RU2844779C2 - Pharmaceutical compositions - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to an ionic complex having modulatory activity with respect to melanocortin-4 receptor, containing (i) a cationic polypeptide, wherein the cationic polypeptide has structural formula: Ac-Arg-cyclo[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys]-NH₂ (SEQ ID No. 1) or a pharmaceutically acceptable salt thereof; and (ii) an anionic filler containing methoxy(polyethylene glycol 2000)-1,2-distearyl-sn-glycero-3-phosphoethanolamine (mPEG-2000-DSPE), wherein the molar ratio of the mPEG-2000-DSPE of the cationic peptide to the anionic excipient is in range of 1:1 to 1:10.

EFFECT: complex can provide a more desirable pharmacokinetic profile after administration as compared with the use of the cationic polypeptide alone.

22 cl, 3 dwg, 4 tbl, 18 ex

Description

related application

[0001] This application claims priority to U.S. Provisional Application No. 61/792,440, filed March 15, 2013. The contents of that application are incorporated herein by reference in their entirety.

State of the art INVENTIONS

[0002] There is often a need to improve the pharmacokinetic characteristics of an active agent (e.g., increase the duration of action of the drug or minimize adverse effects). Drugs, in particular peptide drugs, are generally highly soluble in body fluids and can be rapidly absorbed, resulting in a rapid increase in the level of available drug, as opposed to a gradual release. Compositions that provide a gradual or prolonged release of the drug can help reduce fluctuations in drug concentration after administration, increase the drug load upon administration, increase stability and efficacy both in vivo and in vitro, reduce toxicity, and improve patient compliance due to a decrease in the frequency of administration. Thus, there is a need for pharmaceutical compositions containing an active agent that provide a prolonged release of the active agent.

The essence of the invention

[0003] The present invention relates to an ionic complex comprising a cationic polypeptide and an anionic filler selected from: PEG-carboxylic acid; a fatty acid containing 10 or more carbon atoms; anionic phospholipids; and combinations thereof in different molar ratios. The invention also relates to a pharmaceutical composition comprising the ionic complex of the present invention and a pharmaceutically acceptable carrier. The cationic polypeptide included in the ionic complex has pharmacological activity, and the complex can provide a more desirable pharmacokinetic profile after administration, for example, a delayed release in the case of using a complex of a cationic polypeptide compared to using the cationic polypeptide itself.

[0004] The invention also relates to the use of an ionic complex and a pharmaceutical composition comprising such a complex for treating an individual suffering from a disease or disorder responsive to the pharmacological activity of a cationic polypeptide included in the ionic complex. In one embodiment, the disorder to be treated is responsive to modulation of the melanocortin-4 receptor (MC4R) in an individual in need of treatment. The method comprises administering to the individual an effective amount of an ionic complex comprising, as a cationic polypeptide, an MC4R modulator, for example, a modulator of formula I as provided herein. In a specific embodiment, the disorder responsive to MC4R modulation includes type 1 diabetes, type 2 diabetes, obesity, insulin resistance, metabolic syndrome, male erectile dysfunction, female sexual dysfunction, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, substance abuse disorders including alcoholism-related eating disorders, cachexia, inflammation, and anxiety.

[0005] In some embodiments, the compounds and compositions of the present invention have greater selectivity and potency for MC4R and melanocortin-3 receptor (MC3R) than for melanocortin-1 receptor (MC1R). The compounds and compositions of the present invention can reduce or eliminate such undesirable side effects as increased blood pressure, increased heart rate, adverse effects on sexual arousal, and increased skin pigmentation.

[0006] The ionic complex and pharmaceutical compositions of the present invention containing the complex can improve the pharmacokinetic characteristics of the cationic polypeptide included in the complex. For example, they can increase the duration of the pharmacological action of the cationic polypeptide and at the same time significantly reduce the ratio of the maximum to minimum drug level in its pharmacokinetic profile. Therefore, the therapeutic dose of the cationic peptide in the body can be maintained within the range corresponding to the beneficial effect, thereby reducing the possibility of undesirable side effects that can be associated with a high level of the cationic polypeptide drug used alone. Compositions that can provide a gradual or more prolonged release of the active agent can help to reduce fluctuations in the concentration of the drug after administration, increase the drug load upon administration, increase stability and efficacy both in vivo and in vitro, reduce toxicity and improve patient compliance due to a decrease in the frequency of administration. The ionic complex compositions of the present invention can be used for effective therapeutic administration in various dosage regimens, including administration at least once a day, once a week, once every 2 weeks, once every four weeks, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months.

BRIEF DESCRIPTION OF DRAWINGS

[0007] Fig. 1 is a graph showing the pharmacokinetic profile of each of the identified pharmaceutical compositions after administration to cynomolgus monkeys.

[0008] Fig. 2 is a graph showing the pharmacokinetic profile of each of the identified pharmaceutical compositions after administration to cynomolgus monkeys.

[0009] Fig. 3 is a graph showing the pharmacokinetic profile of each of the identified pharmaceutical compositions after administration to cynomolgus monkeys.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The following is a description of illustrative embodiments of the present invention.

Glossary

[0011] To identify peptides, the traditional nomenclature is used, according to which the N-terminal amino group is depicted on the left and the C-terminal carboxyl group is depicted on the right.

[0012] As used herein, the term "amino acid" includes both natural and unnatural amino acids. Unless otherwise specified, all amino acids and residues thereof included in the compounds described herein may be in the D- or L-configuration.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety.

Symbol Meaning Abu α-aminobutyric acid Ace acyl group Aib α-aminoisobutyric acid Ala or A alanine Arg or R arginine Asn or N asparagine Asp or D aspartic acid Atc 2-aminotetralin-2-carboxylic acid Cha β-cyclohexylalanine sChp 1-amino-4-phenylcyclohexane-1-carboxylic acid Cys or C cysteine hCys homocysteine Dbu 2,4-diaminobutyric acid Dpr 2,3-diaminopropionic acid Gln or Q glutamine Glu or E glutamic acid Gly or G glycine His or H histidine Hyp hydroxyproline Ile or I isoleucine Leu or L leucine Lys or K lysine Met or M methionine 1-Nal (1-naphthyl)alanine 2-Nal (2-naphthyl)alanine Nle norleucine Orn ornithine Pen penicillamine Phe or F phenylalanine Pro or P proline QAla quinolinylalanine or 2-amino-3-(quinolin-3-yl)propanoic acid Sar sarcosine (N-methylglycine) Ser or S serine Tle tert-leucine (tert-butylglycine) TzAla 3-(1,2,4-triazol-1-yl)-L-Ala Thr or T threonine Trp or W tryptophan Tyr or Y tyrosine Val or V valine VNA benzhydrylamine Boc tert-butyloxycarbonyl But tertiary butyl DIPEA N,N-diisopropylethylamine DTT dithiothreitol Fmoc fluorenylmethyloxycarbonyl HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate MCR4 melanocortin-4 receptor Mtt 4-methyltrityl NMP N-methylpyrrolidone OBut tertiary butoxy OPip 2-phenylisopropyl Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Trt trityl TIS triisopropylsilane TFA trifluoroacetic acid

[0014] Unless otherwise noted, all abbreviated names (e.g., Ala) of amino acids in this specification refer to amino acid residues, i.e., to the structure -NH-C(R)(R')-CO-, where each of R and R', independently of the others, is hydrogen or an amino acid side chain (e.g., in the case of Ala, R = CH ₃ and R' = H, or R and R' may be joined to form a cyclic system).

[0015] The designation "Ac" or "NH ₂ " at the terminus of a polypeptide means that the corresponding terminus is acylated or amidated, respectively.

[0016] The term "covalent bond between amino acid side chains" means that each of the side chains of the two amino acid residues in question contains a functional group capable of forming a covalent bond with another functional group. Examples of such bonds include disulfide bridges formed by the side chains of Cys, hCys, or Pen, as well as amide bonds formed by the amino group of the side chain of one amino acid and the carboxyl group of the side chain of another amino acid, such as, for example, Asp, Glu, Lys, Orn, Dbu, or Dpr. When a covalent bond is formed between the side chains of amino acids, cyclization of the polypeptide may occur. Such a cyclic polypeptide may be indicated either by a structural formula or by the abbreviation "c()" or "cyclo()". For example, "-c(Cys-Cys)-" or "-cyclo(Cys-Cys)-" denotes the structure:

while "-c(Asp-Lys)-" or "-cyclo(Asp-Lys)-" denotes the structure:

Ionic complex:

[0017] The invention relates to an ionic complex comprising a cationic polypeptide and an anionic filler selected from: a PEG carboxylic acid; a fatty acid containing 10 or more carbon atoms; a phospholipid; and combinations thereof. The molar ratio of the cationic polypeptide to the anionic filler in the ionic complex is, for example, about 1:1 to 1:10, where the molar ratio is given taking into account the ratio of the charge of the cationic polypeptide to the charge of the anionic filler. Typically, a molar ratio selected from the group consisting of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 is used.

Anionic fillers

Anionic phospholipid:

[0018] As used herein, an "anionic phospholipid" is a phospholipid in which one or more oxygen atoms of a phosphate group(s) are deprotonated to form an organophosphorus oxoanion and a negatively charged lipid. Naturally occurring anionic phospholipids typically have a fatty acid chain length of _C16 or greater. The anionic phospholipid may carry one or more (e.g., 1, 2, 3, 4, 5, 6, or more) negative charges. In some embodiments, the anionic phospholipid is selected from: phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), or phosphatidylserine (PS). Suitable anionic lipids include compounds such as: L-α-phosphatidic acid, 1-oleoyl lysophosphatidic acid, L-α-phosphatidylglycerol, 1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), mPEG-2000-DSPE, mPEG-5000 DSPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), mPEG-2000-DPPE, mPEG-5000-DPPE, 1-(1,2-dihexadecanoylphosphatidyl)inositol-4,5-bisphosphate, trisodium salt, and 1-(1,2-dihexadecanoylphosphatidyl)inositol-3,4,5-trisphosphate, tetrasodium salt, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), L-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoglycerol (PAPG), DSPG (distearoylphosphatidylglycerol), DPPG (dipalmitoyl phosphatidylglycerol), DEPG (Dielaidoyl Phosphatidylglycerol), DOPG (Dioleoyl Phosphatidylglycerol), DEPA (Dielaidoyl Phosphatidic Acid), DOPA (Dioleoyl Phosphatidic Acid), DSP (Distearoyl Phosphatidylserine), DPPS (Dipalmitoyl Phosphatidylserine), DEPS (Dielaidoyl Phosphatidylserine) and DOPS (Dioleoyl Phosphatidylserine), L-α-lysophosphatidylserine, L-α-lysophosphatidylinositol, Tetradecylphosphonic acid, L-α-phosphatidylinositol-4-phosphate, L-α-phosphatidylinositol-4,5-bisphosphate, 1,2-diphytanoyl-sn-glycero-3-phosphate, 1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1'-rac-glycerol) and mixtures thereof.

[0019] Particular anionic phospholipids suitable for use in the present invention include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-conjugated polyethylene glycol, which has the following structure:

where the value of "n" varies with molecular weight. Such anionic phospholipids are referred to herein as mPEG-(MW)DSPE. Suitable examples include mPEG-2000-DSPE and mPEG-5000-DSPE, where DSPE is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine. Other examples include mPEG-2000-DPPE and mPEG-5000-DPPE, where DPPE is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.

[0020] In addition, synthetic anionic phospholipids with a chain length of less than C ₁₆ can be prepared and used. Synthetic lipids containing fatty acids bearing additional anionic groups in the fatty acid chains can also be prepared. An example of such compounds is 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine, which contains a fatty acid with a shorter chain that ends with a carboxyl group.

[0021] These natural and synthetic anionic lipids can be obtained from several commercial sources, such as Avanti Polar Lipids (Alabaster, AL), or Lipoid LLC (Newark, NJ), or Cordon Pharma (Boulder, CO), or NOF Corp America (White Plains, NY).

Fatty acid:

[0022] As used herein, a "fatty acid" is a carboxylic acid with a long aliphatic tail (long aliphatic chain), which may be saturated or unsaturated and contains a carbon chain of 10 or more atoms. The chain of carbon atoms may be linear, branched, saturated, mono- or poly-unsaturated.

[0023] Suitable saturated fatty acids include, but are not limited to: capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, docosanoic acid, tetracosanoic acid, and hexacosanoic acid.

[0024] Suitable unsaturated fatty acids include, but are not limited to: cis-2-decenoic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaydic acid, alpha-linolenic acid, arachidonic acid, cis-parinaric acid, eicosapentaenoic acid, and phytanic acid.

PEG carboxylic acids:

[0025] As used herein, the term "PEG carboxylic acid" refers to a polyethylene glycol (PEG) polymer functionalized with a carboxylic acid. PEG can be functionalized to form a monofunctional (monocarboxylate) or homobifunctional (dicarboxylate) derivative. The functionalized PEG can be linear or branched. The average molecular weight range of a PEG carboxylic acid suitable for use in the present invention can be from about 1,000 to about 100,000.

[0026] Homobifunctional PEGs suitable for use in the present invention can be described generally as COOH-PEG-COOH, or by the chemical formula:

where the value of "n" varies depending on the molecular weight. For example, the PEG carboxylic acid indicated in the examples as PEG-10000 dicarboxylate is a homobifunctional PEG having a molecular weight of 10,000. An average molecular weight range of about 1,000 to 100,000 is suitable for use in the present invention. Typically, the molecular weight ranges from 1,000 to 40,000.

[0027] One type of monofunctional PEG carboxylic acids suitable for use in the present invention can be described generally as mPEG-COOH, or by the chemical formula:

where the value of "n" also varies depending on the molecular weight. For example, mPEG-10000 used in the examples is a monofunctional PEG containing a methoxy group as shown in the formula. An average molecular weight range of about 1,000 to 100,000 is suitable for use in the present invention. Typically, the molecular weight ranges from 1,000 to 40,000.

[0028] Another type of monofunctional PEG carboxylic acids suitable for use in the present invention can be described generally as PEG-COOH or by the chemical formula: HO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -COOH, wherein the value of "n" varies depending on the molecular weight. For example, the PEG carboxylic acid referred to in the examples as PEG-10000 monocarboxylate is a monofunctional PEG having a molecular weight of 10,000. An average molecular weight range of about 1,000 to 100,000 is suitable for use in the present invention. Typically, the molecular weight ranges from 1,000 to 40,000.

[0029] Suitable PEG carboxylic acids include, but are not limited to, PEG-10,000 monocarboxylate, PEG-20,000 monocarboxylate, mPEG-1000 monocarboxylate, mPEG-2000 monocarboxylate, mPEG-5000 monocarboxylate, mPEG-10,000 monocarboxylate, mPEG-20,000 monocarboxylate, mPEG-30,000 monocarboxylate, mPEG-40,000 monocarboxylate, PEG-1000 dicarboxylate, PEG-2000 dicarboxylate, PEG-3500 dicarboxylate, PEG-5000 dicarboxylate, PEG-7500 dicarboxylate, PEG-10000 dicarboxylate and Y-shaped PEG-40000 monocarboxylate. The above Y-shaped PEG carboxylic acids can be represented as follows:

where the value of "n" defines the molecular weight.

[0030] In particular embodiments, the PEG carboxylic acid is selected from the group consisting of PEG-5000 monocarboxylate, PEG-10000 monocarboxylate, PEG-20000 monocarboxylate, mPEG-5000 monocarboxylate, mPEG-10000 monocarboxylate, mPEG-20000 monocarboxylate, PEG-5000 dicarboxylate, and PEG-10000 dicarboxylate.

[0031] Suitable PEG carboxylic acids can be obtained commercially from Nanocs, Inc. (New York, NY), JenKem Technology (Allen, TX), or NOF Corp America (White Plains, NY).

[0032] In some embodiments, the composition may include two or more of the above-described anionic excipients (such as a PEG carboxylic acid, an anionic phospholipid, and a fatty acid). For example, a pharmaceutical composition of the present invention may comprise a PEG carboxylic acid and an anionic phospholipid, a PEG carboxylic acid and a fatty acid, or a fatty acid and an anionic phospholipid. In a further embodiment, the composition may include three or more anionic excipients selected from a fatty acid, an anionic phospholipid, and a PEG carboxylic acid. In particular embodiments, the combination of anionic excipients is selected from stearic acid and mPEG-2000-DspE; DPPA and PEG-10000-dicarboxylate; DPPA and PEG-3350; DPPA; DPPA and mPEG-3350; and mPEG-2000-DSPE. In a further embodiment, any of the anionic phospholipids described herein can be further combined with carboxymethylcellulose (CMC) to form, for example, a combination of mPEG-2000-DspE and CMC.

Cationic peptides:

[0033] As used herein, the term "cationic polypeptide" refers to any polypeptide that bears a positive charge at a pH of about 5.0. A cationic polypeptide may comprise naturally occurring amino acid residues, non-naturally occurring amino acid residues, or a mixture of both. The positive charge of cationic polypeptides is due to the presence of a cationic functional group that is either present in the side chain of an amino acid of the polypeptide sequence, or that is part of the alpha-amino group of an amino acid of the polypeptide sequence, or both. The positive charge of a cationic polypeptide may also be due to the presence of a cationic functional group ligated to the polypeptide at either end of the peptide sequence, or at the side chain of an amino acid included in the sequence. A cationic polypeptide may comprise multiple cationic functional groups. For example, a cationic polypeptide may comprise 1, 2, 3, 4, 5, 6, or more cationic functional groups, each of which may bear a positive charge. Such functional groups include, for example, amino groups (primary, secondary or tertiary), quaternary ammonium groups, guanidino groups, amidino groups, pyridine, imidazole groups, phosphonium groups and sulfonium groups. In a particular embodiment, the cationic group is an amino group, a guanidino group or an imidazole group.

[0034] The cationic polypeptides of the present invention may contain one or more chiral centers and, therefore, may exist as different stereoisomeric forms. All stereoisomers and mixtures thereof are within the scope of the present invention. Racemic compounds can be separated into individual enantiomers by preparative high performance liquid chromatography on a column with a chiral stationary phase, using methods known to those skilled in the art. In addition, intermediate chiral compounds can be separated and used to prepare the chiral compounds of the present invention.

[0035] The cationic polypeptides described herein may exist in one or more tautomeric forms. All tautomers and mixtures thereof are within the scope of the present invention.

[0036] Cationic polypeptides suitable for use in the present invention include, but are not limited to, the following categories of polypeptides and specific polypeptides, as well as the polypeptides represented by formula I. Cationic polypeptides are pharmacologically active.

LHRH (GnRH) agonists:

Leuprolide: Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt (SEQ ID NO: 46)

Buserelin: Pyr-His-Trp-Ser-Tyr-D-Ser(tBU)-Leu-Arg-Pro-NHEt (SEQ ID NO: 47)

Histrelin: Pyr-His-Trp-Ser-Tyr-D-His(Bzl)-Leu-Arg-Pro-NHEt (SEQ ID NO: 48)

Goserelin: Pyr-His-Trp-Ser-Tyr-D-Ser(tBU)-Leu-Arg-Pro-Azagly-NH ₂ (SEQ ID NO: 49)

Deslorelin: Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt (SEQ ID NO: 50)

Nafarelin: Pyr-His-Trp-Ser-Tyr-D-Nal(2)-Leu-Arg-Pro-Gly-NH ₂ (SEQ ID NO: 51)

Triptorelin: Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ (SEQ ID NO: 52)

Avorelin: Pyr-His-Trp-Ser-Tyr-D-Trp(2-Me)-Leu-Arg-Pro-NHEt (SEQ ID NO: 53)

GnRH antagonists:

Abarelix: Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-(Me)Tyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH ₂ (SEQ ID NO: 54)

Cetrorelix: Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH ₂ (SEQ ID NO: 55)

Degarelix: Ac-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Phe(4-(4S)-hexahydro-2,6-dioxo-4-pyrimidinyl(carbonyl)amino)-D-Phe(4-guanidino)-Leu-Lys(iPr)-Pro-D-Ala-NH ₂ (SEQ ID NO: 56)

Ganirelix: Ac-D-Nal(2)-D-Cpa(4)-D-Pal(3)-Ser-Tyr-D-Harg(Et)2-Leu-Harg(Et) ₂ -Pro-D-Ala-NH ₂ (SEQ ID NO: 57)

Somatostatin analogues:

Octreotide: D-Phe-[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-ol (SEQ ID NO: 58)

Lanreotide: D-Nal(2)-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH ₂ (SEQ ID NO: 59)

Vapreotide: D-Phe-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH ₂ (SEQ ID NO: 60)

Pasireotide (Signifor): cyclo[(4R)-4-(2-aminoethylcarbamoyloxy)-L-prolyl-L-phenylglycyl-D-tryptophyl-L-lysyl-4-O-benzyl-L-tyrosyl-L-phenylalanyl] (SEQ ID NO: 60)

Other peptides:

Glucagon

Amilin

Pramlintide

Insulin

Glucagon-like peptide 1 (GLP-1)

GLP-1 agonist

Exenatide

Parathyroid hormone (PTH)

Adrenocorticotropic hormone (ACTH)

Botulinum toxin

Amyloid peptide

Cholecystikinin

Calcitonin

Conotoxin

Prialt

Gastric inhibitory peptide

Insulin-like growth factor, including industrially produced recombinant IGF-1, such as

Increlex

Growth hormone releasing factor

Antimicrobial factor

Glatiramer (Copaxone)

Hematid (peginesatide)

Nasiritid

ANF peptide

Angiotensin peptide

ACTH

Human growth hormone (hGH), including industrially produced recombinant hGH

Melanocortin

Opioid peptide

Dynorphin

Oxytocin

Oxytocin analogue including antagonists

Vasopressin and its analogues and

Somatostatin and its analogues

Cationic polypeptides of formula I:

[0037] Cationic polypeptides suitable for use in the present invention include compounds of formula (I):

or their pharmaceutically acceptable salts,

Where:

R ¹ denotes H or C1-C6 acyl;

R ² is -NR ³ R ⁴ , or -OR ⁵ , where each of R ³ , R ⁴ and R ⁵ independently is H or C1-C6 alkyl;

A ¹ is an amino acid residue selected from Arg, Lys, Orn, His, Nle, Phe, Val, Leu, Trp, Tyr, Ala, Ser, Thr, Gln, Asn, Asp, Glu or TzAla; or

A ¹ denotes a moiety selected from optionally substituted C1-C12 alkyl, optionally substituted C6-C18 aryl, optionally substituted C5-C18 heteroaryl, aralkyl in which the aryl part is optionally substituted C6-C18 aryl and the alkyl part is optionally substituted C1-C12 alkyl, or heteroaralkyl in which the heteroaryl part is optionally substituted C5-C18 heteroaryl and the alkyl part is optionally substituted C1-C12 alkyl;

each of ^A2 and ^A8 independently represents an amino acid residue selected from Cys, hCys, Pen, Asp, Glu, Lys, Orn, Dbu or Dpr, wherein the pairs of ^A2 and ^A8 are selected so as to allow the formation of a covalent bond between the corresponding side chains;

A ³ is absent or represents an amino acid residue selected from Ala, Tle, Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg, His, Phe, Gln, Sar, Gly, Asn or Aib;

A ⁴ is absent or represents an amino acid residue selected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, optionally substituted by His, Trp, Tyr, Lys, Arg, sChp, or a residue X, where X represents an amino acid represented by the following structural formula

A ⁵ represents optionally substituted Phe, optionally substituted 1-Nal, or optionally substituted 2-Nal;

And ⁶ stands for Arg; and

And ⁷ stands for Trp,

where any amino acid residue can be in either the L- or D-configuration.

[0038] In exemplary embodiments, A ³ and A ⁴ are not simultaneously absent. The remaining variables have the meanings and preferred meanings defined above and below in application to formula (I).

[0039] In exemplary embodiments, if A ⁴ is an amino acid, A ³ is other than Aib or Gly. The remaining variables have the meanings and preferred meanings defined above in relation to formula (I).

[0040] In exemplary embodiments, if A ⁴ is His and A ⁵ is D-Phe or 2-Nal, A ³ is other than a D-amino acid or L-Ala. The remaining variables have the meanings and preferred meanings defined above in relation to formula (I).

[0041] In exemplary embodiments, if each of ^A2 and ^A8 is selected from Cys, hCys, or Pen, then: (a) if ^A4 is absent, ^A3 is other than L-His; (b) if ^A3 is absent, ^A4 is other than L-His; and (c) if ^A4 is His, ^A3 is other than Glu, Leu, or Lys. The remaining variables have the meanings and preferred meanings defined above in application to formula (I).

[0042] In exemplary embodiments: 1) A ³ and A ⁴ are not simultaneously absent; 2) if A ⁴ is an amino acid, A ³ is other than Aib or Gly; and 3) if A ⁴ is His and A ⁵ is D-Phe or 2-Nal, A ³ is other than a D-amino acid or L-Ala; 4) if A ² and A ⁸ are each selected from Cys, hCys, or Pen, then: (a) if A ⁴ is absent, A ³ is other than L-His; (b) if A ³ is absent, A ⁴ is other than L-His; and (c) if A ⁴ is His, A ³ is other than Glu, Leu, or Lys. The remaining variables have the meanings and preferred meanings defined above with respect to formula (I).

[0043] In another embodiment of the polypeptides of formula (I), A ⁴ is an L-amino acid. In other embodiments, A ⁴ is absent. The remaining variables have the meanings and preferred meanings defined above with respect to formula (I).

[0044] In exemplary embodiments, A ⁵ can be optionally substituted 1-Nal or optionally substituted 2-Nal, such as optionally substituted D-2-Nal. A ⁵ can be substituted at any of the five aromatic carbon atoms with a substituent selected from F, Cl, Br, I, -CH ₃ , -OH, -CN, amine, -NO ₂ , or -OCH ₃ .

[0045] In a further embodiment of the polypeptides of formula (I), A ⁵ is optionally substituted D-Phe. A ⁵ may be substituted at any of the five aromatic carbon atoms with a substituent selected from F, Cl, Br, I, -CH ₃ , -OH, -CN, amine, -NO ₂ or -OCH ₃ . Suitable examples of A ⁵ include, but are not limited to, a D-amino acid residue selected from:

The remaining variables have the meanings and preferred meanings defined above in application to formula (I).

[0046] In another embodiment of the polypeptides of formula (I), A ⁵ is optionally substituted D-2-Nal. A ⁵ may be substituted at any of the five aromatic carbon atoms with a substituent selected from F, Cl, Br, I, -CH ₃ , -OH, -CN, amine, -NO ₂ , or -OCH ₃ .

[0047] In a further embodiment of the polypeptides of formula (I), A ⁴ is His optionally substituted at any substitutable position with a substituent selected from F, Cl, Br, I, -CH ₃ , -OH, -CN, amine, -NO ₂ , benzyl or -OCH ₃ . The remaining variables have the meanings and preferred meanings defined above in relation to formula (I).

[0048] In a particular embodiment, the compounds of the present invention are polypeptides of formula (I) that have an EC ₅₀ for MC4R of from about 0.01 nM to 10 nM, such as 0.01-3 nM, wherein the ratio EC ₅₀ (MCLR) / EC ₅₀ (MC4R) is at least 10. In another embodiment, the polypeptides of the present invention include a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0049] In a further embodiment, the polypeptides of the present invention comprise a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0050] In another embodiment, the polypeptides of the present invention comprise a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0051] In a further embodiment, the polypeptides of the present invention comprise a polypeptide represented by formula (I), wherein A ⁴ is an amino acid residue selected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, optionally substituted with His, Trp, Tyr, Lys, Arg, sChp, or an X residue. Examples of such peptides include a peptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0052] In exemplary embodiments, the polypeptides of the present invention include a peptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0053] In another embodiment, the polypeptides of the present invention include those represented by formula (I), wherein ^A3 is an amino acid residue selected from Tle, Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg, His, Phe, Gln, Sar, Gly, Asn, or Aib; and ^A4 is an amino acid residue selected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln substituted with His, Trp, Tyr, Lys, Arg, sChp, or an X residue. An example of such polypeptides is a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0054] In a further embodiment, the polypeptides of the present invention comprise a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0055] In a further embodiment, the polypeptides of the present invention comprise a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0056] In a further embodiment, the polypeptides of the present invention comprise a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

[0057] In a further embodiment, the polypeptides of the present invention comprise a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof. In another embodiment, the polypeptides of the present invention include a polypeptide represented by one of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

In an alternative embodiment, cationic peptides suitable for use in the present invention include peptides of formula (II) or pharmaceutically acceptable salts thereof:

The isolated polypeptide has the following structural formula (II):

or a pharmaceutically acceptable salt thereof,

in which:

Where:

R ¹ denotes H or C1-C6 acyl;

R ² is -NR ³ R ⁴ , or -OR ⁵ , where each of R ³ , R ⁴ and R ⁵ independently is H or C1-C6 alkyl;

A ¹ is missing; or

A ¹ is an amino acid residue selected from Arg, Lys, Orn, His, Nle, Phe, Val, Leu, Trp, Tyr, Ala, Ser, Thr, Gln, Asn, Asp, Glu or TzAla; or

A ¹ denotes a moiety selected from optionally substituted C1-C12 alkyl, optionally substituted C6-C18 aryl, optionally substituted C5-C18 heteroaryl, aralkyl in which the aryl part is optionally substituted C6-C18 aryl and the alkyl part is optionally substituted C1-C12 alkyl, or heteroaralkyl in which the heteroaryl part is optionally substituted C5-C18 heteroaryl and the alkyl part is optionally substituted C1-C12 alkyl;

each of ^A2 and ^A8 independently represents an amino acid residue selected from Cys, hCys, Pen, Asp, Glu, Lys, Orn, Dbu or Dpr, wherein the pairs of ^A2 and ^A8 are selected so as to allow the formation of a covalent bond between the corresponding side chains;

A ³ is absent or represents an amino acid residue selected from Ala, Tle, Val, Leu, Ile, Cha, Pro, Ser, Thr, Lys, Arg, His, Phe, Gln, Sar, Gly, Asn, Aib or a Y residue, where Y represents an amino acid selected from the amino acids represented by the following structural formulas

Where:

each of R ¹¹ and R ¹² independently represents H, -CH ₃ , phenyl or benzyl;

each of R ²¹ , R ²² , R ²³ and R ²⁴ independently represents H, -CH ₃ , -CF ₃ , phenyl, benzyl, F, Cl, Br, I, -OCH ₃ or -OH;

each of R ³¹ , R ³² , R ³³ , R ³⁴ , R ⁴¹ , R ⁴² and R ⁴³ independently represents H, -CH ₃ , -CF ₃ , phenyl, benzyl, F, Cl, Br, I, -OCH ₃ or -OH;

A ⁴ is absent or represents an amino acid residue selected from Atc, Ala, QAla, Aib, Sar, Ser, Thr, Pro, Hyp, Asn, Gln, optionally substituted by His, Trp, Tyr, Lys, Arg, sChp, or a residue X, where X represents an amino acid represented by the following structural formula

Where:

each of R ⁵¹ and R ⁵² independently represents H, -CH ₃ , phenyl or benzyl;

each of R ⁶¹ , R ⁶² , R ⁶³ and R ⁶⁴ independently represents H, -CH ₃ , -CF ₃ , phenyl, benzyl, F, Cl, Br, I, -OCH ₃ or -OH;

each of R ⁷¹ , R ⁷² , R ⁷³ , R ⁷⁴ , R ⁸¹ , R ⁸² and R ⁸³ independently represents H, -CH ₃ , -CF ₃ , phenyl, benzyl, F, Cl, Br, I, -OCH ₃ or -OH;

A ⁵ represents optionally substituted Phe, optionally substituted 1-Nal, or optionally substituted 2-Nal;

And ⁶ stands for Arg; and

And ⁷ stands for Trp,

where any amino acid residue can be in either the L- or D-configuration.

In exemplary embodiments, each of A ³ and A ⁴ independently represents an amino acid residue selected from amino acids represented by the following structural formulas:

The remaining variables have the meanings and preferred meanings defined above in application to formula (I).

[0058] The term "alkyl" used alone or as part of a larger moiety such as "hydroxyalkyl", "alkoxyalkyl", "alkylamine" means a linear or branched, saturated aliphatic group containing the indicated number of carbon atoms, typically from 1 to 12 carbon atoms. More specifically, the aliphatic group may contain from 1 to 8, from 1 to 6, or from 1 to 4 carbon atoms. This term is exemplified by such groups as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like.

[0059] "Haloalkyl" is an alkyl group substituted with one or more halogen atoms.

[0060] The term "halogen" includes fluorine, chlorine, bromine or iodine.

[0061] "Cyano" represents the -CN group.

[0062] "Ph" denotes a phenyl group.

[0063] The term "carbonyl" refers to the divalent group -C(O)-.

[0064] The term "aryl", used alone or as part of a larger moiety such as "aralkyl", refers to an aromatic carbocyclic group of 6 to 18 carbon atoms in size, containing a single ring or multiple fused rings. The term "aryl" also includes aromatic carbocycle(s) fused to cycloalkyl or heterocycloalkyl groups. Examples of aryl groups include phenyl, benzo[d][1,3]dioxole, naphthyl, phenanthrenyl, and the like.

[0065] The term "aryloxy" refers to the group -OAr, where O is an oxygen atom and Ar is an aryl group as described above.

[0066] "Aralkyl" is an alkyl in which at least one alkyl hydrogen atom is replaced by an aryl moiety such as benzyl, -(CH ₂ ) ₂ phenyl, -(CH ₂ ) ₃ phenyl, -CH(phenyl) ₂ , etc.

[0067] The term "heteroaryl" used alone or as part of a larger moiety such as "heteroaralkyl" includes a 5- to 18-membered monocyclic, bicyclic, or tricyclic heteroaromatic system containing one to four ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. The term "heteroaryl" also includes heteroaromatic ring(s) fused to cycloalkyl or heterocycloalkyl groups. Specific examples of heteroaryl groups include pyridyl, optionally substituted pyrrolyl, pyrimidinyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolisinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 1,2,3,4-tetrahydroquinolyl, 1,2,3,4-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl, benzoquinolyl, etc.

[0068] The term "heteroaralkyl" refers to an alkyl in which at least one alkyl hydrogen atom is replaced with a heteroaryl moiety such as -CH2 _- pyridinyl, -CH2 _- pyrimidinyl, etc.

[0069] The term "alkoxy" refers to the group -O-R, where R is "alkyl", "cycloalkyl", "alkenyl", or "alkynyl". Examples of alkoxy groups include, for example, methoxy, ethoxy, ethenoxy, and the like.

[0070] "Hydroxyalkyl" and "alkoxyalkyl" are alkyl groups substituted with hydroxyl and alkoxyl groups, respectively.

[0071] The term "amino" refers to -NH ₂ ; "alkylamine" and "dialkylamine" represent -NHR and -NR ₂ , respectively, where R is an alkyl group. The terms "cycloalkylamine" and "dicycloalkylamine" refer to -NHR and -NR ₂ , respectively, where R is a cycloalkyl group. "Cycloalkylalkylamine" is -NHR where R is a cycloalkylalkyl group. "[Cycloalkylalkyl][alkyl]amine" is -N(R) ₂ , where one R is cycloalkylalkyl and the other R is alkyl.

[0072] The term "acyl" refers to R"-C(O)-, wherein R" is H, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl, aryl, alkylaryl, or substituted alkylaryl, and is designated "Ac" in the general formula of a particular embodiment.

[0073] Suitable substituents of "alkyl", "aryl", "heteroaryl" and the like include moieties the introduction of which results in the formation of a stable compound of the present invention. Examples of suitable substituents can be selected from the group consisting of halogen, -CN, -OH, -NH ₂ , (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )haloalkyl, aryl, heteroaryl, (C ₃ -C ₇ )cycloalkyl, (5-7-membered) heterocycloalkyl, -NH(C ₁ -C ₆ )alkyl, -N((C ₁ -C ₆ )alkyl) ₂ , (C ₁ -C ₆ )alkoxy, (C ₁ -C ₆ )alkoxycarbonyl,

More specifically, the substituents can be selected from halogen, -CN, -OH, _-NH2 , ( _C1 - _C4 )alkyl, ( _C1 - _C4 )haloalkyl, ( _C1 - _C4 )alkoxy, phenyl and ( _C3 - _C7 )cycloalkyl. In the context of this invention, the term "substitution" also includes cases of substitution of a hydrogen atom by a deuterium atom. p is an integer equal to 1 or 2.

[0074] The substituted Phe may contain from one to five substituents on any aromatic carbon atoms, wherein the substituents may be selected from F, Cl, Br, I, -CH ₃ , -OH, -CN, amine, -NO ₂ or -OCH ₃ . Examples of suitable substituents include

[0075] Substituted His includes one to three substituents on any substitutable ring atoms, wherein suitable substituents can be selected from F, Cl, Br, I, -CH ₃ , -OH, -CN, amine, -NO ₂ , benzyl, or -OCH ₃ . Examples include 1-methylhistidine and 3-methylhistidine.

[0076] The notation "(amino acid) _n " indicates that the amino acid is repeated n times. For example, the notation "(Pro) ₂ " or "(Arg) ₃ " indicates that the proline or arginine residues are repeated two or three times, respectively.

[0077] Pharmaceutically acceptable salts of the compounds disclosed herein are included in the present invention. For example, a salt of an acid and a compound containing an amino group or other basic group can be prepared by reacting the compound with a suitable organic or inorganic acid to produce pharmaceutically acceptable anionic salt forms. Examples of anionic salts include acetates, benzenesulfonates, benzoates, bicarbonates, bitartrates, bromides, calcium edetates, camsylates, carbonates, chlorides, citrates, dihydrochlorides, edetates, edisylates, estolates, esylates, fumarates, glycopates, gluconates, glutamates, glycol larsanilates, hexyl resorcinates, hydrobromides, hydrochlorides, hydroxy naphthoates, iodides, isethionates, lactates, lactobionates, malates, maleates, mandelates, mesylates, methyl sulfates, mucates, napsylates, nitrates, pamoates, pantothenates, phosphates/diphosphates, polygalacturonates, salicylates, stearates, subacetates, succinates, sulfates, tannates, tartrates, theoclates, tosylates, triethiodides and trifluoroacetates.

[0078] Salts of compounds containing an acidic functional group can be prepared by reaction with a suitable base. Suitable pharmaceutically acceptable salts which can be prepared by reaction with bases which provide pharmaceutically acceptable cations include alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts formed with physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline and basic amino acids such as lysine and arginine.

Pharmaceutical compositions

[0079] The invention also relates to a pharmaceutical composition comprising an ionic complex comprising: a cationic polypeptide; an anionic excipient selected from: a PEG carboxylic acid; a fatty acid containing 10 or more carbon atoms; anionic phospholipids; and combinations thereof; and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition also comprises an additional excipient as described below (e.g., carboxymethylcellulose (CMC)). For example, any of the anionic phospholipids described herein can be further combined with carboxymethylcellulose (CMC), such as combinations of mPEG-2000-DspE and CMC.

[0080] The ratio of the concentrations of the cationic peptide and the anionic excipient is determined as the molar ratio of the charge of the cationic polypeptide to the charge of the anionic excipient. For example, based on one positive charge of the polypeptide, the amount of anionic excipient can vary from 1:1 to 1:10. In the case of the presence of additional positive charges on the polypeptide, the ratio of the anionic excipient can be adjusted accordingly. By varying the amount of anionic excipient in this ratio, the release characteristics of the polypeptide in vivo can be modulated. A higher ratio, as a rule, results in a composition that provides a slower release of the polypeptide at the site of administration than a composition obtained using a lower ratio.

[0081] The term "pharmaceutically acceptable carrier" as used in connection with the pharmaceutical composition of the present invention refers to a biocompatible polar liquid. The polar nature of the liquid helps maintain the complex in ionic form. Biocompatible polar liquids include, but are not limited to, PEG (polyethylene glycol, e.g., polyethylene glycol having an average molecular weight of 100 to 5000), polyol (e.g., propylene glycol (PG), tripropylene glycol, glycerol), ethanol, benzyl alcohol, DMSO, NMP, DMF, water, buffer solutions, and mixtures thereof. It is understood that other diluents and other excipients may be included in the pharmaceutical composition, as described below.

[0082] In some embodiments, a pharmaceutical composition of the present invention forms a drug depot when administered to an individual. In some embodiments, upon administration to an individual, the drug depot slowly releases the active compound. In one aspect, upon administration to an individual, at least a portion of the pharmaceutical composition precipitates to form a drug depot and releases the pharmacologically active compound over time. In some embodiments, the compositions of the present invention provide high concentrations of a polypeptide of structural formula (I) suitable for creating a drug depot that provides long-term, sustained release of therapeutically effective levels of the polypeptide in vivo. The concentration of a polypeptide of structural formula (I) in the composition can vary, for example, in a range of from about 0.0001 mg/mL to about 100 mg/mL.

[0083] The pharmaceutical composition of the present invention may contain auxiliary excipients (also referred to herein as co-excipients). Suitable examples of auxiliary excipients include pH stabilizing buffers, preservatives, surfactants, antioxidants, tonicity agents, and the ionic and non-ionic polymers described herein.

[0084] Such a co-excipient may facilitate the formation of uniform suspensions and dispersions of an ionic complex, or even a hydrophobic anionic excipient such as a lipid or fatty acid. The class of co-excipients includes dispersing and emulsifying agents such as lecithin, soybean oil, castor oil, miglyol, polyethylene glycol (with a molecular weight in the range of 200 to 5000), methylcellulose and carboxymethylcellulose.

[0085] As used herein, the term "surfactant" refers to a surface-active agent, or a substance capable of reducing the surface tension of a liquid in which it is dissolved. Suitable surfactants include polysorbates, poloxamers, triton, sodium dodecyl sulfate, sodium lauryl sulfate, and betaines. Examples of surfactants include polyoxyethylene (20) sorbitan monolaurate (Tween® 20, e.g., Sigma-Aldrich), polyoxyethylene (20) sorbitan monopalmitate (Tween® 40), polyoxyethylene (20) sorbitan monooleate (Tween® 80), poloxamer 188, polyoxyethylene-polyoxypropylene block copolymer (Pluronic® F-68, e.g., Sigma-Aldrich), polyethyleneglycol 660-12-hydroxystearate (Soluto ^1® HS 15 BASF), cocamidopropyl betaine, linoleyl betaine, myristyl betaine, cetyl betaine, polyethoxylated castor oil (Cremophor®, now Kolliphor BASF), and lecithin.

[0086] As used herein, the term "tonicity adjusting agents" refers to substances used to modulate the tonicity of a composition. Tonicity is typically related to the osmotic pressure of a solution, and is typically defined relative to human serum. The composition may be hypotonic, isotonic, or hypertonic. Typically, the composition is preferably isotonic. An isotonic composition is a liquid, or a liquid obtained by diluting a solid form, such as a lyophilized form, and is a solution that has the same tonicity as some other solution to which it is compared, such as saline and blood serum. Tonicity adjusting agents can help reduce pain and irritation in an individual upon administration. Suitable tonicity adjusting agents include dextrose, glycerol, hydroxyethyl starch, lactose, mannitol (eg, D-mannitol), raffinose, sorbitol, sucrose, trehalose, sodium chloride, calcium chloride, magnesium chloride, and potassium chloride.

[0087] The term "buffer" as used herein refers to an excipient that stabilizes the pH of a pharmaceutical composition. Suitable buffers are well known in the art and described in the literature. Examples of suitable buffers include histidine buffers, citrate buffers, succinate buffers, acetate buffers, and phosphate buffers, or mixtures thereof. Most preferred buffers include citrate, L-histidine, or mixtures of L-histidine and L-histidine hydrochloride. Another preferred buffer is an acetate buffer. Regardless of the buffer used, the pH can be adjusted using an acid or base known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, sodium hydroxide, and potassium hydroxide.

[0088] Ionic polymers suitable for use as an auxiliary filler include ionic carboxymethylcellulose (CMC), hyaluronic acid, poly(glutamic acid), poly(aspartic acid), glutamic acid-glycine copolymer, aspartic acid-glycine copolymer, glutamic acid-alanine copolymer, aspartic acid-alanine copolymer, starch glycolate, polygalacturonic acid, poly(acrylic acid), carrageenan, and alginic acid.

[0089] In some embodiments, the average molecular weight range of the CMC is from about 5,000 to 700,000. In some embodiments, methylcellulose and carboxymethylcellulose can help modulate the viscosity of the ionic complex, allowing for the formation of a sustained release drug depot and release of the active compound over time after administration to an individual.

[0090] Nonionic polymers suitable for use as an auxiliary filler include polyethylene glycol having a molecular weight in the range of about 100 to 100,000, such as about 100 to 60,000, such as about 100 to 10,000, such as about 100 to 5,000. Other weight ranges include about 200 to 60,000. Suitable examples of neutral polymers include PEG-3350 and PEG-3400. Polyethylene glycol polymers include both PEG and mPEG, which may be a monomethoxyl or dimethoxyl derivative. The structure of PEG can be depicted as follows:

HO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OH

where the value of "n" varies depending on the molecular weight. For example, PEG-3500 acid has a molecular weight of 3500.

[0091] mPEG can have one of the following two structures:

Monomethoxy mPEG: CH ₃ O-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OH. One end is capped with a methoxy group and the other end has a hydroxyl (OH) group.

Dimethoxy mPEG: CH ₃ O-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OCH ₃ . Both ends are capped with methoxyl groups.

[0092] Diluents are added to the pharmaceutical compositions of the present invention to dissolve or suspend the ionic complex of the present invention. Diluents include biocompatible substances such as non-aqueous low viscosity injection liquids. Non-aqueous low viscosity injection liquids include castor oil, vegetable oil, mineral oil, squalene, monoglycerides, diglycerides, triglycerides, or mixtures of glycerides. In some embodiments, the diluent is Miglyol® 812 (from Sasol GmbH, Germany), Labrafac® WL1349 (caprylic acid triglyceride from Gattefosse company, France), Lipoid MCT (from Lipoid company, Germany), and the like.

[0093] As used herein, the term "drug depot" refers to a precipitate that may form following administration of a pharmaceutical composition of the present invention. The compositions may form a slow-release drug depot and release the active compound over time following administration to an individual. In one aspect, at least a portion of the composition precipitates and releases the pharmacologically active compound over time following administration to an individual.

[0094] A "preservative" is a compound that can be added to a composition to reduce bacterial activity or undesirable chemical changes in the compositions. Examples of preservatives include benzyl alcohol, ethanol, methanol, isopropanol, butylparaben, ethylparaben, methylparaben, propylparaben, catechol, 2-chlorophenol, m-cresol, phenol, resorcinol, xylitol, 2,6-dimethylcyclohexanol, 2-methyl-2,4-pentadiol, polyvinylpyrrolidone, benzethonium chloride, thimerosal, benzoic acid, benzalkonium chloride, chlorobutanol, sodium benzoate, sodium propionate, and cetylpyridinium chloride.

TREATMENT METHODS

[0095] The present invention relates to the use of an ionic complex and a pharmaceutical composition comprising the same for treating a patient suffering from a disease or disorder responsive to the pharmacological activity of a cationic polypeptide included in the ionic complex. In one embodiment, the disorder to be treated is responsive to MC4R modulation in an individual in need of treatment. The method comprises administering to the individual an effective amount of an ionic complex comprising, as a cationic polypeptide, an MC4R modulator, such as that described in formula I herein. In a specific embodiment, the disorder responsive to MC4R modulation includes type 1 diabetes, type 2 diabetes, obesity, insulin resistance, metabolic syndrome, male erectile dysfunction, female sexual dysfunction, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, substance abuse disorders including alcoholism-related eating disorders, cachexia, inflammation, and anxiety.

[0096] In some embodiments, the ionic complex and pharmaceutical composition comprising the cationic polypeptide of Formula I may have higher selectivity and activity for MC4R and melanocortin-3 receptor (MC3R) than for melanocortin-1 receptor (MC1R). The ionic complex and pharmaceutical composition of the present invention may help reduce or eliminate such undesirable side effects as increased blood pressure, increased heart rate, undesirable effects on sexual arousal, and increased skin pigmentation.

[0097] As used herein, the phrase "melanocortin-4 receptor modulation-responsive disorder" refers to any disorder that can be treated by activating (agonist) or inhibiting MC4R. Examples of such disorders are described in detail below.

[0098] As used herein, the term "modulator" refers to compounds that are capable of interacting with a target receptor and influencing its biological function. Examples of modulators include full agonists, partial agonists, neutral antagonists, and inverse agonists.

[0099] As used herein, the term "agonist" refers to any chemical compound, natural or synthetic, that upon interaction (e.g., binding) with a target, here MC4R, increases MC4R signaling activity to a level above background. An agonist may be a superagonist (i.e., a compound capable of producing a higher maximal response than an endogenous agonist of the target receptor and thus having an efficacy greater than 100%), a full agonist (i.e., a compound that produces a maximal response upon binding to and activating the receptor), or a partial agonist (i.e., a compound capable of activating the receptor but unable to elicit a maximal response from the receptor system). Examples of MC4R agonists are described in detail below.

[00100] As used herein, the term "antagonist" refers to any chemical compound that, when interacting with (e.g., binding to) a target, here MC4R, blocks the signaling activity of an MC4R agonist compound in a dose-dependent manner.

[00101] As used herein, the term "inverse agonist" refers to any chemical compound that, when interacting with (e.g., binding to) a target, here MC4R, reduces the background level of MC4R signaling activity in a dose-dependent manner.

[00102] As used herein, the term "effective amount" refers to an amount of a pharmacologically active agent, present as either an ionic complex or a pharmaceutical composition comprising an ionic complex, that is therapeutically or prophylactically sufficient to treat a target disorder. Examples of an effective amount typically range from about 0.0001 mg/kg body weight to about 500 mg/kg body weight. An illustrative range includes doses from about 0.001 mg/kg body weight to about 500 mg/kg. For example, an effective amount can range from about 0.005 mg/kg to about 500 mg/kg. In other examples, the range can be from about 0.0001 mg/kg to about 5 mg/kg. In further examples, an effective amount can range from about 0.01 mg/kg body weight to about 50 mg/kg body weight, or from 0.01 mg/kg body weight to about 20 mg/kg body weight.

[00103] As used herein, the term "other agent" includes any active pharmaceutical ingredient (API) that, when combined with a peptide described herein, enhances the therapeutic effect of the peptide described herein alone or in synergy with the peptide described herein (i.e., their combined effect exceeds additive effect). As used herein, the term "enhanced therapeutic effect" includes an improved therapeutic profile other than synergy. Examples of enhanced therapeutic effects include a decrease in the effective dose of the peptide described herein, an increase in the therapeutic window of the peptide described herein, etc. One or more other agents may be administered. Examples of other agents are described in detail below.

[00104] The other agent may be administered prior to, concurrently with, or subsequent to the administration of the peptide described herein. Accordingly, the peptide described herein and the other agent may be administered together in a single composition, or they may be administered in different compositions, such as simultaneously or sequentially. For example, if the peptide described herein and the other agent are administered sequentially in different compositions, the peptide described herein may be administered before or after the other therapeutic agent. Additionally, the peptide described herein and the other agent may be administered using the same or different administration schedules. For example, the peptide described herein and the other therapeutic agent may have different half-lives and/or may act on different time scales, such that the peptide described herein may be administered more frequently than the other therapeutic agent, or vice versa. Finally, the peptide described herein may be administered prior to the other agent, resulting in further enhancement of therapeutic efficacy resulting from the sequential administration of both therapeutic agents. Both the peptide described here and the other agent can be administered once or over a long period of time.

[00105] An effective amount in the methods or compositions of the present invention can be achieved by co-administering a first amount of a compound having MC4R modulatory activity, or a pharmaceutically acceptable salt thereof, and a second amount of at least one other agent. In one embodiment, each of the peptide described herein and the second agent is administered in a respective effective amount (i.e., each is administered in an amount that is therapeutically effective when administered separately). In another embodiment, each of the peptide described herein and the second agent is administered in an amount that does not provide a therapeutic effect by itself (a subtherapeutic dose). In a further embodiment, a peptide described herein is administered in an effective amount and the other agent is administered in a subtherapeutic dose. In a further embodiment, a peptide described herein can be administered in a subtherapeutic dose while the second agent is administered in an effective amount. In an illustrative embodiment, the combination of a peptide described herein and another agent provides an enhanced therapeutic effect, or a synergistic effect, compared to the peptide described herein or another agent used alone.

[00106] The presence of a synergistic effect can be determined using appropriate drug interaction analysis methods. Suitable methods include, for example, the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe additivity equation (S., Muischnek, H., Arch. Exp. Pathol. Pharmacol. 114: 313-326 (1926)), and the median effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each of the above equations can be used with experimental data to produce an appropriate graph to aid in analyzing the effects of a drug combination. The corresponding graphs constructed using the above equations include concentration-effect curves, isobolograms, and additivity index curves, respectively.

[00107] As used herein, the term "individual" refers to a mammal, preferably a human, but may also refer to an animal in need of veterinary treatment, including pets (such as dogs, cats, etc.), farm animals (such as cows, sheep, pigs, horses, etc.), and laboratory animals (such as rats, mice, guinea pigs, etc.).

[00108] As used herein, the term "treating" includes achieving, partially or substantially, a delay in, suppression of, or prevention of the development of clinical symptoms associated with a target disorder. For example, the term "treating" includes achieving, partially or substantially, one or more of the following results: a partial or complete reduction in body weight (which can be measured, for example, by body mass index, BMI); alleviation or improvement of clinical symptoms or indicators associated with obesity, such as type 2 diabetes, prediabetes, hemoglobin A1C (Hb1Ac) in the blood above 6%, hyperinsulinemia, hyperlipidemia, insulin insensitivity, glucose intolerance, etc.; delaying, suppressing, or preventing the development of obesity and obesity-related indicators; or delaying, suppressing, or preventing, partially or completely, the onset or development of obesity or obesity-related indicators. Delaying, suppressing or preventing the development of obesity includes, for example, delaying, suppressing or preventing the development of obesity in an individual of normal weight. The term "treating" also includes partially or completely reducing the risk of coronary heart disease, stroke and diabetes (e.g., type 2) associated with metabolic syndrome, as well as alleviating or improving clinical symptoms or signs of metabolic syndrome, such as one or more of the five indicators listed above. For example, the term "treating" includes delaying, inhibiting or preventing the development of parameters associated with metabolic syndrome, such as insulin resistance, glucose clearance and parameters of cardiovascular disease including heart rate and blood pressure, joint disease, inflammation, sleep apnea, binge eating and other eating disorders including bulimia, maintenance therapy for weight loss surgery and maintenance therapy for weight loss before orthopedic surgery. The term "prophylactic treatment" refers to treatment undertaken before the onset of clinical symptoms of the target disorder with the aim of preventing, suppressing, or reducing the frequency of its occurrence.

Sensory disorders

[00109] Examples of disorders sensitive to MC4R modulation, and more generally, disorders sensitive to the pharmacological action of the above categories of polypeptides and specific polypeptides, include acute and chronic inflammatory diseases such as general inflammation, inflammatory bowel disease, brain inflammation, sepsis and septic shock; diseases with an autoimmune component such as rheumatoid arthritis, gouty arthritis and multiple sclerosis; metabolic diseases and medical conditions accompanied by weight gain such as obesity, eating disorders and Prader-Willi syndrome; metabolic diseases and medical conditions accompanied by weight loss such as anorexia, bulimia, AIDS-related wasting, cachexia, cancer cachexia and wasting in the frail elderly; diabetes and related conditions and complications such as retinopathy; neoplastic proliferation including skin cancer and prostate cancer; medical conditions of the reproductive or genital organs such as endometriosis and uterine bleeding in women, sexual dysfunction, erectile dysfunction and decreased sexual response in women; diseases or conditions resulting from treatment or insult to the body such as transplant rejection, ischemia and reperfusion injury, treatment of spinal cord injury and acceleration of wound healing, and weight loss caused by chemotherapy, radiation therapy, temporary or permanent immobilization or dialysis; cardiovascular diseases or conditions such as hemorrhagic shock, cardiogenic shock, hypovolemic shock, cardiovascular collapse and cardiac cachexia; pulmonary diseases or conditions such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, asthma and pulmonary fibrosis; measures to increase immune tolerance and prevent threats to the immune system, such as those associated with certain allergies or organ transplant rejection; treatment of dermatological diseases and conditions such as psoriasis, loss of skin pigmentation, acne, keloid formation and skin cancer; behavioural conditions and disorders, central nervous system or neurological conditions and disorders such as anxiety, depression, memory impairment and memory dysfunction, modulation of pain perception and treatment of neuropathic pain; conditions and diseases associated with alcohol consumption, alcohol abuse and/or alcoholism; and renal conditions or diseases such as renal cachexia or natriuresis. Other examples include normalizing or homeostatic activities in the individual, including thyroxine release, aldosterone synthesis and release, regulation of body temperature, blood pressure, heart rate, vascular tone, cerebral blood flow, blood glucose levels, bone metabolism, bone formation or development, ovarian mass, placental development, prolactin and FSH secretion, intrauterine fetal development, parturition, spermatogenesis, sebum and pheromone secretion, neuroprotection and nerve growth, and modulation of motivation, learning ability, and other behavioral parameters. Other examples include binge eating, bulimia, or other eating disorders.

[00110] In exemplary embodiments, disorders responsive to MC4R receptor modulation include type 1 diabetes mellitus, type 2 diabetes, obesity, insulin resistance, metabolic syndrome, cardiovascular disease or low-density lipoprotein/high-density lipoprotein/triglyceride imbalance, non-alcoholic fatty liver disease, and substance abuse disorders.

[00111] In exemplary embodiments, disorders responsive to MC4R receptor modulation include type 1 diabetes, type 2 diabetes, obesity, insulin resistance, or metabolic syndrome.

Obesity

[00112] As used herein, the term "obese" refers to an individual whose body mass index (BMI) is about 30 kg/ ^m2 or greater, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 kg/ ^m2 , or greater. In specific embodiments, an obese individual has a BMI within the range defined as "obese" by the Centers for Disease Control. See URL http://www.cdc.gov/obesity/defining.html, last accessed October 28, 2011. For example, in some embodiments, an adult is considered to be obese if his or her BMI is ≥30.0 kg/ ^m2 .

Diabetes and related disorders

[00113] In exemplary embodiments, individuals who can be treated using the methods of the present invention suffer from diabetes-related disorders or are at increased risk of developing such disorders. The term "diabetes-related disorders" refers to diabetes (including type 1 (OMIM 222100) and type 2 (OMIM 125853)), insulin resistance, and metabolic syndrome.

[00114] In exemplary embodiments, the subject to be treated suffers from diabetes (type 1 or type 2), insulin resistance, or metabolic syndrome. In exemplary embodiments, the disorder comprises diabetes, such as type 2 diabetes. In exemplary embodiments, the subject suffers from type 2 diabetes, or is at increased risk of developing type 2 diabetes, as defined by the World Health Organization and the International Diabetes Federation in Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycemia, published in 2006, which is incorporated herein by reference in its entirety. In exemplary embodiments, the subject suffering from diabetes has a fasting plasma glucose level of ≥126 mg/dL, or a 2-hour plasma glucose level (2 hours after oral administration of 75 g of glucose) of ≥200 mg/dL. In exemplary embodiments, the diabetic or prediabetic individual has elevated levels of glycated hemoglobin, such as may be greater than 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6% or more of total hemoglobin. In exemplary embodiments, a diabetic or prediabetic individual may be identified or further characterized by analyzing genetic polymorphisms (including, for example, polymorphisms that cause an alteration in expression levels, such as an increase or decrease in expression levels, and/or an alteration in coding sequences) in or near one or more of the genes listed below in Table 1:

[00115]

[00116] In exemplary embodiments, other genes that can be used to identify or further characterize individuals to be treated with the methods of the present invention include FTO (OMIM 610966), JAZF 1 (OMIM 606246), and HHEX (OMIM 604420).

[00117] In exemplary embodiments, the subject to be treated with the methods of the present invention suffers from type 1 diabetes. In exemplary embodiments, the subject with type 1 diabetes is characterized by a C-peptide assay, such as such subject having a fasting C-peptide level of less than about 1.0 nmol/L, such as less than 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nmol/L, or less, such as less than 0.33, 0.25, 0.2, or 0.1 nmol/L. In exemplary embodiments, the C-peptide level is measured after oral glucose administration (2 hours after oral administration of 75 g glucose) and an increase of less than 0.54 nmol/L, such as less than 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 nmol/L, is detected. An abnormal fasting glucose level (110-125 mg/dL) or impaired glucose tolerance (2 hours after oral administration of 75 g glucose: 140-199 mg/dL) can be used to identify or further characterize a decrease in beta cell function in individuals with type 1 diabetes. In exemplary embodiments, type 1 diabetes is identified or further characterized by the presence of autoantibodies to islet cell antigens and/or insulin, such as autoantibodies directed against GAD 65 kDa (OMIM 138275) and/or phosphatase-related molecule IA-2.

Insulin resistance

[00118] In exemplary embodiments, the disorder is "insulin resistance," which can be identified by any means known in the art and is characterized by a decreased ability of insulin to lower blood glucose levels. In exemplary embodiments, insulin resistance is determined or further characterized by the presence of one or more polymorphisms (including, e.g., polymorphisms that result in altered expression levels, e.g., increased or decreased expression levels, as well as the formation of variants of gene products of coding sequences, such as proteins) in one or more of the following genes: RETN, PTPN1, TCF1 (OMIM 142410; see, e.g., polymorphism 0011), PPP1R3A (OMIM 600917; see, e.g., polymorphisms 0001, 0003), PTPN1 (OMIM 176885; see, e.g., polymorphism 0001), ENPP1 (OMIM 173335; see, e.g., polymorphism 0006), IRS1 (OMIM 147545; see, e.g., polymorphism 0002), EPHX2 (OMIM 13281 1; see, for example, polymorphism 0001), the leptin gene (OMIM 164160, see, for example, polymorphisms 0001 and 0002), the leptin receptor gene (OMIM 601007; see, for example, polymorphisms 0001, 0002, 0004, and 0005), or the insulin receptor gene (INSR, OMIM 147670; see, for example, polymorphisms 0001-0037).

Metabolic syndrome

[00119] In exemplary embodiments, the disorder is metabolic syndrome. As used herein, the term "metabolic syndrome" refers to a group of symptoms that occur together and increase the risk of coronary heart disease, stroke, and type 2 diabetes. According to the American Heart Association and the National Heart, Lung, and Blood Institute, metabolic syndrome (also referred to as syndrome X) is considered to be present if an individual exhibits three or more of the following symptoms:

1) Blood pressure is equal to or higher than 130/85 mmHg;

2) Fasting blood sugar (glucose) level is equal to or exceeds 100 mg/dL;

3) Large waist circumference (length around the waist):

- for men - 40 inches or more;

- for women - 35 inches or more;

4) Low HDL Cholesterol:

- in men - below 40 mg/dl;

- in women - below 50 mg/dl;

5) Triglyceride levels are equal to or greater than 150 mg/dL.

Metabolic syndrome can be diagnosed by determining an individual's blood pressure, blood glucose levels, HDL cholesterol levels, LDL cholesterol levels, total cholesterol levels, and triglyceride levels.

[00120] In exemplary embodiments, the individual has central obesity (waist circumference ≥80 cm in women; ≥90 cm in Asian and South and Central American men, and ≥94 cm in all other men), a BMI of 30 kg/ ^m2 , elevated triglycerides (≥150 mg/dL, or specific treatment for this lipid abnormality), low HDL cholesterol (<40 mg/dL in men, <50 mg/dL in women, or specific treatment for this lipid abnormality), elevated blood pressure (sBP=130 mmHg or dBP=85 mmHg, or treatment for a previously diagnosed hypertension), or elevated fasting plasma glucose (FPG=100 mg/dL or an existing diagnosis of type 2 diabetes), including combinations thereof. In exemplary embodiments, the subject to be treated with the methods of the present invention suffers from, or is at increased risk for, metabolic syndrome, as defined by the International Diabetes Federation in "The IDF consensus worldwide definition of the metabolic syndrome," published in 2006, which is incorporated herein by reference in its entirety, i.e., the subject has central obesity (as described above and/or a BMI >30 kg/ ^m2 ), and any two of elevated triglycerides, reduced HDL cholesterol, elevated blood pressure, or elevated fasting plasma glucose. In exemplary embodiments, metabolic syndrome is characterized, or further characterized, by the subject having a mutation at a locus selected from 3q27 (see, e.g., OMIM 605552) and/or 17pl2 (see, e.g., OMIM 605572).

Disorders caused by MC4R mutations

[00121] The present invention relates to a method of treating a disorder in an individual suffering from an impaired MC4R response to α-melanocortin-stimulating hormone (α-MSH). The method comprises administering an effective amount of a melanocortin-4 receptor (MC4R) agonist. In an exemplary embodiment, the individual is a heterozygous carrier of an MC4R mutation resulting in an impaired MC4R response to α-melanocortin-stimulating hormone (α-MSH). Since heterozygous carriers retain the ability to respond to the natural MC4R ligand, treatment of MC4R-associated diseases in heterozygous carriers by administering an MC4R agonist does not require knowledge of the type of MC4R mutation.

[00122] In one exemplary embodiment, the disease is obesity, such as MC4R-associated obesity. In another exemplary embodiment, the disorder is metabolic syndrome.

[00123] The human MC4R gene product (hMC4R) is a well-characterized protein encoded by the genomic sequence with GenBank accession number CH471077. Mutations in the MC4R receptor are a cause of severe obesity in childhood. In the population of juveniles with known obesity, the prevalence of MC4R mutation carriers is approximately 2.5%, with the highest prevalence of 6% observed among severely obese children. Humans with MC4R mutations have more or less the same phenotype as described for mice with a mutation in the MC4 receptor gene. Such individuals have marked hyperphagia, hyperinsulinemia, increased fat mass with associated lean body mass, increased bone mineral density, and increased linear growth velocity, without any changes in cortisol, gonadotropin, thyroid hormones, or sex steroid hormones. In contrast to MC4 receptor deletion, hyperphagia and hyperinsulinemia generally tend to decrease with human age. Similar to MC4R knockout mice, heterozygous carriers have an intermediate phenotype compared to homozygous carriers. The resulting hyperphagia observed during a test meal is less severe than that seen in leptin-deficient individuals. The degree of MC4 receptor dysfunction, as determined by in vitro assays, can predict the amount of food ingested during a test meal by an individual carrying a given mutation and correlates with the occurrence and severity of the obesity phenotype. At least 90 different MC4 receptor mutations have been associated with obesity, and other MC4 receptor mutations that cause a similar obesity phenotype may be found.

[00124] Examples of MC4R mutations that cause obesity in humans are described in Farooqi et al., The Journal of Clinical Investigation, July 2000, vol. 106(2), pp. 271-279 and Vaisse et al., The Journal of Clinical Investigation, July 2000, vol. 106(2), pp. 253-262, the relevant portions of which are incorporated herein by reference.

[00125] Other mutations that can cause obesity in humans include R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA, as described in Xiang et al., "Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist", Biochemistry, 2010 Jun 8; 49(22):4583-600, the relevant portions of which are incorporated herein by reference.

[00126] Other examples of mutations that can cause obesity in humans include those listed in the Online Catalog of Mendelian Inheritance, Human (OMIM), a database of human genes and genetic disorders, under accession number 155541 (MC4R) (more specifically, under accession numbers 155541.0001-155541.0023) at the URL http://omim.org/entry/155541. Typical examples of mutations include 4-BP DEL, NT631; 4-BP INS, NT732; TYR35TER; ASP37VAL; SER58CYS; ILE102SER; ASN274SER; 1-BP INS, 112A; 4-BP DEL, 211CTCT; ILE125LYS; ALA175THR; ILE316SER; TYR287TER; ASN97ASP; 15-BP DEL (codons delta88-92); and SER127LEU. The relevant portions of the OMIM database are incorporated herein by reference.

[00127] In exemplary embodiments, the MC4R mutation results in the retention of MC4R signaling activity.

[00128] Mutations in the genomic sequence encoding MC4R can be detected using methods well known to those of ordinary skill in the art. For example, the genomic sequence can be cloned using nucleotide primers, such as, for example, those described in Farooqi et al, The Journal of Clinical Investigation, July 2000, vol. 106 (2), pp. 271-279 and Vaisse et al, The Journal of Clinical Investigation, July 2000, vol. 106 (2), pp. 253-262, after which the cloned sequence can be analyzed using commercially available sequencers and software.

[00129] MC4R activity can be measured using methods well known to those of ordinary skill in the art. For example, cells are transiently transfected with cloned MC4R DNA, the transfected cells are contacted with an MC4R agonist (e.g., α-MSH), and the level of intracellular cAMP, a second messenger of MC4R, is measured using an electrochemiluminescence assay described, for example, in Roubert et al, Journal of Endocrinology (2010) 207, pp. 177-183. A decrease in MC4R signal can be determined by comparing the level of intracellular cAMP produced in response to a particular agonist between wild-type MC4R and mutant MC4R.

[00130] MC4R modulators (e.g., agonists) can also be used to treat patients suffering from other disorders such as decreased tone of natural MC4R agonists. Examples of such patients include individuals heterozygous or homozygous for mutations in genes that play an important role in the leptin-dependent pathway (Nature Clinical Practice Endocrinology and Metabolism, 2006; 2; 6; 318 and NEng J Med: 2007; 356; 3; 237), proopiomelanocortin processing (Nature Genetics, 1998, 155; Cell Metabolism, 2006; 3; 135; Annals Acad Med, 2009, 38; 1; 34), or mutations in genes encoding prohormone convertases.

Modes of administration

[00131] The administration of the ionic complex or pharmaceutical composition described herein used to carry out the methods described herein can be carried out continuously, hourly, four times a day, three times a day, twice a day, once a day, once every two days, twice a week, once a week, once every two weeks, once a month, or once every two months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months or more, or using some other periodic administration regimen. The ionic complex compositions of the present invention can be used for effective therapeutic administration, for example, once a day, once a week, once every 2 weeks, once every four weeks, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months.

[00132] Suitable routes of administration include, but are not limited to, peripheral administration. Examples of peripheral administration include oral, subcutaneous, intraperitoneal, intramuscular, intravenous, rectal, transdermal, buccal, sublingual, inhalation, pulmonary, or transnasal administration. In preferred embodiments, subcutaneous administration is used.

Combination therapy

[00133] Any peptide described herein, whether or not included in an ionic complex (e.g., peptide 1 prior to complexation), can be used to treat any of the diseases responsive to MC4R modulation by administration in combination with one or more other pharmaceutically active compounds (an "other agent"). Such combined administration can be accomplished using a unit dosage form that contains one or more peptides described herein and one or more other agents, such unit dosage forms include tablets, capsules, spray, inhalation powder, injection liquids, and the like. Alternatively, combined administration can be accomplished by administering two different dosage forms, one containing one or more peptides described herein and the other dosage form containing one or more other agents. In this case, the dosage forms can be the same or different. Non-limiting examples of combined treatments are provided below.

[00134] The peptide described herein (e.g., whether or not part of an ionic complex) can be used in combination with one or more other agents suitable for treating various disorders associated with changes in body weight and eating patterns, such as obesity and/or overweight. In particular, the other agent can be an anti-obesity drug that affects energy expenditure, glycolysis, gluconeogenesis, glucogenolysis, lipolysis, lipogenesis, fat absorption, fat storage, fat release, fasting and/or satiety and/or thirst mechanisms, appetite/motivation, food intake, or gastrointestinal motility. Drugs that reduce energy expenditure include, in particular, various pharmacological agents called anorexigenic agents, which are used as adjuncts to behavioral therapy in weight loss programs.

[00135] Typically, the total dose of the anti-obesity agent or drug, when used in combination with one or more of the peptides described herein, may be in the range of 0.1 to 3000 mg/day, preferably about 1 to 1000 mg/day, more preferably about 1 to 200 mg/day, and said dose may be administered in one dose or divided into 2 to 4 doses. However, the exact dose is determined by the attending physician and depends on factors such as the activity of the compound administered, the age, weight, condition of the patient and his ability to respond to treatment.

[00136] One or more of the peptides described herein (e.g., whether or not part of an ionic complex) can be used in combination with one or more other agents used to treat diabetes.

[00137] In addition or alternatively, one or more of the peptides described herein can be further combined with one or more other agents used to treat diseases, disorders, and conditions associated with obesity and/or overweight, such as insulin resistance; impaired glucose tolerance; type 2 diabetes; metabolic syndrome; dyslipidemia (including hyperlipidemia); hypertension; cardiac disorders (e.g., coronary artery disease, myocardial infarction); cardiovascular diseases; non-alcoholic fatty liver disease (including non-alcoholic steatohepatitis); joint diseases (including secondary osteoarthritis); gastroesophageal reflux; sleep apnea; atherosclerosis; stroke; macro and micro vascular diseases; steatosis (e.g., liver); gallstones; and gallbladder disorders.

Another remedy

[00138] One or more other means may be selected, for example, from the group comprising

insulin and insulin analogues;

insulin secretagogues, including sulfonylureas (eg, glipizide), and prandial glucose regulators (sometimes called "short-acting secretagogues") such as, for example, meglitinides (repaglinide and nateglinide);

incretin enhancers: incretin, incretin mimetic, incretin function enhancers such as GLP-1, GIP; GLP-1 agonists (eg, exenatide and liraglutide (VICTOZA)), DPP-4 inhibitors (eg, vildagliptin, saxagliptin, and sitagliptin)

insulin sensitizers, including peroxisome proliferator-activated receptor gamma (PPARγ) agonists such as thiazolidinediones (eg, pioglitazone and rosiglitazone), as well as agents with any combination of activity at PPAR alpha, gamma, and delta;

agents that modulate hepatic glucose balance, such as biguanides (eg, metformin), fructose-1,6-biphosphatase inhibitors, glycogen phosphorylase inhibitors, glycogen synthase kinase inhibitors, and glucokinase activators;

agents aimed at reducing/slowing down the absorption of glucose from the intestine, such as alpha-glucosidase inhibitors (eg, miglitol and acarbose);

agents that antagonize the action of glucagon or reduce its secretion, such as amylin analogues (eg, pramlintide);

agents that prevent glucose reabsorption in the kidneys, such as sodium-dependent glucose transporter 2 (SGLT-2) inhibitors (eg, dapagliflozin);

agents intended to treat complications of long-term hyperglycemia, such as aldose reductase inhibitors (eg, epalrestat and ranirestat);

agents used to treat complications associated with microangiopathies;

anti-dyslipidemic agents such as HMG-CoA reductase inhibitors (statins, eg, rosuvastatin) and other cholesterol-lowering agents;

PPARA agonists (fibrates, such as gemfibrozil and fenofibrate);

agents that promote the excretion of bile acids (eg, cholestyramine);

cholesterol absorption inhibitors (eg, plant sterols (such as phytosterols), synthetic inhibitors);

inhibitors of cholesterol ester transport protein (CETP); inhibitors of the ileal bile acid transport system (IB AT inhibitors);

bile acid binding resins;

nicotinic acid (niacin) and its analogues;

antioxidants such as probucol;

omega-3 fatty acids;

antihypertensive agents including adrenergic receptor antagonists such as beta-blockers (eg, atenolol), alpha-blockers (eg, doxazosin), and mixed alpha/beta-blockers (eg, labetalol);

adrenergic receptor agonists, including alpha-2 agonists (eg, clonidine);

angiotensin-converting enzyme (ACE) inhibitors (eg, lisinopril), calcium channel blockers such as dihydropyridines (eg, nifedipine), phenylalkylamines (eg, verapamil), and benzothiazepines (eg, diltiazem);

angiotensin II receptor antagonists (eg, candesartan); aldosterone receptor antagonists (eg, eplerenone);

centrally acting adrenergic agents such as central alpha agonists (eg, clonidine); and diuretics (eg, furosemide);

hemostasis modulators, including antithrombotic agents such as fibrinolysis activators;

thrombin antagonists;

factor VIIa inhibitors; anticoagulants such as vitamin K antagonists (eg, warfarin), heparin and its low-molecular-weight analogues, factor Xa inhibitors, and direct thrombin inhibitors (eg, argatroban); antiplatelet agents such as cyclooxygenase inhibitors (eg, aspirin), adenosine diphosphate (ADP) receptor inhibitors (eg, clopidogrel), phosphodiesterase inhibitors (eg, silostazol), glycoprotein IIB/IIA inhibitors (eg, tirofiban), and adenosine reuptake inhibitors (eg, dipyridamole);

antiobesity agents such as appetite suppressants (eg, ephedrine), including noradrenergic agents (eg, phentermine) and serotonergic agents (eg, sibutramine), pancreatic lipase inhibitors (eg, orlistat), microsomal transport protein (MTP) modulators, diacylglycerol acyltransferase (DGAT) inhibitors, and cannabinoid receptor antagonists (eg, rimonabant);

agents that modify feeding behavior, such as orexin receptor modulators and melanin-concentrating hormone (MCH) modulators;

neuropeptide Y (NPY)/NPY receptor modulators;

pyruvate dehydrogenase kinase (PDK) modulators;

serotonin receptor modulators;

leptin/leptin receptor modulators;

ghrelin/ghrelin receptor modulators;

agents that enhance beta cell function;

agents that stimulate energy expenditure (eg, beta-adrenergic receptor stimulants, UCP-1 agonists, brown fat modulators and stimulators);

agents that cause adipocyte lysis (eg, antibodies);

nicotine or nicotine addiction medications;

estrogen, natural or synthetic estrogen receptor modulator;

μ-opioid receptor modulator; and

agents modulating monoamine transport, such as selective serotonin reuptake inhibitors (SSR1) (e.g. fluoxetine), norepinephrine reuptake inhibitors (NARIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), triple monoamine reuptake inhibitors (e.g. tesofensine) and monoamine oxidase inhibitors (MAOIs) (e.g. toloxatone and amiflamine), or their pharmaceutically acceptable salts.

[00139] In an exemplary embodiment, the MC4R agonist (e.g., an MC4R agonist that is part of an ionic complex or not part of a complex) and the other agent are administered simultaneously, sequentially, or separately with ingestion of a very low calorie diet (VLCD) or a low calorie diet (LCD).

Method for obtaining an ionic complex

[00140] Furthermore, the invention relates to a method for producing an ionic complex and a pharmaceutical composition of the present invention. The method comprises producing a cationic polypeptide and an anionic filler selected from a PEG carboxylic acid, a fatty acid containing 10 or more carbon atoms, a phospholipid, or a combination thereof. Combining the cationic polypeptide and the anionic filler under conditions that promote the formation of an ionic complex, and producing a pharmaceutical composition containing the ionic complex.

[00141] For example, a mixture of an anionic excipient selected from a PEG carboxylic acid, a fatty acid containing 10 or more carbon atoms, a phospholipid, or a combination thereof, and any other excipient in an aqueous medium (e.g., water) can be uniformly distributed to achieve a well-dispersed state by autoclaving the mixture under suitable conditions. Suitable conditions can include, for example, a time range of about 3 minutes to 25 minutes and a temperature of 121°C to 134°C. An example of suitable conditions includes a time period of about 15 minutes and a temperature of about 121°C. In addition, autoclaving allows for a sterile mixture to be obtained. A sterile aqueous solution of a cationic peptide can then be added to the mixture to obtain a sterile, homogeneous ionic complex of the present invention. Depending on the nature of the anionic excipient, either a clear solution or a homogeneous suspension of the ionic complex can be obtained. For example, PEG-carboxylic acid and mPEG2000-DSPE used in the examples described herein typically yield a clear solution containing the ionic complex, whereas use of the lipid DPP typically results in a homogeneous suspension containing the ionic complex.

[00142] Alternatively, the ionic complex mixture can be prepared by dissolving the excipients in water containing the cationic polypeptide, followed by sterilizing the resulting mixture by filtration through a 0.2 μm filter.

[00143] A method for producing an ionic complex comprising a cationic polypeptide and an anionic filler selected from: a PEG carboxylic acid; a fatty acid containing 10 or more carbon atoms; a phospholipid; and a combination thereof, which comprises:

a) obtaining a mixture of an anionic filler and an aqueous filler-diluent;

b) autoclaving the mixture under conditions sufficient to sterilize the filler;

c) adding to the mixture of excipients a sterile solution containing a cationic polypeptide and an aqueous diluent for the peptide.

In one embodiment, the filler mixture is a suspension. In another embodiment, the filler mixture is a solution.

[00144] In an alternative embodiment, the present invention provides a method for producing an ionic complex comprising a cationic polypeptide and an anionic excipient selected from: a PEG carboxylic acid; a fatty acid containing 10 or more carbon atoms; a phospholipid; and a combination thereof, which comprises:

a) preparation of a solution of anionic filler and aqueous filler-diluent;

b) filtering the solution obtained in step a through a 0.2 micron filter;

c) adding to the excipient solution obtained in step b a sterile solution containing a cationic polypeptide and an aqueous diluent for the peptide.

[00145] In a further embodiment, the invention provides a method for producing an ionic complex comprising a cationic polypeptide and an anionic filler selected from: a PEG carboxylic acid; a fatty acid containing 10 or more carbon atoms; a phospholipid; and a combination thereof, which comprises:

a) obtaining a solution containing an anionic filler, which is an aqueous filler-diluent, and a cationic polypeptide;

b) sterilization of the resulting solution by filtration through a 0.2 micron filter.

Suitable examples of the excipient and vehicle for the cationic polypeptide include polyol (such as propylene glycol, tripropylene glycol, glycerol), ethanol, benzyl alcohol, DMSO, NMP, DMF, water, pH stabilizing buffer solutions and mixtures thereof.

EXAMPLES

Peptide synthesis

[00146] Cationic polypeptides suitable for use in the present invention are prepared by conventional solid-phase peptide synthesis. For example, peptide 1 and other MC4R analogs described herein can be prepared by the methods described in U.S. Patent 8,349,797, the contents of which are incorporated herein by reference. The peptide chain is extended stepwise, starting with a C-terminal amino acid derivative attached to an appropriate solid support resin suitable for peptide synthesis. Rink amide resin MBHA is used as a solid support to synthesize a peptide containing a C-terminal amide functionality. Resins such as 2-chlorotrityl chloride resin, Wang resin, or Merrifield resin capable of forming an ester linkage with the Fmoc-amino acid can be used to synthesize a peptide containing a free C-terminal carboxyl functional group. Most of these ester-linked Fmoc-amino acid-resin conjugates are commercially available from various sources and are widely used when practical.

Synthesis of disulfide-cyclized peptides

[00147] A linear amide derivative of a disulfide-containing cyclic peptide is assembled using Fmoc chemistry on a solid phase peptide synthesizer. The Rink Fmoc-Amide Resin is placed in a reaction vessel and allowed to swell in NMP. It is then treated with 20% piperidine in NMP for 15 min and washed three times with NMP. The resin is tested for a positive score in the Kaiser assay (Kaiser, E., Colescot, R. L., Bossinge, C. D. & Cook, P. I. Anal. Biochem., 1990, 34: 595-598). The resin is resuspended in NMP and mixed with the Fmoc derivative of the first C-terminal amino acid and HOBt. The coupling reaction is initiated by adding HBTU reagent and DIPEA. After stirring for 2-3 hours, completion of the coupling reaction is confirmed by obtaining a negative result in the Kaiser test using a small aliquot of the resin taken from the reaction mixture. The resin is then washed three times with NMP. The Fmoc group is then removed as described previously and the entire cycle is repeated with the Fmoc derivative of the second C-terminal amino acid as described. The same reaction cycle is repeated sequentially for each subsequent amino acid. The chloranil staining test (Vojkovsky, T. Pept. Res, 1995, 8: 236-237) is used instead of the Kaiser test to analyze the removal of the Fmoc protecting group from the proline residue in the peptide sequence and to test the completion of the amino acid-proline coupling reaction (negative result in the chloranil test). In the case of peptides containing an N-terminal acetyl group, after removal of the Fmoc protecting groups, the peptide resin is treated with acetic anhydride and pyridine for 10 minutes. After a negative result in the Kaiser test, the resin is washed with NMP, dichloromethane and dried in a vacuum. Fmoc derivatives of amino acids are used to synthesize these peptides. In the case of amino acids containing three functional groups, the following derivatives are used: Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-hCys(Trt)-OH, Fmoc-Pen(Trt)-OH, Fmoc-Tyr(But)-OH, Fmoc-His(L-Me)-OH, Fmoc-His(3-Me)-OH and Fmoc-Glu(O-But)-OH.

[00148] To cleave the peptide from the resin and to deprotect the side chain functionalities, the peptide-resin conjugate is placed in a mixture of 2% TIS/5% water/5% (w/v) DTT/88% TFA. The solution is stirred for 3.5 h and then filtered. The filtrate is mixed with cold anhydrous ethyl ether. The precipitate is collected by centrifugation. The solvent is decanted and the peptide pellet is resuspended in fresh ether. The ether treatment is repeated two more times. The peptide is dried in vacuo. The crude linear peptide product is diluted to a concentration of 2 mg/mL with 5% acetic acid and 0.5 M iodine/methanol is added dropwise with vigorous stirring until the solution turns persistently pale yellow. The solution is stirred for an additional 10 min. The excess iodine is then quenched by adding 1 M sodium thiosulfate with stirring until the mixture becomes colorless. The cyclic peptide solution is lyophilized and the crude powder is purified by preparative HPLC using a C-18 reversed phase column. Fractions containing the purified product are pooled and lyophilized. The peptides are analyzed by mass spectrometry using the electrospray ionization technique and identified by the correct mass.

Synthesis of lactam-cyclized peptides

[00149] Cyclic lactam-containing peptides are also synthesized using standard solid-phase peptide synthesis methods. For peptides containing a C-terminal Dpr, the Fmoc-Dpr(Mtt)-BHA resin is transferred to the reactor of a solid-phase peptide synthesizer. The Fmoc group is removed as described above and the next Fmoc-protected amino acid, such as Fmoc-Trp(Boc)-OH, is coupled to the resin via standard coupling reactions. The Fmoc protecting group is removed and the remaining amino acids are added individually in the correct sequence, repeating the coupling and deprotection reactions until the amino acid sequence is complete. Fmoc-Glu(OPip) is used to couple glutamic acid. The fully assembled peptide is acetylated at the N-terminus as in the method described previously for disulfide-containing peptides. Orthogonal side chain protecting groups are then removed. For example, orthogonal protecting groups from a peptide-resin conjugate containing Glu, the side chain of which is protected with 2-phenylisopropyl (OPip) ester, or Dpr, the side chain of which is protected with 4-methyltrityl (Mtt), are cleaved by treatment with 1% TFA in dichloromethane. Following deprotection, the peptide-resin conjugate is suspended in NMP and treated with HBTU DIPEA. After cyclization (negative in the Kaiser test), the peptide-resin is washed with dichloromethane and dried. Cleavage of the cyclic peptide from the resin, as well as any remaining protecting groups, is accomplished using trifluoroacetic acid (TFA) in the presence of water and 1,2-ethanedithiol (EDT). The product is precipitated by addition of cold anhydrous ether and collected by centrifugation. Final purification is performed by HPLC on a C-18 reversed phase column. The purified peptide is lyophilized and its mass is determined by mass spectrometry using electron spray technology.

[00150] Certain cationic polypeptides listed in Table 1 were tested to determine their activity using the assays described below. The results of such assays are presented in Table 2.

Peptide testing:

Radioligand binding assays:

[00151] Receptor binding assays to determine the binding constant (K _d ) or inhibitory concentration (IC ₅₀ ) by displacing a radiolabeled ligand from a complex with a receptor by a cyclic peptide of the present invention can be carried out using any method known in the art.

[00152] As an example, cell membrane preparations for binding assays were prepared from CHO-K1 cells transfected to stably express hMC receptor subtypes 1, 3, 4, or 5. Competitive inhibition of [ ¹²⁵ I](Tyr ² )-(Nle ⁴ -D-Phe ⁷ )-alpha-MSH([ ¹²⁵ I]-NDP-α-MSH) binding was performed in polypropylene 96-well plates. Briefly, cell membranes (1-10 μg protein/well) prepared as described above were incubated in 50 mM Tris-HCl, pH 7.4, containing 0.2% BSA, 5 mM MgCl ₂ , 1 mM CaCl ₂ , and 0.1 mg/ml bacitracin, using increasing concentrations test compound and 0.1-0.3 nM [ ¹²⁵ I]-NDP-α-MSH for approximately 120 minutes at 37°C. Ligand-bound [ ¹²⁵ I]-NDP-α-MSH was separated from free [ ¹²⁵ I]-NDP-α-MSH by filtration through GF/C glass fiber filter plates (Unifilter®, Meriden, CT, USA) pre-soaked in 0.1% (w/v) polyethyleneimine (PEI). The filters were washed three times with 50 mM Tris-HCl, pH 7.4, at approximately 0-4°C and then analyzed for radioactivity. Binding assay results were analyzed using computer-assisted nonlinear regression analysis.

Cyclic AMP stimulation assay:

[00153] The agonist or antagonist status of a cyclic peptide of the present invention is determined by functional assays, which can be carried out by any method known in the art.

[00154] Electrochemiluminescence (ECL) analysis

[00155] Peptide-stimulated intracellular cyclic AMP (cAMP) levels were determined in a dose-dependent manner using an electrochemiluminescence (ECL) assay (Meso Scale Discovery, Gaithersburg, MD, USA; hereinafter referred to as "MSD"). Briefly, CHO-K1 cells stably expressing hMC receptor subtypes were suspended in RMPI 1640® assay buffer (RMPI 1640 buffer containing 0.5 mM IBMX and 0.2% protein mixture (MSD Blocker A)). Transgenic CHO-K1 cells stably expressing hMC receptor subtypes 1, 3, 4, or 5 are distributed at approximately 7,000 cells/well into 384-well Multi-Array (MSD) plates containing embedded carbon electrodes and coated with anti-cAMP antibodies. Following addition of increasing concentrations of test compounds, cells are incubated for approximately 40 minutes at 37°C. Cell lysis buffer (HEPES-buffered saline containing _MgCl2 and Triton X-100®, pH 7.3) containing 0.2% protein mixture and 2.5 nM TAG™ ruthenium-labeled cAMP (MSD) is added and cells are incubated for approximately 90 minutes at room temperature. At the end of the second incubation period, reading buffer (Tris-buffered saline containing ECL coreagent and Triton X-100, pH 7.8) is added and cAMP levels in the cell lysates are immediately determined by ECL detection using a Sector Imager 6000 reader® (MSD). The results are processed by computer-assisted nonlinear regression analysis (XL fit; IDBS) and expressed as EC ₅₀ values. The EC ₅₀ is the concentration of the agonist compound required to elicit a response of 50% of the maximal, e.g., 50% of the maximal cAMP level determined by the assay described above.

Analysis by measuring the level of cAMP:

[00156] Cells transfected with human MC4-R are grown to confluence in 96-well plates (approximately 250,000 cells per well). Cells are treated in triplicate with 0.2 mM isobutyl methylxanthine (IBMX) and sorted based on peptide concentration or, alternatively, peptide in the presence of 20 nM NDP-MSH. Cells are treated identically and samples containing only 20 nM NDP-MSH are used as positive controls in a volume of 200 μl. Blank samples containing only buffer are used as negative controls. After incubation for one hour at 37°C, cells are lysed by adding 50 μl of cell lysis buffer. The total amount of cAMP accumulated in 250 μl of the specified incubation medium is quantified using a commercially available low pH cAMP assay kit (Amersham Biosciences) according to the method provided by the kit supplier. A peptide causing cAMP accumulation in the same range or higher than that of alpha-MSH, used as a positive control, is considered an agonist. A graph is plotted using the results obtained for the agonist, the curve is fitted, and EC ₅₀ values are determined. A peptide causing accumulation in the same range as the negative control (empty buffer in the absence of alpha-MSH) is considered ineffective at the concentration tested. A peptide causing reduced accumulation is considered an antagonist if inhibition of the cAMP level is observed compared to that induced by alpha-MSH, also used in the assay. A similar analysis can be performed using cells expressing hMC-1R, hMC-3R and hMC-5R.

Measurement of cAMP accumulation using the β-galactosidase (β-Gal) reporter system:

[00157] An enzyme fragment complementation (EFC) chemiluminescence readout system using β-galactosidase (β-Gal) as a functional reporter system is used. This assay system, which uses different melanocortin receptor systems, is commercially available (Hunter GPCR cAMP Assay System, Discoverx Corp, Fremont, CA). The assay uses the enzyme β-Gal, which is cleaved into two complementary fragments; EA, an enzyme acceptor, and ED, an enzyme donor. In the assay, the ED fragment hybridized to cAMP competes with cAMP produced by cells for binding to cAMP-specific antibodies. EA is then added, forming active β-Gal with unbound ED-cAMP. The resulting active enzyme then converts the chemiluminescent substrate, generating a signal that is recorded on a standard microplate reader.

[00158] Briefly, 10,000 cells per well are seeded in a plate, incubated overnight, and then each well (cells incubated with 10 μl of assay buffer) is incubated with 4-fold serial concentrations of the test compound in cell assay buffer (5 μl) and cAMP-specific antibody (5 μl) for 30 min at 37°C. Cell lysis buffer (20 μl) containing ED-cAMP enzyme fragment conjugate and reporter substrate (Emerald II-Galacton Star, 5:1) is then added, followed by incubation for 60 min at room temperature. Then 20 μl of EA β-Gal fragment reagent are added. After incubation for 120 min at room temperature, chemiluminescence was measured using a plate reader (Envision) and the results were used to calculate the _EC50 values of the test peptides.

[00159] The results are presented in Table 2.

Table 2
EC ₅₀ values (nM) of typical cationic polypeptides used in the present invention

Obtaining the ionic complex and pharmaceutical composition of the present invention

Example 1: Preparation of composition 2A (peptide 1 and mPEG-10000-monocarboxylate)

[00160] A mixture of mPEG-10000 monocarboxylate (5.4 g) and 8.4 g of water is placed in a vial. The vial is stoppered, sealed and autoclaved for 15 min at 121°C to obtain a homogeneous, clear, viscous solution. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (100 mg in 1 ml), pre-filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous, clear, viscous solution. The concentration of peptide 1 according to HPLC is 9.9 mg/ml.

Example 2: Preparation of composition 2B (peptide 1 and PEG-10000-dicarboxylate)

[00161] A mixture of PEG-10000 dicarboxylate (2.7 g) and 2.0 g of water is placed in a vial. The vial is stoppered, sealed and autoclaved for 15 min at 121°C to obtain a homogeneous, clear, viscous solution. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (50 mg in 0.5 ml), pre-filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous, clear, viscous solution. The concentration of peptide 1 according to HPLC is 10.8 mg/ml.

Example 3: Preparation of composition 2C (peptide 1 and mPEG-20000-monocarboxylate)

[00162] A mixture of mPEG-20000 carboxylate (1.8 g) and 3.5 g of a mixture of propylene glycol-ethanol-water (in a ratio of 40:15:45 v/v) are placed in a vial. The vial is stoppered, sealed and autoclaved for 15 min at 121°C to obtain a homogeneous, transparent viscous solution. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (30 mg in 0.5 ml), pre-filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous, transparent viscous solution. The concentration of peptide 1 according to HPLC is 6 mg/ml.

Example 4: Preparation of 2D composition (peptide 1 and sodium DPPA)

[00163] A mixture of sodium DPPA (302 mg) and 8.6 g of a 1:1 propylene glycol-water mixture are placed in a vial. The vial is stoppered, sealed, and autoclaved for 15 min at 121°C to obtain a homogeneous lipid dispersion. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (100 mg in 1 ml), pre-filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous, non-viscous dispersion. The concentration of peptide 1 according to HPLC is 9.6 mg/ml.

Example 5: Preparation of composition 2E (peptide 1 and sodium stearate)

[00164] A mixture of mannitol (300 mg) and sodium stearate (165 mg) in 8.3 g of water is placed in a vial. The vial is stoppered and heated at 75°C for 0.5 h to obtain a clear solution. After cooling, approximately 400 μl of 1.0 N acetic acid is added to adjust the pH to a value in the range of 6-7. The vial is stoppered, sealed and autoclaved for 15 min at 121°C. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (100 mg in 1.0 g of aqueous solution), previously filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous light milky white opaque non-viscous suspension. The concentration of peptide 1 according to HPLC data is 9.7 mg/ml.

Example 6: Preparation of Composition #3B (Peptide 1, Sodium Stearate, and mPEG-2000-DSPE)

[00165] A mixture of mPEG-2000-DspE (1.85 g) and sodium stearate (100.1 mg) in 10.8 ml of water is placed in a vial. The vial is stoppered and heated at 75°C for 1 hour. After cooling, approximately 240 μl of acetic acid is added to adjust the pH to a value in the range of 6-7. The vial is stoppered, sealed and autoclaved for 15 min at 121°C. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), pre-filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous light milky white opaque non-viscous suspension. The concentration of peptide 1 according to HPLC data is 10.1 mg/ml.

Example 7: Preparation of Composition #3C (Peptide 1, Sodium DPPA, and PEG-10000-dicarboxylate)

[00166] A mixture of PEG-10000 dicarboxylate (1.68 g) and sodium DPPA (362.4 mg) in 9.1 mL of water is placed in a vial. The vial is stoppered, sealed and autoclaved for 15 min at 121°C to obtain a homogeneous milky-white opaque suspension. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), pre-filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous milky-white opaque suspension. The concentration of peptide 1 according to HPLC is 10.6 mg/mL.

Example 8: Preparation of composition #3D (peptide 1, sodium DPPA and PEG-3350)

[00167] A mixture of PEG-3350 (1.8 g in 10.8 mL of aqueous solution) and DPPA (363 mg sodium) is placed in a vial. The vial is stoppered, sealed and autoclaved for 15 min at 121°C to obtain a homogeneous milky-white opaque suspension. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), previously filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous milky-white opaque suspension. The concentration of peptide 1 according to HPLC is 10.1 mg/mL.

Example 9: Preparation of Composition #3E (Peptide 1, Sodium DPPA, PEG-3350, and Sodium CMC)

[00168] An aqueous solution of sodium CMC (average MW 90,000) (72 mg in 9.0 mL) is mixed with PEG-3350 (1.8 g). Sodium DPPA (216 mg) is added to the resulting clear solution. The vial is stoppered, sealed and autoclaved for 15 min at 121°C to obtain a homogeneous milky-white opaque suspension. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), pre-filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous milky-white opaque suspension. The concentration of peptide 1 according to HPLC is 10.9 mg/mL.

Example 10: Preparation of composition #3F (peptide 1, mPEG-2000-DSPE and sodium CMC)

[00169] An aqueous solution of sodium CMC (average MW 90,000), (72 mg in 10.8 mL, or 6.7 mg/mL) is mixed with mPEG-2000-DSPE (1.85 g). The vial is stoppered, sealed and autoclaved for 15 min at 121°C to obtain a homogeneous clear solution. Alternatively, the contents of the vial, including a mixture of sodium CMC and mPEG-2000-DSPE, are stirred overnight to obtain a clear solution, which is sterilized by filtration through a 0.2 μm filter. The vial is opened in an aseptic fume hood and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), previously filtered through a 0.2 μm filter, is added. The peptide composition is obtained as a homogeneous clear solution. The concentration of peptide 1 according to HPLC data is 10.5 mg/ml.

Example 11: Preparation of composition #4B (peptide 1 and mPEG-2000-DSPE)

[00170] A mixture of mPEG-2000 (1.855 DspE g) and mannitol (181 mg) in 8.8 mL of water for injection (purged with nitrogen for 10 min) is placed in a vial. The vial is stoppered and gently inverted to completely wet all solid ingredients with water. It is then autoclaved for 15 min at 121 °C. The contents of the vial at this stage are a clear solution. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), previously filtered through a 0.2 μm filter, is added over 1 h. The peptide composition is obtained as a homogeneous clear solution. The concentration of peptide 1 according to HPLC is 8.7 mg/mL.

Example 12: Preparation of Composition #4C (Peptide 1, mPEG-2000-DSPE and CMC)

[00171] An aqueous solution of sodium CMC (average MW 90,000) with a concentration of 8.9 mg/mL is prepared in nitrogen-purged water for injection. 9.8 mL of this solution is placed in a vial and mPEG-2000-DSPE (1.853 g) and mannitol (182 mg) are added. The vial is stoppered and gently inverted to completely wet all solid ingredients with the solvent. It is then autoclaved for 15 min at 121 °C. The contents of the vial at this stage are a clear solution. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), previously filtered through a 0.2 μm filter, is added over 1 h. The peptide composition is obtained as a homogeneous clear solution. The concentration of peptide 1 according to HPLC data is 8.0 mg/ml.

Example 13: Preparation of composition #4D (peptide 1, mPEG-2000-DSPE and DPPA)

[00172] A mixture of mPEG-2000-DSPE (1.86 g), sodium DPPA (363 mg), and mannitol (60.2 mg) in 9 mL of water for injection (purged with nitrogen for 10 min) is placed in a vial. The vial is stoppered and gently inverted to completely wet all solid ingredients with water. It is then autoclaved for 15 min at 121 °C. The content of the vial at this stage is a clear milky-white homogeneous suspension. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), previously filtered through a 0.2 μm filter, is added over 1 h to obtain a homogeneous clear milky-white opaque non-viscous suspension. The concentration of peptide 1 according to HPLC data is 9.2 mg/ml.

Example 14: Preparation of composition #4E (peptide 1, mPEG-2000-DSPE and CMC)

[00173] An aqueous solution of sodium CMC at a concentration of 7.2 mg/mL is prepared in nitrogen-purged water for injection. To 10 mL of this solution placed in a vial, mPEG-2000-DSPE (0.94 g) and mannitol (181.7 mg) are added. The vial is stoppered and gently inverted to completely wet all solid ingredients with solvent. It is then autoclaved for 15 min at 121 °C. The contents of the vial at this stage are a clear, colorless solution. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), pre-filtered through a 0.2 μm filter, is added over 1 h. The peptide composition is obtained as a homogeneous clear solution. The concentration of peptide 1 according to HPLC data is 9.6 mg/ml.

Example 15: Preparation of composition #4F (peptide 1, mPEG-2000-DSPE and DPPA)

[00174] A mixture of mPEG-2000-DSPE (0.954 g), sodium DPPA (183.7 mg), and mannitol (61.7 mg) in 9.6 mL of water for injection (purged with nitrogen for 10 min) is placed in a vial. The vial is stoppered and gently inverted to completely wet all solid ingredients with water. It is then autoclaved for 15 min at 121 °C. The contents of the vial at this stage are a clear, milky-white, homogeneous suspension. After cooling, the vial is transferred to an aseptic fume hood. The vial is opened and a sterile aqueous solution of peptide 1 (120 mg in 1.2 g), previously filtered through a 0.2 μm filter, is added over 1 h to obtain a homogeneous, clear, milky-white, opaque, non-viscous suspension. The concentration of peptide 1 according to HPLC data is 9.9 mg/ml.

Example 16: Preparation of a composition containing peptide 1, mPEG-2000-DSPE, CMA and D-mannitol

The scale of the method described below, calculated for a composition volume of 10 ml, can be easily reduced to a final volume of 1 ml, as shown in the table above.

An aqueous solution of sodium CMC (average MW 90,000) (80 mg) and D-mannitol (220 mg) in 7 ml of water for injection is placed in a pre-weighed vial with a stirrer and a stopper. Solid sodium mPEG-2000-DSPE (1 g) is added to the above solution. The vial is stoppered and sealed. The vial is then placed in a water bath at 60°C and its contents are stirred. Alternatively, the vial can be placed in an oven maintained at 60°C. The contents of the vial may initially be cloudy, but within 1-2 hours they turn into a clear homogeneous solution. The vial is then allowed to cool to room temperature. The vial is then opened and an aqueous solution of peptide 1 (100 mg in 1 ml, based on the content of pure peptide) is added. The contents may momentarily become cloudy, but upon stirring they turn into a clear homogeneous solution. The vial is weighed and the total contents are brought to 10.1 g by adding the required amount of water for injection. The resulting composition is then mixed and filtered through a 0.2 μm filter inside an aseptic fume hood. The peptide composition is obtained as a clear, homogeneous solution and analyzed by HPLC, confirming the peptide concentration of 1 10 mg/mL.

Example 17: Preparation of a composition containing peptide 1, sodium DPPA and PEG-3350

The scale of the method described below, calculated for a composition volume of 10 ml, can be easily reduced to a final volume of 1 ml, as shown in the table above.

Add 1.2 g of PEG-3350 and 8 ml of water for injection to a pre-weighed stoppered vial. Dissolve the contents by stirring and add 302 mg of sodium DPPA. Stopper the vial, seal it and autoclave it for 15 min at 121°C to obtain a homogeneous opaque milky-white suspension. After cooling, transfer the vial to an aseptic fume hood. Open the vial and add a sterile aqueous solution of peptide 1 (100 mg in 1 ml of water, based on the content of pure peptide), pre-filtered through a 0.2 μm filter. Bring the total contents of the vial to 10.1 g by adding the required amount of water for injection. After stirring, obtain a peptide composition in the form of a homogeneous milky-white opaque suspension. The obtained peptide is analyzed by HPLC, confirming the concentration of peptide 1 10 mg/ml.

Example 18: Administration of formulations to cynomolgus monkeys for pharmacokinetic analysis

[00175] Cynomolgus monkeys (average body weight range of 3 to 7 kg) are randomly assigned to groups of 6 animals for each formulation described above and for formulation 3A. All monkeys in the groups are administered a single bolus dose of drug at 0.5 mg/kg body weight subcutaneously in the scapular region. Blood samples are collected at various time points beginning 30 minutes after dosing and up to 36 or 48 hours. Plasma is obtained and the concentration of peptide drug is analyzed by LC-MS/MS. The results of the pharmacokinetic analysis are presented in Table 3 below, and a graphical representation of the pharmacokinetic profile is shown in Figure 1, Figure 2, and Figure 3.

As can be seen from the results of comparative pharmacokinetic analysis of examples of ionic complexes of peptide 1, a significant decrease in Cmax is observed (Cmax of peptide 1 is 1100 ng/ml, and Cmax of complex 3D is 228 ng/ml). Many other ionic complexes of the present invention are characterized by a similar range of reduced Cmax values compared to peptide 1. In addition, some of these ionic complexes have much higher Tmax values (Tmax of peptide 1 is 0.5 h, while for many ionic complexes Tmax reaches several hours).

The disclosures of all patents, published applications, and references cited herein are incorporated herein in their entirety.

Although the present invention has been particularly shown and described with reference to illustrative embodiments thereof, those skilled in the art can make various changes in form and details of the invention without departing from the scope thereof as defined in the appended claims.

Claims (54)

1. An ionic complex with modulatory activity in relation to the melanocortin-4 receptor, containing (i) a cationic polypeptide, wherein the cationic polypeptide is represented by the structural formula: Ac-Arg-cyclo[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys]-NH ₂ (SEQ ID No. 1) or a pharmaceutically acceptable salt thereof; and (ii) an anionic filler containing methoxy(polyethylene glycol 2000)-1,2-distearyl- sn -glycero-3-phosphoethanolamine (mPEG-2000-DSPE), where the molar ratio of mPEG-2000-DSPE cationic peptide to anionic filler is in the range of 1:1 to 1:10. 2. The ionic complex according to claim 1, further comprising a fatty acid selected from the group consisting of: lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaydic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid and phytanic acid. 3. The ionic complex of claim 2, wherein the fatty acid is stearic acid. 4. The ionic complex according to claim 1, further comprising a phospholipid selected from the group consisting of the compounds: L-α-phosphatidic acid, 1-oleoyl lysophosphatidic acid, L-α-phosphatidylglycerol, 1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, 1,2-distearoyl-sn-glycero-3-phosphatidic acid, mPEG-5000-DSPE, 1-(1,2-dihexadecanoylphosphatidyl)inositol-4,5-bisphosphate, trisodium salt, and 1-(1,2-dihexadecanoylphosphatidyl)inositol-3,4,5-triphosphate, tetrasodium salt, 1-palmitoyl-2-oleoylphosphatidylglycerol, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoglycerol, DSPG, DPPG, DEPG, DOPG, DEPA, DOPA, DSPS, DPPS, DEPS, DOPS, L-α-lysophosphatidylserine, L-α-lysophosphatidylinositol, tetradecylphosphonic acid, L-α-phosphatidylinositol-4-phosphate, L-α-phosphatidylinositol-4,5-bisphosphate, 1,2-diphytanoyl-sn-glycero-3-phosphate, 1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1'-rac-glycerol), mPEG-5000-DSPE and mixtures thereof. 5. The ionic complex according to claim 1, further comprising a phospholipid selected from the compounds: L-α-phosphatidic acid, 1-oleoyl lysophosphatidic acid, tetradecylphosphonic acid, L-α-phosphatidylglycerol, L-α-phosphatidylinositol, L-α-phosphatidylserine, 1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, 1,2-distearoyl-sn-glycero-3-phospho-L-serine, distearoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-distearoyl-sn-glycero-3-phosphatidic acid, mPEG-5000-DSPE, 1-(1,2-dihexadecanoylphosphatidyl)inositol 4,5-bisphosphate, trisodium salt, and 1-(1,2-dihexadecanoylphosphatidyl)inositol 3,4,5-triphosphate, tetrasodium salt, and mixtures thereof. 6. The ionic complex according to claim 1, additionally containing 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid. 7. The ionic complex of claim 1, further comprising a PEG carboxylic acid selected from the compounds: PEG-10000 monocarboxylate, PEG-20000 monocarboxylate, mPEG-1000 monocarboxylate, mPEG-5000 monocarboxylate, mPEG-10000 monocarboxylate, mPEG-20000 monocarboxylate, mPEG-30000 monocarboxylate, mPEG-40000 monocarboxylate, PEG-1000 dicarboxylate, PEG-2000 dicarboxylate, PEG-3500 dicarboxylate, PEG-5000 dicarboxylate, PEG-7500 dicarboxylate, PEG-10000 dicarboxylate and Y-shaped PEG-40000 monocarboxylate. 8. The ionic complex according to claim 7, wherein the PEG carboxylic acid is selected from the compounds: PEG-5000 monocarboxylate, PEG-10000 monocarboxylate, PEG-20000 monocarboxylate, mPEG-5000 monocarboxylate, mPEG-10000 monocarboxylate, mPEG-20000 monocarboxylate, PEG-5000 dicarboxylate and PEG-10000 dicarboxylate. 9. The ionic complex according to claim 1, additionally containing stearic acid, DPPA, PEG-10000 dicarboxylate and CMC. 10. A pharmaceutical composition having a modulatory activity with respect to the melanocortin-4 receptor, containing an ionic complex according to any one of claims 1-9 and a pharmaceutically acceptable carrier. 11. The pharmaceutical composition according to claim 10, wherein the pharmaceutically acceptable carrier is selected from the group consisting of PEG, polyol, ethanol, DMSO, NMP, DMF, benzyl alcohol, water, pH-stabilizing buffer solutions and mixtures thereof. 12. The pharmaceutical composition according to claim 11, wherein the pharmaceutically acceptable carrier is PEG with an average molecular weight of 100 to 5000. 13. The pharmaceutical composition according to claim 11, wherein the polyol is selected from propylene glycol, tripropylene glycol, glycerin and mixtures thereof. 14. The pharmaceutical composition of claim 10, wherein the concentration of the cationic polypeptide is in the range of about 0.01 mg/mL to about 100 mg/mL. 15. The pharmaceutical composition of any one of claims 10-14, wherein the molar ratio, adjusted for the charge ratio of the cationic polypeptide to the charge of the anionic filler, is selected from about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. 16. An ionic complex with modulatory activity in relation to the melanocortin-4 receptor, containing: i) a cationic polypeptide represented by the following structural formula: ii) methoxy(polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG-2000-DSPE), and where the molar ratio of the cationic peptide to mPEG-2000-DSPE is from 1:3 to 1:6. 17. An ionic complex containing: i) a cationic polypeptide represented by the following structural formula: ii) methoxy(polyethylene glycol 2000)-1,2-distearyl- sn -glycero-3-phosphoethanolamine (mPEG-2000-DSPE), and iii) an additional component selected from stearic acid or a pharmaceutically acceptable salt thereof (e.g. sodium stearate), 1,2-dipalmitoyl- sn -glycero-3-phosphatidic acid (DPPA) or a pharmaceutically acceptable salt thereof (e.g. sodium DPPA salt) and carboxymethylcellulose (CMC), where the molar ratio of cationic peptide to mPEG-2000-DSPE is from 1:1 to 1:10. 18. A method for obtaining an ionic complex containing: (i) a cationic polypeptide, wherein the cationic polypeptide is represented by the structural formula: Ac-Arg-cyclo[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys] _-NH2 (SEQ ID No. 1), or a pharmaceutically acceptable salt thereof; and (ii) an anionic filler containing methoxy(polyethylene glycol 2000)-1,2-distearyl- sn -glycero-3-phosphoethanolamine (mPEG-2000-DSPE); wherein the molar ratio of mPEG-2000-DSPE cationic polypeptide to anionic filler is in the range of from about 1:1 to about 1:10, wherein the method comprises: (a) obtaining a mixture of an anionic filler and an aqueous filler-diluent; (b) autoclaving the mixture under conditions sufficient to sterilize the excipient; and (c) adding to the mixture of excipients a sterile solution containing a cationic polypeptide and an aqueous diluent for the peptide, thus obtaining an ionic complex. 19. A method for producing an ionic complex according to claim 18, wherein the filler mixture is a suspension. 20. A method for obtaining an ionic complex according to claim 18, wherein the filler mixture is a solution. 21. A method for obtaining an ionic complex containing: (i) a cationic polypeptide, wherein the cationic polypeptide is represented by the structural formula: Ac-Arg-cyclo[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys] _-NH2 (SEQ ID No. 1), or a pharmaceutically acceptable salt thereof; and (ii) an anionic filler containing methoxy(polyethylene glycol 2000)-1,2-distearyl- sn -glycero-3-phosphoethanolamine (mPEG-2000-DSPE); wherein the molar ratio of mPEG-2000-DSPE cationic polypeptide to anionic filler is in the range of from about 1:1 to about 1:10, wherein the method comprises: (a) obtaining a solution of an anionic filler and an aqueous filler-diluent; (b) filtering the solution obtained in step a through a 0.2 micron filter; and (c) adding to the excipient solution obtained in step b a sterile peptide solution containing a cationic polypeptide and an aqueous diluent for the peptide, thus obtaining an ionic complex. 22. A method for obtaining an ionic complex containing: (i) a cationic polypeptide, wherein the cationic polypeptide is represented by the structural formula: Ac-Arg-cyclo[Cys-D-Ala-His-D-Phe-Arg-Trp-Cys] _-NH2 (SEQ ID No. 1), or a pharmaceutically acceptable salt thereof; and (ii) an anionic filler containing methoxy(polyethylene glycol 2000)-1,2-distearyl- sn -glycero-3-phosphoethanolamine (mPEG-2000-DSPE); wherein the molar ratio of mPEG-2000-DSPE cationic polypeptide to anionic filler is in the range of from about 1:1 to about 1:10, wherein the method comprises: (a) obtaining a solution of an anionic filler, an aqueous filler-diluent and a cationic polypeptide; and (b) sterilization of the resulting solution by filtration through a 0.2 micron filter, thus obtaining an ionic complex.