PPI-149) and a carrier polymer (preferably with instructions for using an insoluble complex to treat a subject with symptoms that can be treated with an LHRH analogue). Packaging formulations are provided for treating subjects with symptoms that can be treated with LHRH analogs, which contain an insoluble complex of carboxymethylcellulose). In another embodiment, the present invention provides a syringe with a lumen, wherein the LHRH analog (preferably PPI-149) and the carrier polymer (preferably carboxymethylcellulose) are included in the lumen.

The complex of the present invention is prepared by combining the peptide compound and the carrier polymer under conditions that form an insoluble complex of the peptide compound and the carrier polymer. Accordingly, another aspect of the present invention relates to a method of preparing a pharmaceutical formulation. In one embodiment the method provides a peptide compound and a carrier polymer; Bonding the peptide compound and the carrier polymer under conditions to form an insoluble complex of the peptide compound and the carrier polymer; Preparation of a pharmaceutical formulation containing an insoluble complex. For example, a solution of the peptide compound and a solution of the carrier polymer are combined until an insoluble complex of the peptide compound and the carrier polymer precipitates in solution. In some embodiments, the solution of peptide compound and carrier polymer is an aqueous solution. Also, if the peptide compound or carrier molecule (or both) is not substantially water soluble prior to combining the two, then the peptide is added to a water-miscible solvent such as an alcohol (eg ethanol) prior to combining the two components of the complex. Compounds and / or carrier polymers can be dissolved. In another embodiment of the method of making the water-insoluble complex, the solution of the peptide compound and the carrier polymer solution are combined and heated until the water-insoluble complex of the peptide compound and the carrier polymer precipitates in solution. The amount of peptide compound and carrier polymer required to form the water insoluble complex may vary depending on the particular peptide compound and carrier polymer used to form the complex, the particular solvent used and / or the process used. However, in general, the peptide compound will be exceeded relative to the carrier polymer on a molar basis. Often, peptide compounds will be exceeded on a weight / weight basis, as demonstrated in the examples. In some embodiments, the carrier polymer, preferably carboxymethylcellulose sodium and the peptide compound, preferably PPI-149, is combined in a carrier polymer: peptide compound ratio of 0.2: 1 (w / w). In various other embodiments, the ratio of carrier polymer to peptide compound (w / w) can be, for example, 0.5: 1, 0.4: 1, 0.3: 1, 0.25: 1, 0.15: 1 or 0.1: 1. . Non-limiting examples of conditions and processes for preparing the water-insoluble composite of the present invention are further described in Examples 1-5 and 8-9.

Once the peptide compound / polymer complex precipitates in solution, the precipitate can be removed from the solution by means known in the art, such as by filtration (eg, through a 0.45 micron nylon membrane), centrifugation. The recovered paste is dried (eg in a vacuum or in an oven at 70 ° C.) and then the solid is ground by means known in the art (eg, hammer or gore grinding, or grinding with mortar and pestle). It can be crushed and powdered. After milling or crushing, the powder can be sieved through a screen (preferably 90 micron screen) to obtain a uniform particle distribution. Moreover, the recovered paste can be frozen and lyophilized to dry. The complex in powder form may be dispersed in a carrier solution to form a suspension or semi-solid dispersion suitable for injection. Thus, in various embodiments, the pharmaceutical formulations of the invention are dry solids, suspensions or semi-solid dispersions. Examples of suitable liquid carriers for use in suspensions are saline, glycerin solutions and lecithin solutions.

In another embodiment, the pharmaceutical formulation of the invention is a sterile preparation. For example, after forming an insoluble complex, the complex can be optimally sterilized by gamma irradiation or electron beam sterilization. Therefore, the method of preparing the pharmaceutical formulation of the present invention described above may further include sterilization of the insoluble complex by gamma irradiation or electron beam irradiation. Preferably, the formulation is sterilized by gamma irradiation using a gamma radiation dose of at least 15 KGy. In another embodiment, the formulation is sterilized by gamma irradiation using a gamma radiation dose of at least 19 KGy or at least 24 KGy. As demonstrated in Example 11, the formulations of the present invention have the stability to withstand gamma irradiation.

In addition, to prepare sterile pharmaceutical formulations, the insoluble complex can be separated using conventional sterilization methods (eg, aseptically using a sterile starting material). Thus, in another embodiment of the method of making the aforementioned pharmaceutical formulation, a sterile process is used to form the insoluble complex.

The method for forming the water-insoluble composite of the present invention is further described in Examples 1-5 and 8-9. Pharmaceutical formulations, including powders, suspensions, semi-solid dispersions, dry solids (eg lyophilized solids) and their sterilized forms, prepared according to the methods of the present invention, are also included in the present invention. Included.

Another feature of the present invention relates to a method of using the pharmaceutical formulation of the present invention for treating a subject suffering from a condition that can be treated by a pharmaceutically active peptide compound included in an insoluble complex. Thus, in a preferred embodiment, the present invention provides a method of treating a subject having a condition that can be treated with an LHRH analog, comprising administering to the subject a pharmaceutical formulation containing an insoluble complex of the LHRH analog and a carrier polymer. .

Although the preferred method of administration is parenteral, in particular intramuscular (i.m.) injections and subcutaneous / intradermal (s.c./i.d.) Injections, the pharmaceutical formulations may be administered to the subject in a manner suitable to achieve the desired therapeutic result. In addition, the formulation may be administered orally to the subject. Other suitable parenteral methods include intravenous injection, oral administration, transdermal release and administration to the rectal, vaginal, intranasal or respiratory tract route. i.m. Or s.c./i.d. When the formulations that provide sustained release for a few weeks to several months by the method are selectively administered, the extinction of the drug by other physiological mechanisms (ie, the dosage form may be extended as long as they are observed by im or sc / id injections). It can be seen that the sustained release of the medicament may not be sustained for the same time due to the therapeutic effect may be extinguished at the site of release not observed for a period of time.

Pharmaceutical formulations contain a therapeutically effective amount of LHRH analogues. A "therapeutically effective amount" means an amount effective for unit administration, for the time necessary to achieve the desired result. The therapeutically effective amount of LHRH may vary depending on factors such as disease state, age, individual weight, and the ability of the LHRH analogue (either alone or in combination with one or more other agents) to elicit an individual desired response. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also sufficient to compensate for the shortcomings of toxic or detrimental effects of antagonists as a therapeutically beneficial effect. The non-limiting range of therapeutically effective amounts of LHRH analogs is 0.01-10 mg / kg. The preferred dosage of LHRH analog PPI-149, which continues to decrease in plasma testosterone concentration for 28 days, is about 0.1-10 mg / kg, more preferably 0.3-1.2 mg / kg (free) in a suspension volume of about 1 ml or more. Shown as peptide). It will be appreciated that the value of the dosage may vary depending on the depth at which the symptoms are alleviated. For a particular subject, the particular dosing regimen should be timed according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, the dosage ranges listed herein are merely examples. It is to be understood that it is not intended to limit the scope or practice of the disclosed compositions.

The therapeutic methods of the present invention can be used to treat a variety of symptoms, diseases and disorders having the desired clinical effect by administering LHRH analogs. Examples of diseases and disorders include hormone-dependent cancers such as prostate cancer, breast cancer, ovarian cancer, uterine cancer and testicular cancer, benign prostatic hyperplasia, precocious adolescence, endometriosis and uterine fibroids. Accordingly, the present invention provides methods for treating these diseases and disorders by administering the pharmaceutical formulations of the present invention. In addition, LHRH analogs can be used to alter conception. Thus, the method of the present invention can also be used for in vitro fertilization and contraception purposes.

In a particularly preferred embodiment, the method is used to treat prostate cancer and the LHRH analogue used in the formulation is an LHRH antagonist, most preferably PPI-149, in which the method is administered after intramuscular or subcutaneous administration. Allow LHRH analogs to be sustained in vivo for at least 4 weeks. LHRH analogues prepared according to the present invention, preferably PPI-149, can be used to inhibit the growth of prostate cancer cells by administering LHRH analogues to subjects with prostate cancer. Furthermore, subjects with prostate cancer prior to initiation of treatment with LHRH agonists are administered in accordance with the present invention to pre-administer LHRH antagonists, preferably PPI-149, to inhibit testosterone spikes with the use of LHRH agonists. LHRH antagonist, preferably PPI-149, may be used. Methods of inhibiting LHRH agonist-induced testosterone spikes and treatment of other prostate cancers using LHRH antagonists, which may use the formulations of the present invention, are described in US patent application Ser. No. 08 / 573,109, filed Dec. 15, 1995. ("Methods For Treating Prostate Using LHRH Antagonists") and U.S. Patent Application Serial No. 08 / 755,593, filed November 25, 1996 ("Methods For Treating Prostate Using LHRH Antagonists"). The contents are cited in the published PCT application WO 97/22357. The entire contents of PCT applications published with US applications are specifically incorporated herein by reference.

Special methods of incorporating pharmaceutically active peptide compounds and carrier polymers are listed in Examples 1-5 and 8-9 below. It was also demonstrated that LHRH antagonist-containing complexes can provide sustained release of pharmaceutically active peptides in vivo (Example 6) and can inhibit LHRH-agonist induced testosterone spikes (Example 7). The test results are described. It is to be understood that the following examples further illustrate, but do not limit, the present invention. The contents of all references, patents, and published patent applications cited herein are hereby incorporated by reference.

Example   One

A 100 ml solution of LHRH antagonist PPI-149 was prepared by dissolving 6.25 mg / ml PPI-149 in water. The same sample (minimum 100 mL) of USP Carboxymethylcellulose Sodium (CMC) (low viscosity grade, Hercules Chemical Company) was prepared at 0.125% w / v and mixed until dissolved. The same PPI-149 and CMC solution are mixed (CMC: peptide ratio represents 0.2: 1 (w / w)) to obtain a solid material. The solid material is stirred overnight and then collected by filtration on a 0.45 micron nylon filter. Solution filtrates representing at least 95% of PPI-149 compounds are converted to solid complexes by HPLC evaluation and removed from solution. The recovered white paste is washed twice with water, then transferred to a glass bottle and dried in vacuo. Drying for 72 hours yields 633 mg of white powder. The solid material is powdered with a mortar and pestle. Elemental analysis shows 57% of the peptides in the complex.

Example   2

25 mg of PPI-149 is dissolved in 1 ml of water. 1 ml of 0.5% carboxymethylcellulose solution is added thereto and mixed to form a silvery white solid. The mixture is heated to reflux for 5 minutes to form a flocculated white precipitate. This material is separated by centrifugation / tilting. The solid is resuspended in water and collected by repeated centrifugation. HPLC evaluation of the solution filtrate showed at least 90% of the PPI-149 mixture, which was converted to a solid complex. The white precipitate is dried in vacuo and the solids are ground with a mortar and pestle. Elemental analysis shows 77% peptide in the complex.

Example   3

50 mg of PPI-149 is dissolved in 2 ml of 5% mannitol and mixed with 2 ml of 0.5% carboxymethylcellulose (low viscosity, USP, spectral quality chemicals). Stirring the mixture immediately yields a white precipitate. The suspension is frozen and lyophilized to give the PPI-149 sustained release complex.

Example   4

25 mg PPI-149 is dissolved in 1 ml of water, and 1 ml of 0.5% sodium alginate is added thereto. [See USP (Spectrum)] When mixed, the mixture immediately forms a white precipitate. The material is separated by centrifugation / tilting. The solid is resuspended in water and collected by repeated centrifugation. The white precipitate is dried in vacuo. Elemental analysis yields a 66% peptide.

Example   5

25 mg of PPI-149 is dissolved in 1 ml of water and the pH is adjusted to 11.0 by addition of ammonia. 1 ml of 0.5% alginic acid is added thereto. [See USP (Spectrum)] When mixed, the mixture immediately forms a white precipitate. This material is separated by centrifugation / tilting. The solid is resuspended in water and collected by repeated centrifugation. The white precipitate is dried in vacuo. Elemental analysis yields a peptide of 79% content.

Example   6

According to the above embodiment, an insoluble complex of LHRH antagonist PPI-149 and carboxymethyl cellulose was prepared. Suspensions of the PPI-149 / CMC complex are prepared and a single dose is injected intramuscularly in rats and dogs. The dose of rats is 50 μg / kg / day × 60 days and the dose to dogs is 40 μg / kg / day × 28 days. Plasma testosterone concentrations (ng / ml) are measured at various time points as measured by the activity of LHRH antagonists in animals. Representative results are shown in the graph of FIG. 1, which indicates that intramuscular injection of the PPI-149 / CMC complex of plasma testosterone concentrations (indicated by “□” in FIG. 1) for at least 42 days in mice and at least 28 days in dogs. Demonstrates sustained suppression and demonstrates sustained release of LHRH antagonists. Plasma concentrations (ng / ml) of PPI-149 are also measured in animals (marked with “■” in FIG. 1). Initial spikes of PPI-149 were observed for the first approximately 8 days, after which PPI-149 was essentially undetectable in plasma. Although PPI-149 could not be detected in plasma for more than about 8 days, the resulting testosterone concentrations demonstrate that PPI-149 has therapeutic activity in vivo during the course of the experiment.

Example   7

According to the above embodiments, an insoluble complex of LHRH antagonist PPI-149 and carboxymethylcellulose is prepared. Suspensions of the PPI-149 / CMC complex are prepared and a single dose is injected into the rat muscle on day 0. On day 30, the LHRH agonist Lupron ^™ (leuprolide) is injected into the rat. As measured by the activity of LHRH antagonists in animals, plasma testosterone concentrations (ng / ml; indicated by “□” in FIG. 2) are measured at various time points. Plasma concentrations (ng / ml) of PPI-149 are also measured in animals (indicated by “■” in FIG. 2). Representative results shown in the graph of FIG. 2 show that rapid pretreatment of PPI-149 / CMC complex rapidly reduces plasma testosterone to castration levels and, moreover, blocks LHRH agonist-induced testosterone spikes. Prove it. Although no PPI-149 can be detected in plasma for about 8 days or more, testosterone concentration results demonstrate that PPI-149 still has therapeutic activity in vivo during the course of the experiment.

Example   8

In this example, an insoluble complex is formed between the LHRH analogue PPI-258 and carboxymethylcellulose (CMC). PPI-258 has the following structure: Acetyl-D-naphthylalanyl-D-4-Cl-phenylalanyl-D-pyridylalanyl-L-seryl-L-tyrosyl-D-asparaginyl- L-leucil-LN ^e -isopropyl-lysyl-L-propyl-D-alanyl-amide. To prepare the PPI-258 / CMC residue, 174.8 mg (148.6 mg net weight) of PPI-258 is added to 29.72 mL of water and the material is stirred to suspend and dissolve the peptides. To this stirred solution is added 1.85 mL of 2% sodium CMC solution (Hercules). Solid precipitate is observed immediately. The suspension is heated to reflux to become translucent and then appear as a white precipitate. The reaction is refluxed for 5 minutes, then cooled and centrifuged to separate the solids. The solid is washed with water and dried in vacuo overnight. The dried powder is ground with a mortar and pestle and sieved with a 90 micron stainless steel screen. Sieved powder (90 micron sieve) is collected and characterized. The total yield is 198.4 mg of dry solids, which resulted in 110.8 mg of granulated powder after the grinding process. Characterization provides the following compositional composition of the complex: peptide PPI-258-80%, CMC-18.8%, water-6.6%.

Example   9

In this example, an insoluble complex is formed between the LHRH analogue Cetrorelix ^™ (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-prolyyl-D-alanyl-amide. To prepare Cetrorelix / CMC retentate, 102.8 mg (87 mg net weight) of Cetrorelix ^™ is added to 17.4 mL of water and the material is stirred to suspend and dissolve the peptides. To this stirred solution is added 1.1 mL of 2% sodium CMC solution (Hercules). A massive white precipitate is observed immediately. Heating and cooling the suspension while refluxing for 5 minutes yields a solid white precipitate. The solids are separated by centrifugation, washed with water and dried in vacuo overnight. The dried powder is ground with a mortar and pestle and sieved with a 90 micron stainless steel screen. The powder is collected and characterized. The total yield is 95 mg of dry solid which is produced as 60 mg of granulated powder after the grinding process. Characterization provides the composition of the following compositions of the complex: peptide Cetrorelix ^™ -75%, CMC-20.7%, water-6.5%.

Example   10

In this example, the sustained release of three different LHRH analogs, PPI-149, PPI-258 and Cetrorelix ^™ , prepared with the CMC reservoir formulation described in the three examples described above, is tested in vivo. Excipients in three different formulations are tested with saline, glycerin (15% glycerin / 4% dextrose) and lecithin. Sprag-Doleley rats (25 males, weight range 300-325 g) are used and the efficacy of LHRH analogs is determined based on a decrease in plasma testosterone concentrations.

Dosage and method of administration are as follows:

The actual dose of peptide is 300 μg / kg / day for 30 days, which is a single 200 μl given in 2.7 mg / rat upon intramuscular (IM) or subcutaneous (SC) injection. The total volume required to inject 5 rats / group was 1.3 ml at a concentration of 13.5 mg / ml activating peptide. The injection volume was kept constant and the weight of the powder was adjusted for the total peptide content as follows:

A single 200 μl intramuscular, or subcutaneous injection of the test article is injected under anesthesia into the upper flank of the left hind limb or skin between the shoulder blades on day 0, respectively.

To test plasma testosterone concentrations, about 0.4 ml of blood is obtained in the post-orbital sinus at 1 day and 3, 7, 14, 21, 28 and 35 days after administration. Blood is treated with plasma and frozen on dry ice while measuring testosterone plasma levels by standard methods.

Representative results, shown in FIGS. 3A-3C, show a sustained release of LHRH analogs PPI-149, PPI-258 and Centrolix ^™ prepared as a CMC retention formulation for 50 days with reduced plasma testosterone concentrations in male Sprague-Doleley rats. Demonstrate that the reaction is maintained at low concentrations for at least 28 days (shown in FIGS. 3A, 3B and 3C, respectively). These results indicate that all three formulations are effective in reducing plasma testosterone concentrations in vivo and maintaining reduced plasma testosterone concentrations for a given time.

Example   11

In this example, the PPI-149-CMC formulation is exposed to gamma irradiation for sterilization, followed by evaluation of the physical and chemical properties of the irradiated formulation. The data described below indicate that γ-irradiation is a viable sterilization means of PPI-149-CMC residues.

Peptide stability

Two separate PPI-149-CMC lots of about 40 mg each were separated (filled in air overhead space) into various Type I vials and sealed with rubber stoppers and aluminum seals. The vial is then subjected to various small amounts of gamma-irradiation. At each level of γ-irradiation exposure to each of the two lots, two vials are analyzed for peptide purity (expressed in%). As a result, it can be seen that PPI-149-CMC consistently shows a decrease of 2% or less (measured by HPLC impurity shape) in peptide purity at γ-irradiation of 24 KGy or less. Secondary studies with higher doses of gamma exposure are done with additional experimental lots of PPI-149-CMC. PPI-149-CMC showed very good chemical stability when exposed to high γ-irradiation.

The following preemptive studies were performed to compare the degradation profiles obtained after PPI-149-CMC γ-irradiation with those obtained after autoclaving PPI-149 injections (1 mg / ml). Two samples: a) PPI-149-CMC exposed to 19 KGy γ-irradiation; b) Prepare PPI-149 solution (1 mg / ml) subjected to pressurized heating (121 ° C./20 min). HPLC chromatograms of the two samples show that the decomposition patterns of the two samples are quantitatively similar (representing a similar relationship between the retention times of the major peaks).

Gamma-Stores Pressure Stability with Irradiation

Pressure-storage preemptive studies are done after glass via-gamma-irradiation. Sealed vials consisting of two experimental lots of PPI-149-CMC are exposed to 19 KGy gamma-irradiation and stored at 25 ° C., 37 ° C. and 50 ° C. for up to one month. The chemical stability data of these preemptive studies indicate that the pressure-storage stability following the γ-irradiation at a dose of 19 KGy does not lead to significant chemical instability even under high pressure-storage conditions (eg 1 week at 50 ° C). Indicates. The data indicate storage of PPI-149-CMC up to 28 days at 50 ° C. or below at γ-irradiation of 19 KGy or less, consistently showing a reduction of 2% or less in peptide purity (measured by HPLC impurity shape). . Although there was a clear difference in the initial moisture content between the two lots studied, no significant difference in peptide purity was measured in the initial preemptive stability samples or samples stored for up to one month.

PPI -149- CMC  Particle Size Analysis

A particle size method using laser beam scattering has been developed and applied to the particle size study of PPI-149-CMC. To illustrate the practicality of the method, preemptive experiments are performed to study the effect of gamma-irradiation on the particle size of PPI-149-CMC. In this experiment, it is necessary to understand in advance that the amorphous solid material is pre-treated upon storage, causing the particles to solidify. Two samples of PPI-149-CMC from the experimental lot are filled into a Type I vial, sealed with a gray butyl rubber stopper and sealed with an aluminum seal. Particle evaluation is performed before and after exposure to a gamma dose of 15.5 KGy. Particle evaluation was performed by laser light scattering (using a Malvern Mastersizer S us station Four ^TM which the lens device in). A 20 mg sample for particle size analysis by laser beam scattering is dispersed in about 0.5 ml of demineralized water with vigorous shaking and then ultrasonically crushed in a room temperature bath for 5 minutes. After the background coefficients are manipulated, a method limit experiment is performed. The sample dispersion is dipped into a continuous feed reservoir (about 60 mL nominal volume) until an opacity of about 20% is obtained. In the experiment, the mixer rotation speed was maintained at 2700 rpm (plus background check). At this speed no vortex-induced bubbles were generated, but a sufficiently stable dispersion was maintained. Analysis of the data obtained after 8 scans revealed a standard deviation of <0.03% as the extreme value of the data points taken. No significant change was observed when sample dispersion was maintained in the reservoir for 15 minutes and then re-manipulated, and no particle dissolution appeared during the experiment.

Samples are analyzed using the experimental parameters given above. Eight scans are performed to determine average particle diameter data. Looking at two distinct size distributions, both were sharply cut at the height-end particle size, with no particle aggregation. One lot of PPI-149-CMC had a significantly lower average volume diameter before gamma irradiation than after sample-irradiation. In these preemptive studies, it can be seen that there is some particle solidification that occurred during the sterilization process.

Example   12

In this example, various preemptive experiments are conducted to study the effects of gamma-irradiation and temperature / humidity pressure on the solid form of PPI-149-CMC.

X-ray powder diffraction

In an initial experiment, two 60 mg samples of PPI-149-CMC were filled into a Type I vial (under the air overhead space), sealed with a gray butyl rubber stopper and sealed with an aluminum seal. Next, one sample is exposed to a gamma-irradiation of 19.0 KGy. The solid form of the two 60 mg samples is then studied by X-ray powder diffraction. The diffraction patterns before and after exposure to gamma irradiation of 19.0 KGy are compared.

In the next study, a 60 mg sample of PPI-149-CMC (post gamma-irradiation) was placed in a Type I vial and placed in a line-equilibrium constant humidity warmer at 50 ° C./75% relative humidity for 5 days. Immediately after removal from the warmer, the sample vessel is sealed with a gray butyl rubber stopper and sealed with an aluminum seal. The X-ray powder diffraction pattern of this compressed sample is compared to other samples in the same lot kept at room temperature in a closed container. Samples are analyzed using a graphite monochromator and a Siemens D 500 automatic powder diffractometer equipped with a Cu (λ = 1.54 μs) X-ray source operated at 50 kV, 40 mA. The two-theta scan range is 4-40 ° using a step scan window of 0.05 ° / 1.2 seconds. The beam voids are fixed at numbers (1) 1 °, (2) 1 °, (3) 1 °, (4) 0.15 ° and (5) 0.15 ° widths. Two-theta calibration is done using the NBS Mica Standard (SRM 675). Samples are analyzed using a zero background sample plate.

The data reveal that prior to gamma irradiation, PPI-149-CMC does not have an apparent crystal or pseudo-crystal structure. Indeed, it exhibits an amorphous solid X-ray powder diffraction pattern characteristic (a wide hump between 2-20 ° 20, with no noticeable peaks in the diffraction pattern). After the PPI-149-CMC sample-irradiation produces a diffraction pattern very similar to the non-irradiated sample, and gamma-irradiation treatment (at dosages below 19 KGy) does not induce solid-phase polymorphic changes in the material. Indicates. In a similar way, the PPI-149-CMC's temperature / humidity compression sample produces a diffraction pattern very similar to the two non-irradiated and irradiated samples, which causes the PPI-149-CMC to transiently change solid-phase polymorphic changes in the material Strongly suggests no tendency to lead.

Hygroscopic

Preemption studies on PPI-149-CMC (post-irradiation) are conducted to determine equilibrium moisture absorption (measured by weight gain) at a constant temperature (25 ° C.) under various relative humidity conditions. Analyzing equilibrium moisture (% water) as a function of relative humidity (% RH) indicates that the moisture content gradually increases to about 80% relative humidity. PPI-149-CMC can absorb moisture significantly at high relative humidity (95% RH). No significant care is taken with regard to protection from moisture at relative humidity of 80% RH or less; Therefore, the manufacturing step can be carried out under ambient humidity conditions (avoid supply of extreme humidity).

Example   13

In this example, dissolution studies on PPI-149-CMC are conducted. Experiments are performed using inhalation and non-inhalation conditions. PPI-149-CMC has a solubility (indicated by free peptide) of about 100 μg / ml at 25 ° C. in 0.1 M phosphate buffered saline at pH 3.3. Under inhalation conditions (defined as <10% saturated solubility of the system at a given temperature), PPI-149-CMC dissolves quickly (with measurement as free peptide) without stirring. In a similar experiment, three samples: PPI-149-CMC alone, PPI-149-CMC in the presence of 10% additional (weight) PPI-149 (labeled as free peptide, but injected with PPI-149 associated with acetate) and Equilibrium solubility of PPI-149-CMC is measured at 25 ° C. in 0.1 M phosphate buffered saline at pH 7.3 using PPI-149-CMC in the presence of 50% additional (weight) carboxymethylcellulose sodium USP (free peptide Measured and displayed). All three samples show apparently similar peptide equilibrium solubility. Since the buffer system chosen is close to physiological conditions, the presence of additional free carboxymethylcellulose or peptide species present in PPI-149-CMC does not appear to affect solubility.

Example   14

In this example, the pharmacokinetics, pharmacology and safety of repeated subcutaneous (SC) and intramuscular (IM) doses of PPI-149-CMC are characterized in dogs.

In a first study conducted over three months, 40 PPI-149-CMCs were taken at 1.2 mg / kg (1 day), 0.3 or 0.6 mg / kg (29 days) and 1.2 mg / kg (57 days) in various reconstituted excipients. Male beagle dogs are evaluated by monthly IM or SC injection. Eight groups of five dogs are allocated to the studies shown below:

a. Reconstitution excipients are used to reconstitute PPI-149-CMC as a special suspension. They contain the following components (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: The reconstituted excipient used in clinical studies is 0.9% sodium chloride USP. c. All doses are expressed in peptide (PPI-149) content. d. Three animals were sacrificed at 85 days for complete anatomical and microscopic histology.

This study was designed to evaluate the efficacy of PPI-149-CMC at the initial dose in other excipients during the first month of treatment. During the second month of the study, the dog received a lower PPI-149-CMC dose in an attempt to measure an effective "maintenance" dose. Three months were planned to evaluate the long-term safety and efficacy characteristics of PPI-149-CMC.

The IM or SC dose of PPI-149-CMC formulated as one of the reconstituted excipients, or the IM dose of the comparator, may be used as a daily dose in the upper flank of the right hind limb (IM) or in the middle-shoulder bone area (SC). Administration. The material is placed into a 1 'tuberculin syringe with 23 g short bevel needle. Immediately before injection, wipe the injection site with an alcohol swab. The volume to be injected is based on the specific dosage of peptide / kg body weight. It can be seen that all doses are related to the amount of PPI-149 peptide administered.

Obvious signs of toxic or pharmacological effects and changes in general behavior and appearance are observed at least twice daily for each animal during the entire study period. Record any abnormal clinical observations.

To determine PPI-149 and testosterone concentrations twice a week by whole cell count (CBC), serum chemistry and radioimmunoassay, blood is collected at various times prior to and after the first dose.

Three months after the study, nine animals were sacrificed and their tissues collected for total pathological and histopathological analysis. An excipient control group, one in the IM-administered group and one in the SC-administered group, were selected as animals to be sacrificed. Tissues collected for total pathology and histopathology at the three-month sacrifice included the site of administration (SC or IM), kidney line, aorta, bone, bone marrow, brain, diaphragm, epididymis, esophagus, eye with optic nerve, heart, kidney, colon (Cecum, colon), liver with gallbladder, lungs with bronchus, lymph nodes, pancreas, mucous glands, urethra with prostate, salivary glands, sciatic nerve, skeletal muscle, skin, small intestine (duodenum, jejunum, ileum), spinal cord, spleen , Thyroid gland with stomach, testicles, thymus, parathyroid gland, tongue, respiratory tract, bladder and gross disorders.

There were no significant changes in hematology or blood chemistry at baseline during the study on treated or control animals. In the total and histological evaluation of the three-month sacrifice, there were no significant differences between the PPI-149-CMC treated dogs and the comparative (excipient-treated) animals, except for the changes in testicles and prostate expected from these LHRH antagonists. Did.

In PPI-149-CMC pharmacokinetics, all dogs treated with 1.2 mg / kg PPI-149-CMC resuspended in various reconstituted excipients and administered with IM or SC had peaked plasma concentrations on the first 2 days. A similar plasma PPI-149 pharmacokinetic shape, slowly decreasing exponentially over the next month. PPI-149-CMC appears similar to the plasma distribution of PPI-149 when suspended in any one of the three reconstituted excipients used in the study.

For PPI-149-CMC endocrine gland efficacy, testosterone concentrations when castrated (<0.6 mg / ml) were observed within the first 24 hours of administration of PPI-149-CMC to all dogs, and concentrations were generally administered with reconstituted excipients. Regardless of the method or choice, it remained within range when castrated for the first month. Twenty six (26) of the 35 dogs (75%) had testosterone concentrations when castrated in blood samples obtained immediately before the second dose of PPI-149-CMC on day 29. These results indicate that an initial dose of 1.2 mg / kg in dogs successfully induces rapid, long-lasting suppression (> 28 days) of plasma testosterone. In the second month of administration, when studying the efficacy of a "maintenance" dose (lower than the initial dose), 30 out of 35 dogs were given 0.3 or 0.6 mg / kg of PPI-149-CMC. It shows the result of maintaining the concentration of testosterone when castrated for 20 days or more. At the end of the second month of treatment (day 57), 21 of 35 dogs (60%) remained at the concentration when castrated, while 14 animals had a normal range (> 0.6% mg / ml) of testosterone. At the beginning of the third month a dose of 1.2 mg / kg was administered. The plasma concentration of PPI-149 lasted for the next 28 days, while the plasma concentration of testosterone was at the concentration when it was castrated again. At the end of the third month (day 85), the plasma concentration of testosterone indicates that 30 out of 35 PPI-149-CMC-treated dogs are at concentration when castrated.

In summary, thirty-five (35) dogs use various reconstituted excipients for IM or SC administration, with 1.2 mg / kg PPI-149-CMC per day, 0.3 or 0.6 mg / kg PPI-149- per day 29 1.2 mg / kg PPI-149-CMC was administered at CMC and 57 days. Of these 35 dogs, 19 animals (54%) had a range of plasma testosterone concentrations when castrated during the entire course of treatment. Thus, administration of PPI-149-CMC at 28-day intervals can result in complete suppression of plasma testosterone that is rapid (with concentrations when all animals castrated within 24 hours) and long-term (maintained during the administration). have.

A study similar to the one described above is conducted for 6 months in dogs to further evaluate the long-term safety and efficacy characteristics of PPI-149-CMC. Animals were given an initial dose of 1.2 mg / kg PPI-149-CMC and five doses (0.3 mg / kg, 0.6 mg / kg or 1.2 mg / kg) at 28-day intervals by IM or SC. did. Plasma testosterone and PPI-149 concentrations were evaluated by radioimmunoassay at regular intervals. Representative results are shown in FIGS. 4 (for SC treatment) and 5 (for IM treatment), illustrating plasma testosterone concentrations (“□”) and PPI-149 concentrations (“■”). The particular dose used for each dose of PPI-149-CMC is shown in the graph. The results illustrated in FIGS. 4 and 5 further suggest that administration of PPI-149-CMC at 28-day intervals can result in complete suppression of long-lasting plasma testosterone to maintain rapid and slowed plasma testosterone concentrations for 6 months. Prove it.

Equal Description

Those skilled in the art will recognize, or be able to recognize, various equivalents to the specific embodiments of the invention described herein without using routine experimentation. Such equivalents will be completed by the following claims.

A pharmaceutical composition for treating a condition treatable with a pharmaceutically active peptide, comprising an insoluble complex consisting of a pharmaceutically active peptide and a carrier polymer of the invention, provides sustained release of the pharmaceutically active peptide over several weeks. It is possible to contain a high concentration of the peptide compound in the complex (high peptide loading) and to maintain the safety of the complex even after sterilization by γ-ray irradiation.

Claims (90)

A pharmaceutical composition containing an insoluble complex of a pharmaceutically active peptide compound and a carrier polymer. The pharmaceutical composition of claim 1, wherein the formation of the water-insoluble complex is at least partially regulated by the ionic interaction between the pharmaceutically active peptide compound and the carrier polymer. The pharmaceutical composition according to claim 2, wherein the pharmaceutically active peptide compound is cationic and the carrier polymer is anionic. The pharmaceutical composition according to claim 2, wherein the pharmaceutically active peptide compound is anionic and the carrier polymer is cationic. The pharmaceutical composition of claim 1, wherein the formation of the water-insoluble complex is at least partially regulated by hydrophobic interaction between the pharmaceutically active peptide compound and the carrier polymer. The pharmaceutical composition of claim 1, wherein the single dose of insoluble complex provides sustained release of the pharmaceutically active peptide to the subject for at least one week after administering the pharmaceutical composition to the subject. The pharmaceutical composition of claim 1, wherein the single dose of insoluble complex provides the subject with sustained release of the pharmaceutically active peptide for at least two weeks after administering the pharmaceutical composition to the subject. The pharmaceutical composition of claim 1, wherein the single dose of insoluble complex provides sustained release of the pharmaceutically active peptide to the subject for at least three weeks after administering the pharmaceutical composition to the subject. The pharmaceutical composition of claim 1, wherein the single dose of insoluble complex provides sustained release of the pharmaceutically active peptide to the subject for at least 4 weeks after administering the pharmaceutical composition to the subject. The pharmaceutical composition of claim 1, wherein the pharmaceutically active peptide compound is a polyvalent cationic or anionic peptide. The pharmaceutical composition according to claim 1, wherein the peptide is 5 to 20 amino acids in length. The pharmaceutical composition according to claim 1, wherein the peptide is 8 to 15 amino acids in length. The pharmaceutical composition according to claim 1, wherein the peptide is 8 to 12 amino acids in length. The pharmaceutical composition of claim 1, wherein the carrier polymer is an anionic polymer. The pharmaceutical composition according to claim 1, wherein the carrier polymer is an anionic polyhydric alcohol derivative or fragment thereof or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 1, wherein the carrier polymer is an anionic polysaccharide derivative or fragment thereof or a pharmaceutically acceptable salt thereof. The pharmaceutical composition of claim 1, wherein the carrier polymer is carboxymethylcellulose or a pharmaceutically acceptable salt thereof. 2. An alginate, anionic acetate polymer, anionic acrylic polymer, xanthan gum, anionic carrageenan derivative, anionic polygalacturonic acid derivative, sodium starch glycorate, and fragments thereof, wherein the carrier polymer is known. Pharmaceutical composition, characterized in that it is selected from derivatives and pharmaceutically acceptable salts. 2. A pharmaceutical composition according to claim 1 in the form of a dry solid. 2. A pharmaceutical composition according to claim 1 in the form of a liquid suspension or semi-solid dispersion. A pharmaceutical composition containing an insoluble complex consisting essentially of a pharmaceutically active peptide compound and a carrier polymer. A pharmaceutical composition containing an insoluble complex of an LHRH analog and a carrier polymer. The pharmaceutical composition of claim 22, wherein the formation of the water-insoluble complex is at least partially regulated by the ionic interaction between the LHRH analog and the carrier polymer. The pharmaceutical composition of claim 22, wherein the formation of the water insoluble complex is at least partially regulated by hydrophobic interaction between the LHRH analog and the carrier polymer. The pharmaceutical composition of claim 22, wherein the single dose of insoluble complex provides sustained release of the LHRH analog to the subject for at least one week after administering the pharmaceutical composition to the subject. The pharmaceutical composition of claim 22, wherein the single dose of insoluble complex provides sustained release of the LHRH analog to the subject for at least two weeks after administering the pharmaceutical composition to the subject. The pharmaceutical composition of claim 22, wherein the single dose of insoluble complex provides sustained release of the LHRH analog to the subject for at least three weeks after administration of the pharmaceutical composition to the subject. The pharmaceutical composition of claim 22, wherein the single dose of insoluble complex provides sustained release of the LHRH analog to the subject for at least 4 weeks after administration of the pharmaceutical composition to the subject. The pharmaceutical composition of claim 22, wherein the LHRH analog is an LHRH antagonist. The pharmaceutical composition of claim 29, wherein the LHRH antagonist contains a peptide compound and the residue of the peptide compound corresponding to the amino acid at position 6 of the native mammalian LHRH has a D-asparagine structure. The pharmaceutical composition according to claim 29, wherein the LHRH antagonist contains a peptide compound or a pharmaceutically acceptable salt thereof having the structure: A-B-C-D-E-F-G-H-I-J From here, 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 (N-O) 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 Ile, F is

ego     In this expression           R and X are independently H or alkyl;           L contains a small polar moiety; G is Leu or Trp, H is Lys (iPr), Gln, Met or Arg, I is Pro, J is Gly-NH ₂ or D-Ala-NH ₂ . 32. The pharmaceutical composition of claim 31, wherein F is selected from D-Asn, D-Gln, and D-Thr. 32. The pharmaceutical composition of claim 31, wherein F is D-Asn. 32. The pharmaceutical composition of claim 31, wherein E is tyrosine or N-methyl-tyrosine. The method of claim 29, wherein the LHRH antagonist has the structure: Ac-D-Nal ¹ , 4-Cl-D-Phe ² , D-Pal ³ , N-Me-Tyr ⁵ , D-Asn ⁶ , Lys (iPr) ⁸ , D-Ala ¹⁰ -LHRH having a pharmaceutical composition. The pharmaceutical composition of claim 22, wherein the carrier polymer is an anionic polymer. 23. The pharmaceutical composition of claim 22, wherein the carrier polymer is an anionic polyhydric alcohol derivative or fragment thereof or a pharmaceutically acceptable salt thereof. 23. The pharmaceutical composition of claim 22, wherein the carrier polymer is an anionic polysaccharide derivative or fragment thereof or a pharmaceutically acceptable salt thereof. 23. The pharmaceutical composition of claim 22, wherein the carrier polymer is carboxymethylcellulose or a pharmaceutically acceptable salt thereof. 23. The method of claim 22, wherein the alginate, anionic acetate polymer, anionic acrylic polymer, xanthan gum, anionic carrageenan derivative, anionic flipgalacturonic acid derivative, sodium starch glycorate, and fragments thereof are known. Pharmaceutical composition, characterized in that it is selected from derivatives and pharmaceutically acceptable salts. The pharmaceutical composition according to claim 22, which is in dry solid form. The pharmaceutical composition according to claim 22, which is in the form of a liquid suspension or semi-solid dispersion. Subjects with symptoms that can be treated with an LHRH analogue containing an insoluble complex of an LHRH analogue and a carrier polymer, packaged with instructions for using an insoluble complex to treat a subject having symptoms that can be treated with an LHRH analogue. Packaged formulations for treatment. 44. The method of claim 43, wherein the LHRH analogue has the structure: Ac-D-Nal ¹ , 4-Cl-D-Phe ² , D-Pal ³ , N-Me-Tyr ⁵ , D-Asn ⁶ , Lys (iPr) ⁸ , D-Ala ¹⁰ -LHRH, wherein the carrier polymer is carboxymethylcellulose or a pharmaceutically acceptable salt thereof. A syringe having the lumen, wherein the lumen is improved by including a suspension of an insoluble complex of an LHRH analog and a carrier polymer. 46. The method of claim 45, wherein the LHRH analogue has the structure: Ac-D-Nal ¹ , 4-Cl-D-Phe ² , D-Pal ³ , N-Me-Tyr ⁵ , D-Asn ⁶ , Lys (iPr) ⁸ , D-Ala ¹⁰ -LHRH, characterized in that the carrier polymer is carboxymethylcellulose or a pharmaceutically acceptable salt thereof. Method for preparing a pharmaceutical formulation comprising the following steps Providing a peptide compound and a carrier polymer; To form an insoluble complex of a peptide compound and a carrier polymer Binding the peptide compound and the carrier polymer under the conditions; Preparing a pharmaceutical formulation containing the insoluble complex. 48. The method of claim 47, wherein the solution of the peptide compound and the solution of the carrier polymer are combined until the water-insoluble complex of the peptide compound and the carrier polymer precipitates. 49. The method of claim 48, wherein the solution of the peptide compound and the solution of the carrier polymer are aqueous solutions. 49. The process according to claim 48, wherein the solution of the peptide compound and the solution of the carrier polymer are heated by bonding until the water-insoluble complex of the peptide compound and the carrier polymer precipitates. 48. The method of claim 47, further comprising sterilizing the insoluble composite by gamma irradiation or electron beam irradiation. 48. The process of claim 47, wherein the water-insoluble complex is formed under a sterile process. 48. The method of claim 47, wherein the peptide compound is cationic and the carrier polymer is anionic. 48. The method of claim 47, wherein the peptide compound is anionic and the carrier polymer is cationic. 48. The method of claim 47, wherein the peptide compound is a polyvalent cationic or anionic peptide. 48. The method of claim 47, wherein the peptide compound is an LHRH analog. 59. The method of claim 56, wherein the LHRH analogue is an LHRH antagonist. 58. The method of claim 57, wherein the LHRH antagonist contains a peptide compound and the residue of the peptide compound corresponding to the amino acid at position 6 of the native mammalian LHRH has a D-asparagine structure. The method of claim 57, wherein the LHRH antagonist contains a peptide compound or a pharmaceutically acceptable salt thereof having the structure: A-B-C-D-E-F-G-H-I-J From here, 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 (N-O) 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 Ile, F is

ego     In this expression           R and X are independently H or alkyl;           L contains a small polar moiety; G is Leu or Trp, H is Lys (iPr), Gln, Met or Arg, I is Pro, J is Gly-NH ₂ or D-Ala-NH ₂ . 60. The method of claim 59, wherein F is selected from D-Asn, D-Gln, and D-Thr. 60. The method of claim 59, wherein F is D-Asn. 60. The process of claim 59, wherein E is tyrosine or N-methyl-tyrosine. 59. The method of claim 57, wherein the LHRH antagonist has the structure: Ac-D-Nal ¹ , 4-Cl-D-Phe ² , D-Pal ³ , N-Me-Tyr ⁵ , D-Asn ⁶ , Lys (iPr) ⁸ , D-Ala ¹⁰ -LHRH characterized in that the manufacturing method. 48. The method of claim 47, wherein the carrier polymer is an anionic polymer. 48. The method of claim 47, wherein the carrier polymer is an anionic polyhydric alcohol derivative or fragment thereof or a pharmaceutically acceptable salt thereof. 48. The method of claim 47, wherein the carrier polymer is an anionic polysaccharide derivative or fragment thereof or a pharmaceutically acceptable salt thereof. 48. The method of claim 47, wherein the carrier polymer is carboxymethylcellulose or a pharmaceutically acceptable salt thereof. 48. The composition of claim 47, wherein the alginate, anionic acetate polymer, anionic acrylic polymer, xanthan gum, anionic carrageenan derivative, anionic polygalacturonic acid derivative, sodium starch glycorate, and fragments thereof are known. , Derivatives and pharmaceutically acceptable salts. 48. The method of claim 47, wherein the pharmaceutical formulation is in dry solid form. 48. The method of claim 47, wherein the pharmaceutical formulation is in the form of a liquid suspension or a semi-solid dispersion. A pharmaceutical formulation prepared according to the process of any one of claims 47-70. A method of treating a subject having symptoms that can be treated with an LHRH analog, comprising administering to the subject a pharmaceutical formulation containing an insoluble complex of the LHRH analog and a carrier polymer. The method of claim 72, wherein the single dose of insoluble complex provides sustained release of the LHRH analog to the subject for at least one week after administering the pharmaceutical composition to the subject. The method of claim 72, wherein the single dose of insoluble complex provides sustained release of the LHRH analog to the subject for at least two weeks after administering the pharmaceutical composition to the subject. The method of claim 72, wherein the single dose of the water-insoluble, non-covalent complex provides the subject with sustained release of the LHRH analog for at least 3 weeks after administering the pharmaceutical composition to the subject. The method of claim 72, wherein the single dose of the water-insoluble, non-covalent complex provides the subject with sustained release of the LHRH analog for at least 4 weeks after administering the pharmaceutical composition to the subject. 73. The method of claim 72, wherein the LHRH analogue is an LHRH antagonist. 78. The method of claim 77, wherein the LHRH antagonist has the structure: Ac-D-Nal ¹ , 4-Cl-D-Phe ² , D-Pal ³ , N-Me-Tyr ⁵ , D-Asn ⁶ , Lys (iPr) ⁸ , D-Ala ¹⁰ -LHRH characterized in that the treatment method. 73. The method of claim 72, wherein the carrier polymer is an anionic polymer. 73. The method of claim 72, wherein the carrier polymer is an anionic polyhydric alcohol derivative or fragment thereof or a pharmaceutically acceptable salt thereof. 73. The method of claim 72, wherein the carrier polymer is an anionic polysaccharide derivative or fragment thereof or a pharmaceutically acceptable salt thereof. 73. The method of claim 72, wherein the carrier polymer is carboxymethylcellulose or a pharmaceutically acceptable salt thereof. 73. The method of claim 72, wherein the alginate, anionic acetate polymer, anionic acrylic polymer, xanthan gum, anionic carrageenan derivative, anionic polygalacturonic acid derivative, sodium starch glycorate, and fragments thereof are known. , Derivatives and pharmaceutically acceptable salts. 73. The method of claim 72, wherein the pharmaceutical agent is administered to the subject by parenteral routes. 73. The method of claim 72, wherein the pharmaceutical formulation is administered orally to the subject. 73. The method of claim 72, wherein the pharmaceutical formulation is administered by intramuscular injection or subcutaneous / dermal injection. 73. The method of claim 72, wherein the condition treatable with the LHRH analogue is a hormone dependent cancer. 88. The method of claim 87, wherein the hormone dependent cancer is prostate cancer. 73. The method of claim 72, wherein the condition treatable with the LHRH analogue is selected from benign prostatic hyperplasia, colic, endometriosis and uterine fibroids. 73. The method of claim 72, wherein the LHRH analog is administered for in vitro fertilization or contraception purposes.

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