WO2009009035A1

WO2009009035A1 - Somatostatin analog and uses thereof

Info

Publication number: WO2009009035A1
Application number: PCT/US2008/008341
Authority: WO
Inventors: Sun H. Kim
Original assignee: Ipsen Pharma S.A.S.
Priority date: 2007-07-06
Filing date: 2008-07-02
Publication date: 2009-01-15

Abstract

Claimed is a somatostatin agonist according to the formula: cyclo(4(OH)Pro-Phe- DTrp-Lys-Pty-Phe), or a pharmaceutically acceptable salt thereof, and uses thereof, which has interesting pharmaceutical properties.

Description

Attorney Docket No. 106P

SOMATOSTATIN ANALOG AND USES THEREOF

BACKGROUND OF THE INVENTION

Somatostatin (SRIF), a tetradecapeptide discovered by Brazeau et al. {Science 179:77- 79, 1972), has been shown to have potent inhibitory effects on various secretory processes in tissues such as pituitary, pancreas and gastrointestinal tract. SRIF also acts as a neuromodulator in the central nervous system. These biological effects of SRIF, all inhibitory in nature, are elicited through a series of G protein-coupled receptors, of which five different subtypes have been characterized (SSTR-I, SSTR-2, SSTR-3, SSTR-4 and SSTR- 5). These five subtypes have similar affinities for the endogenous SRIF ligands but have differing distribution in various tissues. SRIF binds to the five distinct receptor (SSTR) subtypes with relatively high and equal affinity for each substype.

SRIF produces a variety of effects, including modulation of hormone release, e.g. , growth hormone, glucagon, insulin, amylin, and neurotransmitter release. Some of these effects have been associated with its binding to a specific SRIF receptor. For example, the inhibition of growth hormone has been attributed to the somatostatin type-2 receptor (SSTR- 2) (Raynor et al, Molecular Pharmacol. 43:838, 1993; Lloyd et al., Am. J. Physiol. 268:G102, 1995), while the inhibition of insulin has been attributed to the somatostatin type- 5 receptor (SSTR-5) (Rossowski, W. J. et al, Biochem. Biophys. Res. Commun. 197:366-371, 1993). Activation of types 2 and 5 have been associated with growth hormone suppression and more particularly with GH secreting adenomas (acromegaly) and TSH secreting adenomas. Activation of type 2 but not type 5 has been associated with treating prolactin secreting adenomas.

As is well known to those skilled in the art, SRIF and analogs thereof are useful in the treatment of a great variety of diseases and/or conditions. An exemplary but by no means exhaustive list of such diseases and/or conditions would include: Cushings Syndrome {see Clark, R. V. et al, Clin. Res. 38:943A, 1990); gonadotropinoma {see Ambrosi, B. et al, Acta Endocr. (Copenh.) 122:569-576, 1990); hyperparathyroidism {see Miller, D. et al, Canad. Med. Ass. J. 145:227-228, 1991); Paget's disease {see Palmieri, G. M. A. etal, J. of Bone and Mineral Research 7:S240, 1992); VIPoma {see Koberstein, B. et al, Gastroenterology 28:295-301, 1990; Christensen, C, Acta Chir. Scand. 155:541-543, 1989); nesidioblastosis and hyperinsulinism {see Laron, Z., IsraelJ. Med. ScL 26:1-2, 1990; Wilson, D. C. et al, Med. Sci. 158:31-32, 1989); gastrinoma {see Bauer, F. E. et al, Europ. J. Pharmacol. 183:55, 1990); Zollinger-Ellison Syndrome (see Mozell, E. et al., Surg. Gynec. Obstet. 170:476-484, 1990); hypersecretory diarrhea related to AIDS and other conditions (see Cello, J. P. et al, Gastroenterology 98:A163, 1990); elevated gastrin-releasing peptide levels (see Alhindawi, R. et al, Can. J. Surg. 33:139-142, 1990); diarrhea associated with chemotherapy (see Petrelli, N. et al, Proc. Amer. Soc. Clin. Oncol. 10:138, 1991); irritable bowel syndrome (see O'Donnell, L. J. D. et al, Aliment. Pharmacol. Therap. 4:177-181, 1990); pancreatitis (see Tulassay, Z. et al, Gastroenterology 98:A238, 1990); Crohn's Disease (see Fedorak, R. N. et al, Can. J. Gastroenterology 3:53-57, 1989); systemic sclerosis (see Soudah, H. et al, Gastroenterology 98:A129, 1990); thyroid cancer (see Modigliani, E. et al, Ann. Endocr. 50:483-488, 1989); psoriasis (see Camisa, C. et al, Cleveland Clinic J. Med. 57:71-76,

1990); hypotension (see Hoeldtke, R. D. et al, Arch. Phys. Med. Rehabil 69:895-898, 1988; Kooner, J. S. et al, Brit. J. Clin. Pharmacol. 28:735-736, 1989); panic attacks (see Abelson, J. L. et al, Clin. Psychopharmacol. 10:128-132, 1990); sclerodoma (see Soudah, H. et al, Clin. Res., Vol. 39, p. 3O3A, 1991); small bowel obstruction (see Nott, D. M. et al, Brit. J. Surg. 77:A691, 1990); gastroesophageal reflux (see Branch, M. S. et al, Gastroenterology 100:A425, 1991); duodenogastric reflux (see Hasler, W. et al, Gastroenterology 100:A448, 1991); Graves' Disease (see Chang, T. C. et al, Brit. Med. J. 304:158, 1992); polycystic ovary disease (see Prelevic, G. M. et al, Metabolism Clinical and Experimental 41 : 76-79, 1992); upper gastrointestinal bleeding (see Jenkins, S. A. et al, Gut. 33:404-407, 1992; Arrigoni, A. et al , American Journal of Gastroenterology 87:1311, 1992); pancreatic pseudocysts and ascites (see Hartley, J. E. et al, J. Roy. Soc. Med. 85:107-108, 1992); leukemia (see Santini et al, 78:429A, 1991); meningioma (see Koper, J. W. et al., J. Clin. Endocr. Metab. 74:543-547, 1992); and cancer cachexia (see Bartlett, D. L. et al, Surg. Forum. 42:14-16, 1991). Because of the short half-life of native somatostatin, various somatostatin analogs have been developed (Raynor et al, Molecular Pharmacol. 43:838, 1993). Hence, there is a need for broad-spectrum somatostatin analogs that are more active or are more metabolically stable than native somatostatin. SUMMARY OF THE INVENTION

The present invention relates to somatostatin peptidomimetics, a process for their production and pharmaceutical preparations containing them. More particularly, the present invention features a peptide of the formula: cyclo(4(OH)Pro-Phe-DTrp-Lys-Pty-Phe), or a pharmaceutically acceptable salt thereof.

The peptide of the invention can be used in any application in which agonistic activity to somatostatin receptors is required. In one aspect, the peptide of the invention can be used to inhibit the release of growth hormone or insulin in a subject (e.g., a mammal such as a human patient). Thus, the peptide is useful in the treatment of physiological conditions in which the suppression of the release of growth hormone or insulin is of benefit. The peptide of the invention can also be used in enhancing wound healing or promoting angiogenesis. Moreover, due to the increased hydrophobicity of the peptide of the invention, the peptide of the invention may be able to cross the blood-brain barrier and may be useful in the treatment of various central-nervous-system disorders, such as Alzheimer's disease. In another aspect of the invention is featured a method of treating a disease or condition in a human or other animal in need thereof, which comprises the step of administering the compound of the formula: cyclo(4(OH)Pro-Phe-DTrp-Lys-Pty-Phe), or a pharmaceutically acceptable salt there of, to said human or other animal, wherein said disease or condition is selected from the group consisting of Cushings Syndrome, gonadotropinoma, hyperparathyroidism, Paget's disease, VIPoma, nesidioblastosis, hyperinsulinism, gastrinoma, Zollinger-Ellison Syndrome, hypersecretory diarrhea related to AIDS and other conditions, irritable bowel syndrome, pancreatitis, Crohn's Disease, systemic sclerosis, thyroid cancer, psoriasis, hypotension, panic attacks, sclerodoma, small bowel obstruction, gastroesophageal reflux, duodenogastric reflux, Graves' Disease, polycystic ovary disease, upper gastrointestinal bleeding, pancreatic pseudocysts, pancreatic ascites, leukemia, meningioma, cancer cachexia, acromegaly, restenosis, hepatoma, lung cancer, melanoma, inhibiting the accelerated growth of a solid tumor, decreasing body weight, treating insulin resistance, Syndrome X, prolonging the survival of pancreatic cells, fibrosis, hyperlipidemia, hyperamylinemia, hyperprolactinemia and prolactinomas. In another aspect of the invention, a labeled peptide of the invention can be used either in vivo to detect cells having somatostatin receptors (e.g. , cancer cells) or in vitro as a ligand in a somatostatin receptor binding assay. In such an aspect, the peptide may be labeled with radioactivity. In another aspect of the invention, the peptide may also be used as a vector to target cells with radioactive isotopes. In yet another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of cyclo(4(OH)Pro-Phe-DTrp-Lys-Pty-Phe) or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or diluent. In this aspect, the composition is useful for treatment of physiological conditions in which the suppression of the release of growth hormone or insulin is of benefit. Also in this aspect, the composition comprising the peptide of the invention can also be used in enhancing wound healing or promoting angiogenesis. Moreover, due to the increased hydrophobicity of the peptide of the invention, the composition comprising the peptide of the invention may be useful in the treatment of various central-nervous-system disorders, such as Alzheimer's disease.

DETAILED DESCRIPTION OF THE INVENTION

One skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrations of the invention and are not meant to be construed as limiting the full scope of the invention in any way.

The nomenclature for the somatostatin receptor subtypes is in accordance with the recommendations of IUPHAR, in which SSTR-4 refers to the receptor originally cloned by Bruno et al, and SSTR-5 refers to the receptor cloned by O'Carroll et al.

With the exception of the N-terminal amino acid, all abbreviations of amino acids in this disclosure stand for the structure of -NH-CH(R)-CO-, wherein R in the immediately foregoing formula is the side chain of an amino acid (e.g., CH₃ for Ala). For the N-terminal amino acid, the abbreviation stands for the structure of (R¹R²J-N-CH(R)-CO-, wherein R is a side chain of an amino acid and R¹ and R² are as defined herein.

Abbreviations of the common amino acids are in accordance with the recommendations of IUPAC-IUB. The following are abbreviations of certain α-amino acids as may appear herein:

Pro = proline Lys = lysine; Phe = phenylalanine; Pty = -o-phenyltyrosine

Tip = tryptophan; Additional abbreviations used herein include:

DBU, 1 ,8-diazabicyclo(5.4.0)undec-7-ene; DCM, dichloromethane; DIC, dicyclohexylcarbodiimide; DIEA, diisopropylethylamine; DMF, dimethylformamide; Fmoc, 9-Fluorenylmethoxycarbonyl MTBD, l,3,4,6,7,8-Hexahydro-l-methyl-2H-pyrimido(l,2-a)pyrimidine;

NPS, 2-nitrophenylsulfonyl;

TBTU, O-Benzorri-azol-l-yl-N,N,N\N'-terramethyluronium tetrafluoroborate; and TFA, trifluoroacetic acid.

• Peptide Synthesis

The synthesis of short peptides is well examined in the peptide art. See, e.g., Stewart et ai, Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984) and Bodanszky, M. et ai, The Practice of Peptide Synthesis (Springer- Verlag, 1984). The following describes the synthesis of cyclo(4(OH)Pro-Phe-DTrp-Lys-Pty-Phe).

1) dl-N-t-butoxycarbonyl-o-phenyltyrosine

To a suspension of 50% NaH dispersed in mineral oil (1.0 g) in 10 ml of pyridine was added dropwise a solution of N-t-butoxycarbonyl-tyrosine methylester (1.0 g) in 10 ml of pyridine and the mixture was stirred for 15min. CuBr.dimethylsulfide (1.0 g) was added to it and after stirring for 30 min., a solution of iodobenzene (1.3 g) in 1 ml of pyridine was added and the mixture was refluxed overnight. The dark reaction mixture was poured into aqueous NH₄CL (100 ml) and it was treated with ethylacetate. The mixture was filtered through celite pad and ethylacetate layer was separated, dried over anhydrous MgSO₄. After evaporation of volatile substances, the residue was chromatographed over silica gel (50 g) using chloroform as an eluent. Appropriate fractions were pooled and solvent was removed in vacuo to give dl-N-t-butoxycarbonyl-o-phenyltyrosine methylester. Pale yellow solid, 0.4 g TLC (silica gel;chloroform/acetone = 9:1) Rf= 0.88.

Small amount of the compound was treated with trifluoroacetic acid yielded dl-o- phenyltyrosine methylester, M/e, 272. dl-N-t-butoxycarbonyl-o-phenyltyrosine methylester was treated with 2N-NaOH in methanol to provide dl-N-butoxycarbonyl-o-phenyltyrosine. TLC (silica gel;chloroform/methanol/acetic acid = 9:1:0.1) Rf = 0.46. 2) N-t-butoxycarbonyl-o-phenyltyrosine

To the mixture of dl-t-butoxycarbonyl-o-phenyltyrosine methylester (0.9 g) in 5 ml of acetone and 5 ml of 0.2 M-phosphate buffer (pH 7.5) was added 0.5 ml of Chiroclec-BL and the mixture was incubated at 37 ⁰C over weekend. The mixture was filtered, washed with water and acetone, and the filtrate was acidified with 0.2 N-HCL (pH 3-4), extracted with ethylacetate. Ethylaceteate extract was dried (MgSO₄), and after evaporation of solvent, the residue was chromatographed over silica gel (30 g) using chloroform/acetone = 19: 1), followed by chlorofrorm/methano = 9: 1) as eluents.

Earlier fraction gave D-t-butoxycarbonyl-o-phenyltyrosine methylester (0.29 g) and later fraction t-butoxycarbonyl-o-phenyltyrosine (0.26 g).

3) dl-o-phenyltyrosyl-phenylalanine ethylester trifluoroacetic acid salt

To an ice-cooled mixture of dl-t-butoxycarbonyl-o-phenyltyrosine (550 mg) and phenylalanine ethylester, HCL (410 mg) in dimethylformide (DMF, 10 ml) was added diethyl cyanophosphonate (0.3 ml) followed by diisopropylethylamine (DIEA, ImI) and the mixture was stirred at room temperature overnight. Volatile substances were removed in vacuo to dryness and the residue was partitioned between chloroform and water. Organic layer was dried (MgSO₄) and sovent was removed in vacuo to dryness. It was treated with 50% trifluoroacetic acid (TFA) in dichloromethane for 30 min. and volatile substances were removed in vacuo to dryness.

4) N-e-benzyloxycarbonyl-lvsyl-dl-o-phenyltyrosyl-phenylalanine ethylester.TFA salt

To an ice-cooled mixture of dl-o-phenyltyrosyl-phenylalanine ethylester trifluoroacetic acid salt (860 mg) and N-t-butoxycarbonyl-e-benzyloxycarbonyl-lysine (670 mg), hydroxybenzotriazole (HOBT, 280 mg) in DMF/dichloromethane (1 :3, 10 ml), were added 1 ml of DIEA, followed by EDC(N-(3-dimethylaminopropyl)-N-ethylcarbodiimide, 400 mg), and the mixture was stirred at room temperature overnight. Volatile substances were removed in vacuo to dryness and the residue was partitioned between chloroform and water. Chloroform layer was washed with aqueous NaHCO₃ and dried over MgSO₄. After evaporation of solvent, the residue was treated with 50% TFA in dichloromethane for 30 min. and volatile substances were removed in vacuo to dryness.

In an analogous manner, t-butyloxycarbonyl-hydroxyprolyl-phenylalanyl-D- tryptophanyl methylester was made and it was treated with 2N-NaOH in ethylalcohol to give t-butyloxycarbonyl-hydroxyprolyl-phenylalanyl-D-tryptophan.

5) N-t-butoxycarbonyl-hvdroxyprolyl-phenylalnyl-D-tryptophanyl-ε- benzyloxycarbonyl-lysyl-dl-o-phenyltyrosyl-phenylalnine and hydroxyprolyl- phenylahiyl-P-trvptophanyl-ε-benzyloxycarbonyl-lvsyl-dl-o-phenyltyrosyl- phenylalnineTFA salt To an ice-cooled mixture of N-e-benzyloxycarbonyl-lysyl-dl-o-phenyltyrosyl- phenylalanine ethylester (350 mg), t-butyloxycarbonyl-hydroxyprolyl-phenylalanyl-D- tryptophan (350 mg), HOBT (90 mg), 0.5 ml of DIEA in DMF/dichloromethane (1:3, 10 ml) was added EDC (130 mg) and the mixture was stirred at room temperature overnight.

Solvents were removed in vacuo to dryness. The residue was partitioned between ethylacetate and water. Ethylacetate layer was washed with aqueous NaHCO₃, dried over MgSO₄ and solvent was removed in vacuo to give 1.2 g of N-t-butoxycarbonyl- hydroxyprolyl-phenylahiyl-D-tryptophanyl-e-benzyloxycarbonyl-lysyl-dl-o-phenyltyrosyl- phenylalanyl ethylester. It was dissolved in 10 ml of ethanol, treated with 2 ml of 2N-NaOH for 30 min. After evaporation of ethanol, the water layer containing yellow solid was acidified with 5% aqueous KHSO₄ (pH 2-3), extracted with ethylacetate and the extract was then dried (MgSO₄) and solvent was removed in vacuo to dryness.

It was treated with 50% TFA in dichloromethane containing triethylsilane (0.2 ml) for 30 min. and volatile substances were removed in vacuo to dryness, 0.9 g.

6) cyclo(hvdroxyprolyl-phenylalanyl-D-trvptophanyl-ε-benzyloxycarbonyl-lvsyl-dl- o-phenvltvrosvl-phenvlalanvl)amide and cvclofhydroxyprolvl-phenylalanyl-D- tivptophanyl-lvsyl-o-phenyltyrosyl-phenylalanyl)amide

To a solution of crude hydroxyprolyl-phenylalnyl-D-tryptophanyl-e- benzyloxycarbonyl-lysyl-dl-o-phenyltyrosyl-phenylalnineTFA salt (0.9 g) in DMF (30 ml) were added HATU (N-((dimethylamino)-lH-l,2,3-triazolo(4,5-b)pyridine-l-ylmethylene)-N- methylmethaminium hexafluorophosphate N-oxide, 0.4 g), HOAT(7-aza-l-hydroxy- benzotriazole, 0.15 g), DIEA (0.7 ml), and the mixture was stirred at room temperature overnight. Volatile substances were removed in vacuo to dryness. The residue was partitioned between ethylacetate and water. Ethylacetate layer was washed with aqueous NaHCO₃ and dried over MgSO_4. Solvent was removed in vacuo to give 0.5 g of cyclo^ydroxyprolyl-phenylalanyl-D-tryptophanyl-e-benzyloxycarbonyl-lysyl-dl-o- phenyltyrosyl-phenylalanyl)amide. It was dissolved in 20 ml of ethylalcohol, treated with 300 mg of 10% Pd-C and hydrogenation was carried out under 32 psi overnight. The reaction mixture was filtered through celite pad, washed with ethylalcohol and solvent was removed in vacuo dryness. Crude mixture was subjected to preparative high performance liquid chromatography (HPLC) using a C]₈ column and 0.1% aqueous TFA and CH₃CN as the mobile phase. Earlier fractions gave cyclo(hydroxyprolyl-phenylalanyl-D-tryptophanyl- lysyl-o-phenyltyrosyl-phenylalanyl)amide, 21 mg as a white solid. Mass spec, (electrospray) = 961.3. Later fractions gave cyclo(hydroxyprolyl-phenylalanyl-D-tryptophanyl-lysyl-D-o- phenyltyrosyl-phenylalanyl)amide, 17 mg as a white solid. Mass spec, (electrospray) = 961.4.

Peptides can be made by solid phase method using oxime resin. Alternatively, linear peptide sequences are synthesized by solid phase using 2-chlorotrityl resin and after treatment with mild acid, N-terminal free peptide acid is cyclized in solution. Final removal of the side chain protecting group of lysine yields the desired cyclic peptides.

• Functional Expression of the Cloned Human Somatostatin Receptors

The genomic clones containing the human somatostatin receptors (hSSTR-1 to hSSTR-5) (Yamada, Y. et al, Proc. Natl. Acad. ScL USA. 89:251-255, 1992; Yasuda, K. et al., J. Biol. Chem. 267:20422-20428, 1992; Yamada, Y. et al., Mol. Pharmacol. 42:2136- 2142, 1992; and Rohrer, L. et al, Proc. Natl. Acad. ScL USA. 90:4196-4200, 1993) were provided by Dr. Graeme I. Bell of the University of Chicago. The hSSTR-1, hSSTR-2, hSSTR-3, hSSTR-4 and hSSTR-5 cDNAs were isolated as a 1.5-kb Pstl-Xmnl fragment, 1.7- kb Bamm-Hinάlll fragment, 2.0-kb Ncol-HindOl fragment, 1.4-kb Nhel-Ndel fragment, and a 1.2-kb HindUl-Xbal fragment, respectively, each containing the entire coding region of the full-length receptors. These fragments were independently subcloned into the corresponding restriction endonuclease sites in the mammalian expression vector pCMV5, downstream from the human cytomegalovirus (CMV) promoter, to produce the expression plasmids pCMV5/hSSTR-l, pCMV5/hSSTR-2, _PCMV5/hSSTR-3, pCMV5/hSSTR-4 and pCMV5/hSSTR-5. For transfection into CHO-Kl cells, a plasmid, pRSV-neo (American Type Culture Collection, Rockville, MD), carrying the neomycin mammalian cell selectable marker was added.

• Receptor Expression and Transfection

Transfections were performed by the calcium phosphate method. CHO-Kl cells were maintained in α-minimum essential medium (α-MEM; Gibco) supplemented with 10% fetal calf serum and transfected with each of the expression plasmids using calcium phosphate precipitation. Clones that had inherited the expression plasmid were selected in α-MEM supplemented with 500 μg mL^"' of geneticin (G418; Gibco). Independent CHO-Kl clones were picked by glass-ring cloning and expanded in culture in the selective media. Membranes were prepared from the isolated clones and hSSTR expression was initially assessed for binding with (¹²⁵I)TyT¹ '-SRIF and (¹²⁵I)MK-678 (for SSTR-2).

• Radioligand Binding Assays

Cell membranes of the five somatostatin receptor types were obtained from homogenates (Polytron setting 6, 15 sec) of the corresponding CHO-Kl cells, in ice-cold Tris-HCl (50 mM) and centrifuged (39,000 g, 2 x 10 min.), with an intermediate resuspension in fresh buffer. The final pellets were resuspended in Tris-HCl (10 mM) for assay. Aliquots of the membranes were incubated (30 min. at 37⁰C) with 0.05 nM (¹²⁵I)Tyr"-SRIF (types 1, 3, 4, 5) or (¹²⁵I)MK-678 (type 2) in 50 nM HEPES (pH 7.4) containing BSA (10 mg/ml); MgCl₂ (5 mM), Trasylol (200 kIU mL^"1), bacitracin (0.02 mg/ml), and phenylmethanesulfonyl fluoride (0.02 mg/ml). The final assay volume was 0.3 ml and incubations were terminated by rapid filtration through GF/C filters pre-soaked in 0.3% poly(ethylenimine) using a Brandel rapid filtration module. Each tube and filter was then washed with aliquots of cold buffer (3 x 5 ml).

Specific binding was defined as the total radioligand bound minus that bound in the presence of 1.0 μM SRIF. The following total radioligand binding and non-specific binding (nsb) values were typically obtained with these assay systems: hSSTR-1, 7,000 cpm total versus 3,500 cpm nsb; hSSTR-2, 9,000 cpm total versus 1,000 cpm nsb; hSSTR-3, 8,000 cpm total versus 1,000 cpm nsb; hSSTR-4, 6,000 cpm total versus 3,500 cpm nsb; and hSSTR-5, 7,500 cpm total versus 3,500 cpm nsb. The binding affinities are expressed as Ki values (nM) for each of the five receptor subtypes. Ki values derived for the compound of the present invention are provided in the following table:

TABLE 1. Human somatostatin receptor subtype specificity of cyclo(4(OH)Pro-Phe- DTrp-Lys-Pty-Phe) Ki CnM) SEM hSSTRl 280 95 hSSTR2 3.26 0.62 hSSTR3 36.63 1.3 hSSTR4 1073 223.0 hSSTR5 0.70 0.11

Administration and Use

The peptide of this invention can be provided in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g. , hydrochloric acid, sulfuric acid, or phosphoric acid), and polymeric acids (e.g. , tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids). A typical method of making a salt of the peptide of the present invention is well known in the art and can be accomplished by standard methods of salt exchange. Accordingly, the TFA salt of a peptide of the present invention (the TFA salt results from the purification of the peptide by using preparative HPLC, eluting with TFA containing buffer solutions) can be converted into another salt, such as an acetate salt, by dissolving the peptide in a small amount of 0.25 N acetic acid aqueous solution. The resulting solution is applied to a semi-prep HPLC column (Zorbax^®, 300 SB, C-8). The column is eluted with: (1) 0.1 N ammonium acetate aqueous solution for 0.5 hours; (2) 0.25 N acetic acid aqueous solution for 0.5 hours; and (3) a linear gradient (20% to 100% of solution B over 30 min.) at a flow rate of 4 ml/min (solution A is 0.25 N acetic acid aqueous solution; solution B is 0.25 N acetic acid in acetonitrile/water = 80:20). The fractions containing the peptide are collected and lyophilized to dryness. As is well known to those skilled in the art, the known and potential uses of peptides with SSTR receptor agonist activity are varied and multitudinous.

Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient cyclo(4(OH)Pro-Phe-DTrp-Lys- Tyr(phenyl)-Phe) in association with a pharmaceutically acceptable carrier.

The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. In general, an effective dosage for the activities of this invention is in the range of 1 x 10^~7 to 200 mg/kg/day, preferably 1 x 10^ to 100 mg/kg/day which can be administered as a single dose or divided into multiple doses.

The compound of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents. Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Preparations may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. Preparations can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.

Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.

Further, the compound of this invention can be administered in a sustained release composition such as those described in the following patents and patent applications. U.S. Patent No. 5,672,659 teaches sustained release compositions comprising a bioactive agent and a polyester. U.S. Patent No. 5,595,760 teaches sustained release compositions comprising a bioactive agent in a gelable form. U.S. Patent No. 5,821,221 teaches polymeric sustained release compositions comprising a bioactive agent and chitosan. U.S. Patent No. 5,916,883 teaches sustained release compositions comprising a bioactive agent and cyclodextrin. The teachings of the foregoing patents and applications are incorporated herein by reference. The use of immediate or of sustained release compositions depends on the type of indications targeted. If the indication consists of an acute or over-acute disorder, a treatment with an immediate form will be preferred over the same with a prolonged release composition. On the contrary, for preventive or long-term treatments, a prolonged release composition will generally be preferred. It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not to limit the scope of the invention. Other aspects, advantages, and modifications are within the claims. Also, the contents of each references cited herein is incorporated by reference in its entirety.

Claims

What is claimed is:

1. A peptide of the formula cyclo(4(OH)Pro-Phe-DTrp-Lys-Pty-Phe), or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising an effective amount of a peptide according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

3. A method of binding one or more of human somatostatin subtype receptors in a human or other animal, which comprises administering an effective amount of a peptide according to claim 1 or a pharmaceutically acceptable salt thereof to the human or other ^• animal.

4. A method of binding one or more of human somatostatin subtype receptors -2 and -5 in a human or other animal, which comprises administering an effective amount of a peptide according to claim 1 or a pharmaceutically acceptable salt thereof to the human or other animal.

5. A method of eliciting a somatostatin agonist response in a human or other animal in need thereof, which comprises administering an effective amount of a peptide according to claim 1 or a pharmaceutically acceptable salt thereof to the human or other animal.

6. A method of treating a disease or condition in a human or other animal in need thereof, which comprises administering a peptide of claim 1 or a pharmaceutically acceptable salt thereof, to said mammal, wherein said disease or condition is selected from the group consisting of Cushings Syndrome, gonadotropinoma, hyperparathyroidism, Paget's disease, VIPoma, nesidioblastosis, hyperinsulinism, gastrinoma, Zollinger-Ellison Syndrome, hypersecretory diarrhea related to AIDS and other conditions, irritable bowel syndrome, pancreatitis, Crohn's Disease, systemic sclerosis, thyroid cancer, psoriasis, hypotension, panic attacks, sclerodoma, small bowel obstruction, gastroesophageal reflux, duodenogastric reflux, Graves' Disease, polycystic ovary disease, upper gastrointestinal bleeding, pancreatic pseudocysts, pancreatic ascites, leukemia, meningioma, cancer cachexia, acromegaly, restenosis, hepatoma, lung cancer, melanoma, inhibiting the accelerated growth of a solid tumor, decreasing body weight, treating insulin resistance, Syndrome X, prolonging the survival of pancreatic cells, fibrosis, hyperlipidemia, hyperamylinemia, hyperprolactinemia, prolactinomas, diabetic neuropathy, macular degeneration, hypercalcemia of malignancy, postprandial portal hypertension, and complications of portal hypertension.

7. A method of treating a central-nervous-system disease or condition in a human or other animal in need thereof, which comprises administering a peptide of claim 1 or a pharmaceutically acceptable salt thereof, to said mammal.

8. A method according to claim 4, wherein said disease or condition is Alzheimer's disease.

9. A method of imaging cells containing somatostatin receptors in vivo in a human or other animal, which comprises administering a peptide of claim 1 or a pharmaceutically acceptable salt thereof, to said mammal.

10. A method of imaging cells containing somatostatin receptors in vitro, which comprises administering a peptide of claim 1 or a pharmaceutically acceptable salt thereof, in a somatostatin receptor binding assay.