EP1843779A2 - Compositions containing the anti-angiogenic phscn-peptide - Google Patents

Abstract

Described herein are compositions/formulations of the Cys-containing anti-angiogenic peptide Pro-His-Ser-Cys-Asn (preferably in its capped form as Ac-PHSCN-NH2) or acid addition salts thereof or analogue thereof, that comprise at least one additional compound that stabilizes the peptide or analogue against spontaneous tandem dimerization or higher oligomerization. Preferred formulations include an acidic buffer such as citrate, glycine as an excipient and bulking agent. Optional additional components of the formulation are a reducing agent, a non-thiol biocompatible anti-oxidant, a lyoprotectant (typically one or more sugars, one or more amino acids, one or more methylamine, one or more lyotropic salts, and/or one or more polyols. Also provided is an article of manufacture or kit comprising the formulation in solution or in lyophilized form. A method of inhibiting angiogenesis in a subject, comprising administering to the subject the peptide in the above formulation is also disclosed.

Description

IMPROVED FORMULATIONS OF ANTI-ANGIOGENIC PEPTIDES

This application claims priority from U.S. Provisional Application 60/648,391, filed 01 February 2005.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to compositions comprising improved formulations of the Cys-containing anti-angiogenic peptide Pro-His-Ser-Cys-Asn that prevent degradation and spontaneous oxidative dimerization or oligomerization of the peptide.

Description of the Background Art Most forms of cancer are manifest as, or derived from, solid tumors (Shockley et al, Ann.

N. Y. Acad. Sci. 1991 , 617:367-82). These types of tumors have generally proven resistant in the clinic to biological therapeutics such as monoclonal antibodies and irnmunotoxins. Anti-angiogenic therapy for the treatment of cancer developed from the recognition that solid tumors require angiogenesis {i.e., new blood vessel formation) for sustained growth (Folkman, Ann. Surg. 1972, 175:409-16; Folkman, MoI. Med. 1995, 1:120-22; Folkman, Breast Cancer Res. Treat. 1995,

36:109-18; Hanahan et at, Cell 1996, 86:353-64). Efficacy of anti-angiogenic therapy in animal models has been demonstrated in many studies, for example, Millauer et ah, Cancer Res. 1996, 56:1615-20; Borgstrom et al, Prostate 1998, 35:1-10; Benjamin et al, J. Clin. Invest. 1999, 103:159-65; Brewer GJ et al, Integr Cancer Ηier. 2002, 1:327-37; van Golen KL et al, Neoplasia 2002, 4:373-9; Cox C et al, Arch Otolaryngol Head Neck Surg. 2003, 129:781-5. In the absence of angiogenesis, internal cell layers of solid tumors are inadequately nourished. Further, angiogenesis (i.e., aberrant vascularization) has now also been shown to be required for the growth of non-solid, hematological tumors and has been implicated in numerous other diseases, including ocular neovascular disease, macular degeneration, rheumatoid arthritis, etc. In contrast, normal tissue does not require angiogenesis except under specialized circumstances such as wound repair, the proliferation of the uterine internal lining during the menstrual cycle, etc. Accordingly, a requirement for angiogenesis is a significant difference between tumor/cancerous tissue and normal tissue. Importantly, the dependence of tumor cells on angiogenesis, when compared to normal cells, is quantitatively greater than differences in cell replication and cell death. The latter differences are typically exploited in cancer therapy. Tumor angiogenesis can be initiated by cytokines such as vascular endothelial growth factor (VEGF) and/or a fibroblast growth factor (FGF), which bind to specific receptors on endothelial cells ("ECs") in the local vasculature, under hypoxic conditions. The activated ECs secrete enzymes which remodel the associated tissue matrix and modulate expression of adhesion molecules such as integrins. Following matrix degradation, ECs proliferate and migrate toward the tumor, which results in the generation and maturation of new blood vessels.

Interestingly, protein fragments, such as endostatin, kringle 5 and PEX, which inhibit angiogenesis, are produced by degradation of matrix proteins (O'Reilly et ah, Cell 1997, 8#:277-85; O'Reilly et ah, Cell, 1994, 79:315-28; Brooks etah, Cell, 1998, 92:391-400). Accordingly, these protein fragments may inhibit neoangiogenesis, thus preventing tumor growth and metastasis.

However, protein fragments have significant drawbacks associated with their use. They are difficult and expensive to produce in large quantities, show poor pharmacological properties, are susceptible to degradation, etc. One approach has been to identify short peptide fragments of these larger proteins or of the longer fragments or subunits, which shorter peptides retain a significant portion of the anti-angiogenic activity of the parent protein.

Although the search for peptides that inhibit angiogenesis has provided compounds with significant effectiveness in preventing growth of new blood vessels, molecules with superior activity profiles are still needed. Accordingly, novel peptides are needed to fully explore the potential of peptides in preventing angiogenesis and detecting aberrant vascularization. The novel peptides may have longer plasma half-lives, be more resistant to degradation, have increased bio-availability, higher affinity, greater selectivity, etc. in comparison to peptides described in the art (Livant, U.S. Pats. No. 6,001,965 and 6,472,369). Such novel peptides may be effective in inhibiting cell migration, invasion and proliferation and treating or preventing various diseases associated with undesired angiogenesis and aberrant vascularization. Examples of such peptides, primarily derivatives of the capped pentapeptide Ac-PHSCN-NH₂ (also referred to herein as ATN- 161) are described in commonly assigned U.S. Patent applications Ser. No. 10/723,144 filed 25 November 2003 (published as US20040162239A1, Allan et a ) and Ser. No. 10/722,843, filed 25 November 2003 (published as US 2005002081 OAl, Ternansky et ah), which are hereby incorporated by reference in their entirety What is needed in the art is a method of preventing degradation of Ac-PHSCN-NH₂ under both solution phase and solid phase conditions. One approach to improving the activity profile of an anti-angiogenic peptide is to exploit the method of its formulation as a means to extend shelf life and enhance the resistance to degradation. In the case of peptides that include Cys, it is also important to prevent spontaneous oxidative dimerization or higher degrees of oligomerization or polymerization through disulfide bond formation. The present invention is directed to such improved formulations for anti-angiogenic peptide Ac-PHSCN-NH₂ and its anti-angiogenic derivatives.

SUMMARY OF THE INVENTION

The present inventors and their colleagues have developed compositions comprising improved formulations for Cys-containing peptides and salts thereof that preserve the biological/biochemical potency of the peptides, particularly by inhibiting disulfide bond formation that leads to undesired dimerization.

Acid addition salts OfAc-PHSCN-NH₂ useful in the formulations of the present invention are described in co-pending applications by Trenansky et ah, both entitled "Acid Addition Salts of Ac-PHSCN-NH₂", United States Provisional Application Serial No. 60/649,308, filed February 1, 2005, and International Application No. (to be assigned), filed February 1, 2006 (Attorney Docket No. 9715-022-228), each of which is incorporated by reference herein in its entirety. The above application discloses acid addition salts OfAc-PHSCN-NH₂, methods of making acid addition salts OfAc-PHSCN-NH₂, pharmaceutical compositions of acid addition salts OfAc-PHSCN-NH₂, methods of using acid addition salts OfAc-PHSCN-NH₂ and pharmaceutical compositions thereof to treat diseases associated with angiogenesis and aberrant vascularization and methods of preventing degradation of Ac-PHSCN-NH₂ by salt formation. Thus, the novel formulations of the present application include those using the new salts described by Ternansky et at, supra.

The present invention is thus directed to a formulation comprising a peptide Pro-His-Ser- Cys-Asn (PHSCN), an analogue thereof of a salt of the peptide or analogue, and at least one additional compound that stabilizes the peptide or analogue against spontaneous tandem dimerization or higher oligomerization. Preferably, the peptide is capped at both termini, with an acetyl group at the N-terminus and amide group at the C-terminus. The additional compound inhibits, prevents or reverses disulfide bond formation between sulfhydryl groups of Cys residues.

Also provided is the above formulation wherein the additional compound is a biocompatible acid buffer with a pK of about 5. Preferably, in the presence of the buffer, the pH of the solution is greater than about 3.0 and less than about, or equal to, 7.5. A preferred acid buffer is citrate, acetate or 2-(N-morpho- lino)ethanesulfonic acid (MES). In the above formulation, the acid buffer is preferably citrate at a concentration of about 25 mM. The buffer may be supplemented with glycine as an excipient and bulking agent, preferably at a concentration of about 50 mg/ml. The buffer may comprise citrate and acetate and may also comprise Tris. In a preferred embodiment, the above formulation comprises (i) the peptide, capped peptide or analogue, (ii) about 50 mM citrate, and (iii) about 50 mg/ml glycine.

A preferred embodiment of the formulation is in a container or vial in lyophilized form having 100 mg peptide, capped peptide, salt or analogue, 50 mM citrate, 50 mg/ml glycine lyophilized from 2 mL of a pH 5.0 solution. The above formulation may further comprise or more reducing agents; preferred agents are dithiothreitol β-mercaptoethanol, or glutathione. Preferably, the concentration of the reducing agent or agents does not exceed about 10 mM.

The above formulation may comprise a non-thiol biocompatible anti-oxidant.

The formulation may comprise a lyoprotectant present in an lyoprotecting amount, for example, about 50-600 mole lyoprotectant: 1 mole peptide. The lyoprotectant is one or more sugars, one or more amino acids, one or more methylamine, one or more lyotropic salts, and/or one or more polyols. In a preferred formulation above,

(a) the sugar is sucrose or trehalose; (b) the amino acid is monosodium glutamate or histidine;

(c) the methylamine is betaine; (d) the lyotropic salt is magnesium sulfate; and (e) the polyol is a trihydric or higher sugar alcohol.

The formulation above may comprise one or more polyols selected from the group consisting of glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, propylene glycol, and a combination thereof. In one formulation, the lyoprotectant is a non-reducing sugar, preferably trehalose or sucrose. In all the above formulations the peptide is preferably capped at its N- and C-termini, most preferably the N-terminal cap is an acyl group and the C terminal cap is an amide group such that the peptide terminates in CO-NH₂.

The formulation above is preferably sterile and formulated for in vivo administration. The present invention includes an article of manufacture or kit comprising (a) a container which contains a formulation as above, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstituting solution for the lyophilized formulation; and (c) optionally, instructions for (i) use of the solution or (ii) reconstitution and/or use of the lyophilized formulation.

The article or kit may further comprise one or more of (i) another buffer, (ii) a diluent, (iii) a filter, (iv) a needle, or (v) a syringe. The container is preferably a bottle, a vial, a syringe or test tube; it may be a multi-use container, the formulation is preferably lyophilized.

The present invention is directed to a method of inhibiting angiogenesis in a subject, comprising administering to the subject a formulation as above , wherein the peptide, analogue or salt is administered in an anti-angiogenic effective amount. Such inhibition of angiogenesis is exploited in a method for the treatment of cancer and or Crohn's disease. The invention provides use of a composition as above in a medicament for administration to a subject to inhibit undesired angiogenesis, such as in the treatment of cancer or Crohn's disease. Also included is use of a composition as above in the manufacture of a medicament for administration to a subject to inhibit undesired angiogenesis and, thereby, to treat cancer and Crohn's disease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present inventors have devised an improved formulation for a Cys-containing peptide,

PHSCN, most preferably, the terminally capped pentapeptide, Acetyl-PHSCN-NH₂, and various salts thereof, and or analogues of capped or uncapped PHSCN and salts of the derivatives and analogues. Various other capping functions are discussed below. An "analogue" of PHSCN refers to a molecule, natural or non- natural, that is structurally and/or functionally substantially similar to PHSCN. Examples include conservative amino acid substitution variants, addition variants of not more than about 20 residues (to the exclusion of native polypeptides or their structural domains), and chemically modified peptides. Other analogues are peptidomimetics and aptamers.

The peptide to be formulated is preferably pure, or essentially pure and, desirably, essentially homogeneous {i.e., free from contaminating peptides or proteins, etc.) "Essentially pure" means a peptide preparation wherein at least 90% by weight is the peptide based on total weight of the preparation, preferably at least 95% by weight. An "essentially homogeneous" preparation means a peptide preparation comprising at least 99% by weight of peptide, based on total weight of the peptide in the preparation. CAPPING GROUPS

As noted above, the PHSCN peptide is preferably capped at its N and C termini with an acyl (abbreviated "Ac") -and an amido (abbreviated "Am") group, respectively, for example acetyl (CH₃CO-) at the N terminus and amido (CO-NH₂) at the C terminus. N-terminal Capping Groups

A broad range of N-terminal capping functions, preferably in a linkage to the terminal amino group, is contemplated, for example: formyl;

C_1-10 alkanoyl, such as acetyl, propionyl, butyryl; C_1-1O alkenoyl, such as hex-3-enoyl;

C_1-10 alkynoyl, having from 1 to 10 carbon atoms, such as hex-5-ynoyl; aroyl, such as benzoyl or 1-naphthoyl; heteroaroyl, such as 3-pyrroyl or 4-quinoloyl; alkylsulfonyl, such as methanesulfonyl; arylsulfonyl, such as benzenesulfonyl or sulfanilyl; heteroarylsulfonyl, such as pyridine-4-sulfonyl;

C_1-10 substituted alkanoyl, such as 4-aminobutyryl;

C_1-I0 substituted alkenoyl, such as 6-hydroxy-hex-3-enoyl;

C_1-10 substituted alkynoyl, such as 3-hydroxy-hex-5-ynoyl; substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphth-2-oyl; substituted heteroaroyl, such as 2,4-dioxo-l,2,3,4-tetrahydro-3-methyl-quinazolin-6-oyl; substituted alkylsulfonyl, such as 2-aminoethanesulfonyl; substituted arylsulfonyl, such as 5-dimethylamino-l-naphthalenesulfonyl; substituted heteroarylsulfonyl, such as l-methoxy-6-isoquinolinesulfonyl; carbamoyl or thiocarbamoyl; substituted carbamoyl (R'-NH-CO) or substituted thiocarbamoyl (R' -NH-CS) wherein R' is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, or substituted heteroaryl; substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH-CS) wherein R' is alkanoyl, alkenoyl, alkynoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynoyl, substituted aroyl, or substituted heteroaroyl, all as above defined. C-terminal Capping Groups

The C-terminal capping function can either be in an amide or ester bond with the terminal

1 0 1 carboxyl. Capping functions that provide for an amide bond are designated as NR R wherein R and R² may be independently drawn from the following group: hydrogen;

C_1-I0 alkyl, such as methyl, ethyl, isopropyl;

Ci-io alkenyl, such as prop-2-enyl;

Ci-io alkynyl, such as prop-2-ynyl;

Ci-io substituted alkyl such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, halogenoalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl;

C_1-IO substituted alkenyl such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, halogenoalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl; Ci-io substituted alkynyl such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, halogenoalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl;

C₁-_Io aroylalkyl such as phenacyl or 2-benzoylethyl; aryl, such as phenyl or 1-naphthyl; heteroaryl, such as 4-quinolyl;

C_1-IO alkanoyl such as acetyl or butyryl; aroyl, such as benzoyl; heteroaroyl, such as 3-quinoloyl;

OR' or NR'R" where R' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, sulfinyl, or SO₂-R' ' ' or SO-R' ' ' where R" ' is substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl.

Capping functions that provide for an ester bond are designated as OR, wherein R may be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted heteroaralkyloxy. Either the N- terminal or the C-terminal capping function, or both, may be of such structure that the capped molecule functions as a prodrug (a pharmacologically inactive derivative of the parent drug molecule) that undergoes spontaneous or enzymatic transformation within the body in order to release the active drug and that has improved delivery properties over the parent drug molecule (Bundgaard H, Ed: Design of Prodrugs, Elsevier, Amsterdam, 1985).

Judicious choice of capping groups allows the addition of other activities on the peptide. For example, the presence of a sulfhydryl group linked to the N- or C-terminal cap will permit conjugation of the derivatized peptide to other molecules.

A "lyoprotectant" is a molecule which, when combined with a protein or peptide of interest, significantly prevents or reduces chemical and/or physical instability of the protein or peptide upon lyophilization and subsequent storage. Exemplary lyoprotectants include sugars such as sucrose or trehalose; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher sugar alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronics; and combinations thereof. The preferred lyoprotectant is a non-reducing sugar, such as trehalose or sucrose. The lyoprotectant is added to the pre-lyophilized formulation in a "lyoprotecting amount" which means that, following lyophilization of the peptide in the presence of the lyoprotecting amount of the lyoprotectant, the peptide essentially retains its physical and chemical stability and integrity upon lyophilization and storage.

The "diluent" of interest is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

A "preservative" is a compound which can be added to the diluent to essentially reduce bacterial action in the reconstituted formulation, thus facilitating the production of a multi-use reconstituted formulation, for example. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkoniurn chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.

A "bulking agent" is a compound which adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol and xorbitol.

One important purpose of the present formulation is to stabilize these peptide or analogue against spontaneous dimerization.. This is preferably accomplished by formulating the peptides at low pH that will prevent disulfide bond formation between two Cys residues. Such acid buffers are preferably biocompatible. Examples include citrate, acetate, 2-(N-morpholino)ethanesulfonic acid (MES), or any similar buffer with a pK of about 5, wherein in the presence of the buffer, the pH of the solution is <7.5 but preferably not below 3.0. A preferred acidic formulation comprises citrate, preferably at about 25mM. The buffer is preferably supplemented with glycine (GIy) as an excipient and bulking agent. A preferred concentration is about 50 mg/ml GIy. Another advantage of GIy is that it is an accepted excipient for intravenous infusion or injection in humans.

Other amino acids or compounds can be used in place of GIy. Examples of desirable acids or combinations are citrate + acetate, acetate and Tris, and the like. The goal is to keep the pH low once the peptide is in lyophilized form. The lyophilized powder is then reconstituted with water. The present invention includes, in addition to lyophilized compositions, stabilized liquid pharmaceutical compositions comprising a peptide as disclosed herein, preferably capped, whose effectiveness as a therapeutically active component is normally compromised during storage in liquid formulations as a result of dimerization and higher order oligomerization, aggregation, etc. The stabilized liquid pharmaceutical composition comprises an amount of an amino acid base sufficient to decrease aggregate formation during storage, where the amino acid base is an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. It is understood that the composition comprises a buffering agent to maintain pH of the liquid composition within an acceptable range for stability of the peptide, where the buffering agent is an acid substantially free of its salt form, an acid in its salt form, or a mixture of an acid and its salt form. The amino acid base serves to stabilize the peptide against aggregate formation during storage of the liquid pharmaceutical composition, while use of an acid buffering agent (in either of it forms) results in a liquid composition having an osmolarity that is nearly isotonic. The liquid pharmaceutical composition may additionally incorporate other stabilizing agents, more particularly methionine, a nonionic surfactant such as polysorbate 80, and EDTA, to further increase stability of the peptide. Such liquid pharmaceutical compositions are said to be stabilized, as addition of amino acid base in combination with an acid substantially free of its salt form, an acid in its salt form, or a mixture of an acid and its salt form, results in the increased storage stability relative to liquid compositions formulated in the absence of the combination of these two components. Methods for increasing the stability of the peptide in a liquid pharmaceutical composition (and for increasing storage stability of such a composition) comprise incorporating into the liquid composition an amount of an amino acid base sufficient to decrease aggregate formation of the polypeptide during storage, and a buffering agent that is an acid substantially free of its salt form, an acid in its salt form, or a mixture of an acid and its salt form.

The acid addition salts Of Ac-PHSCN-NH₂ (see Ternansky et at, supra) may be formed from both organic and inorganic acids. Exemplary organic acids include generally, carboxylic acids and sulfonic acids, such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, glycolic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, fumaric acid, oxalic acid, lactic acid, 4- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, t-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid and muconic acid. Other organic acids are also known to the skilled artisan. In some embodiments, the acid addition salt of Ac-PHSCN-NH₂ is formed from methanesulfonic acid, acetic acid, glycolic acid, (+) camphorsulfonic acid, mandeleic acid, salicyclic acid, succinic acid or combinations thereof.

Exemplary inorganic acids include hydrofluoric acid, perchloric acid, hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, phosphorous acid, hydroiodic acid, chloric acid, thiocyanic acid, hypophosphorous acid, nitrous acid, cyanic acid, chromic acid, sulfurous acid, and hydrazoic acid. Other inorganic acids are known to those of skill in the art. In some embodiments, the acid addition salt OfAc-PHSCN-NH₂ is formed from hydrobromic acid, nitric acid, hydrochloric acid, phosphoric acid or combinations thereof. In other embodiments, the acid addition salt of Ac-PHSCN-NH₂ is formed from hydrochloric acid.

Generally, the acid addition salts OfAc-PHSCN-NH₂ may be made any conventional method known to those of skill in the art. These methods include saturating solutions OfAc-PHSCN-NH₂ with gaseous acids, adding solutions of acids to solutions OfAc-PHSCN-NH₂, etc. In some embodiments, an acid addition salt OfAc-PHSCN-NH₂ is made by adding slightly more than 1 equivalent {e.g., 1.05 equivalent) of the acid to a solution OfAc-PHSCN-NH₂ dissolved in distilled water. The acid addition salt is typically isolated as a solid from the aqueous mixture.

The acid addition salt OfAc-PHSCN-NH₂ is generally, considerably more stable than the free base in both the solid and solution phase. The acid addition salt is believed to prevent oxidative dimerization of Ac-PHSCN-NH₂ mediated by cysteine. Acid addition salts which prevent degradation of Ac-PHSCN-NH₂ are formed from, for example, methanesulfonic acid, acetic acid, glycolic acid, sulfuric acid, (+) camphorsulfonic acid, mandeleic acid, salicyclic acid, succinic acid, hydrobromic acid, hydrochloric acid, nitric acid and phosphoric acid.

The formulation of the present invention preferably comprises one or more reducing agent, such as dithiothreitol (DTT) at a low concentration, preferably not exceeding about 10 mM, β-mercaptoethanol, glutathione (GSH) or other Cys-containing reducing agents. Other non-thiol containing anti-oxidants such as metal binding compounds (EDTA, EGTA) that are biocompatible may also be used.

Another embodiment of the invention is directed to formulations that increase half-life of the peptide after parenteral injection. Examples of these include formulation of Ac-PHSCN-NH₂ with a cyclodextrins, cremaphor or liposomal formulations.

"Cyclodextrin" refers to a cyclic molecule containing six or more cc-D-glucopyranose units linked at the 1,4 positions by α linkages as in amylose. β-Cyclodextrin or cycloheptaamylose contains seven oc-D-glucopyranose units. As used herein, the term "cyclodextrin" also includes cyclodextrin derivatives such as hydroxypropyl and sulfobutyl ether cyclodextrins. Such derivatives are described for example, in U.S. Patents No. 4,727,064 and 5,376,645. One preferred cyclodextrin is hydroxypropyl β -cyclodextrin (HβCD) having a degree of substitution of from about 4.1-5.1 as measured by Fourier Transform Infrared Spectroscopy. Such a cyclodextrin is available from Cerestar (Hammond, Ind., USA) under the name Cavitron 82003™. In one preferred embodiment, the Ac-PHSCN-NH₂ or derivative is formulated in an aqueous solution containing a cyclodextrin. In another embodiment, the peptide of this invention is formulated as a lyophilized powder containing a cyclodextrin or as a sterile powder containing a cyclodextrin. Preferably, the cyclodextrin is (HβCD) or sulfobutyl ether β-cyclodextrin; more preferably, the cyclodextrin is hydroxypropyl- β-cyclodextrin. Typically, in an injectable solution, the cyclodextrin will comprise about 1 - 25% (w/w), preferably, about 2 - 10%, more preferably, about 4 - 6%, of the formulation. Additionally, the weight ratio of the cyclodextrin to the peptide will preferably be from about 1:1 to about 10:1. In another embodiment of the invention, Ac-PHSCN-NH₂ or its derivative is formulated to render the peptide orally or intranasally bioavailable.

Other useful components in the present formulation include polyols such as mannitol, which may act as stabilizers, and further help the bulk powder form a cake. Preferably, the polyol has at least three hydroxyl groups, and may be in the form of a mixture of two or more polyols.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to, or following, lyophilization and reconstitution. Alternatively, sterility, of the entire mixture may be accomplished by autoclaving the ingredients prior to addition of the peptide at about 12O⁰C for about 30 minutes, for example. After the peptide, lyoprotectant and other optional components are mixed together, the formulation is lyophilized. Many different freeze-drivers are available for this purpose such as Hull50™ (Hull, USA) or GT20™ (Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplished by freezing the formulation and subsequently subliming ice from the frozen content at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or the collapse temperature of the formulation. Typically, the shelf temperature for the primary drying will range from about -30 to 25°C (provided the product remains frozen during primary dying) at a suitable pressure, ranging typically from about 50 to 250 mTorr. The formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will mainly dictate the time required for drying, which can range from a few hours to several days (e.g., 40-60 hrs). A secondary drying stage may be carried out at about 0-40°C, depending primarily on the type and size of container and nature of the peptide. For example, the shelf temperature throughout the entire water removal phase of lyophilization may be from about 15- 30°C. (e.g., about 2O⁰C). The time and pressure required for secondary drying will be that which produces a suitable lyophilized cake, dependent e.g., on the temperature and other parameters. The secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g., 10-15 hours). The pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation and vial size.

In some instances, it may be desirable to lyophilize the peptide formulation in the container in which reconstitution of the peptide is to be carried out in order to avoid a transfer step. The container in this instance may, for example, be a 3, 5, 10, 20, 50 or 100 cc vial. As a general proposition, lyophilization will result in a lyophilized formulation in which the moisture content thereof is less than about 5%, preferably less than about 3%.

Reconstitution of the Lvophilized Formulation

At the desired stage, typically when it is time to administer the peptide to the patient, the lyophilized formulation may be reconstituted with a diluent such that the peptide concentration in the reconstituted formulation is at least 10 mg/mL, for example from about 10 mg/mL to about 1000 mg/mL, more preferably from about 50 mg/mL to about 500 mg/mL, and most preferably from about 100 mg/mL to about 500 mg/mL. Such high peptide concentrations in the reconstituted formulation are considered to be particularly useful where subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration, such as intravenous (i.v.) administration, lower concentrations of the peptide in the reconstituted formulation may be desired (for example from about 1-100 mg/mL, or from about 5-50 mg/mL peptide in the reconstituted formulation). In certain embodiments, the peptide concentration in the reconstituted formulation is significantly higher than that in the pre-lyophilized formulation. For example, the peptide concentration in the reconstituted formulation may be about 2-40 times, preferably 3-10 times and most preferably 3-6 times (e.g., at least three fold or at least four fold) that of the pre-lyophilized formulation.

Reconstitution generally takes place at a temperature of about 25°C to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, for example, on the type of diluent, amount of excipient(s) and peptide. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline, PBS), sterile saline solution, Ringer's solution or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives have been described above, with aromatic alcohols such as benzyl or phenol alcohol being the preferred preservatives. The amount of preservative employed is determined by assessing different preservative concentrations for compatibility with the peptide and preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it can be present in an amount from about 0.1-2.0% and preferably from about 0.5-1.5%, but most preferably about 1.0-1.2%. Preferably, the reconstituted formulation has less than 6000 particles per vial which are

>10 μm in size. Articles of Manufacture

In another embodiment of the invention, an article of manufacture is provided which contains the lyophilized formulation of the present invention and provides instructions for its reconstitution and/or use. The article of manufacture comprises a container. Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the lyophilized formulation and a label on, or associated with, the container may indicate directions for reconstitution and/or use. For example, the label may indicate that the lyophilized formulation is reconstituted to peptide concentrations as described above. The label may further indicate that the formulation is useful or intended for subcutaneous administration. The container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the reconstituted formulation. The article of manufacture may further comprise a second container comprising a suitable diluent (e.g., BWFI). Upon mixing of the diluent and the lyophilized formulation, the final protein concentration in the reconstituted formulation will generally be at least 50 mg/mL. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Therapeutic kits may have a single container which contains the formulation of the Ac-PHSCN-NH₂ pharmaceutical compositions with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have distinct container for each component. Preferably, therapeutic kits of the invention include a formulation of Ac-PHSCN-NH₂ or an acid addition salt thereof as disclosed herein packaged for use in combination with the co-administration of a second compound (such as a chemotherapeutic agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator) or a pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient. The components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution. The components of the kit may also be provided as solids, which may be converted into liquids by addition of suitable solvents, which are preferably provided in another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid. Usually, when there is more than one component, the kit will contain a second vial or other container, which allows for separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, a therapeutic kit will contain apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the agents of the invention which are components of the present kit. The present formulation is one that is suitable for administration of the peptide by any acceptable route such a oral (enteral), subcutaneous, intramuscular, intravenous transdermal, Administration may be by infusion pump. All modes and routes of administration disclosed by Ternansky et al., supra, are understood to be useful with the present formulations and are incorporated by reference as if written here in full. The present invention is also directed to formulations of the peptide suitable for administration by inhalation. Many drugs currently administered by inhalation come primarily as liquid or solid aerosol particles of respirable size. For biotherapeutic drugs, this may present a problem, as many of these medicaments are unstable in aqueous environments for extended periods of time and are rapidly denatured if micronized by high shear grinding or other comminution methods when presented as dry powders. Additionally, a number of these medicaments do not survive long enough in the lung as they are extracted quickly from the lung environment after they are administered as inhalation aerosols. Significant drug loss could also occur by deactivation either as a result of reactivity of the medicament with device and container surfaces, or during aerosolization, particularly in high shear, energy intensive, nebulized systems (Mumenthaler, M, et al., Pharm. Res., 11:12-20 (1994). To overcome these instability problems, many drug and excipient systems contain biodegradable carriers, such as poly(lactide-co-glycolides, have been developed for biotherapeutic proteins and peptides (Liu, R. et al., Biotechnol. Bioeng., 57:177-184 (1991). Most therapeutic peptides are poorly absorbed through biologic membranes even upon formulation with penetration enhancer. In numerous therapies, drug dosimetry is increased by orders of magnitude to achieve minimum systemic concentrations required for efficacy. In other cases the drug product is formulated with exotic absorption promoters in order to improve permeability across an absorption barrier, often leading to toxic consequences. The mode of drug administration to the body has also gradually expanded from oral and parenteral to transdermal, rectal and the pulmonary routes of administration, i.e., nose and lung. Success with these drug delivery approaches have been mixed due to lack of acceptance of the newer, complex molecules that must be used for treating difficult diseases, e.g., infections, malignancies, cardiovascular, endocrine, neurologic diseases, and a variety of diseases of immunological compromise. Thus, the present invention exploits the existence of a fluid propelled formulation system comprising the peptide (drug) that is stable and protected by a rate-limiting carrier, easily manufactured, and therapeutically effective when administered as fluid dispersed particles to the lung of a patient. As noted above, the present invention includes formulations of the peptide for oral or nasal inhalation. Such formulations include, but are not limited to, modulated release aerosol particles, and to medicinal, respirable aerosol particles comprising polysaccharide vesicles which are associated with, and, may form a part of, a construct with or entrap a selected medicament, here, the peptide, and provide slow release thereof, as disclosed in U.S. Pat. 6,551,578, incorporated by reference in its entirety. In this approach the peptide is formulated so that it is suitable for administration by oral and nasal inhalation. A stable, colloidal dispersion of the peptide in a fluid, e.g., air, hydrocarbon gases, chlorofluorocarbon (CFC) propellants or non-CFC propellants, such as tetrafluoroethane (HFA- 134a) and heptafluoropropane (HFA-227) are intended.

For purposes of the formulations of this invention that are intended for inhalation into the lungs, the peptide is associated with the naturally occurring polysaccharide polymer to which it is destined to be combined. By "associate" or "associated" in this context is meant that the peptide is present as a matrix or a part of a polymeric construct along with the polysaccharide polymer or is encapsulated as a microsphere in a polysaccharide polymer or in polysaccharide polymeric construct particle, or is on a surface of such particle, whereby a therapeutically effective amount or fraction (e.g., 95% or more) of the peptide is particulate. Typically, the construct particles have a diameter of less than about 10 μm, and preferably less than about 5 μm, in order that the particles can be inhaled into the respiratory tract and/or lungs of the patient being treated.

A suitable polymeric construct is one which will incorporate therein or encapsulate the selected peptide and provide a controlled or modulated release of the medicament therefrom to the sites of action or application of the patient's body, e.g., from the lung to the local surrounding environment of the subject.

A suitable polysaccharide is a polymer selected from the group of an alginate salt, where the cation is, e.g., Li⁺, Na⁺, K⁺, Ca⁺⁺, NH³⁺, NH⁴⁺ etc., such as sodium alginate, calcium alginate, sodium-calcium alginate, ammonium alginate, sodium-ammonium alginate, or calcium-ammonium alginate. A preferred alginate modulating releasing agent is ammonium calcium alginate. These materials are typically used in injectable implants and microsphere preparations for controlled release. A commercial form of ammonium calcium alginate is Keltose™, manufactured and distributed by International Specialty Products (Wayne, NJ). As used herein, "alginate" means alginic acid, or any of its salts; or other naturally occurring polysaccharide or carbohydrate based polymers such as gum arabic, pectin, galacturonic acid, gum karaya; gum Benjamin, plantago ovata gum; agar; carrageenan; cellulose; gelatin; or a mixture of any of the foregoing polymers. Alginates are pharmaceutical excipients generally regarded as safe and used to prepare a variety of well documented pharmaceutical systems (U.S. Pats. No. 6,166,084; 6,166,043; 6,166,042; 6,166,004; and 6,165,615). Alginates are naturally occurring polymers comprising polysaccharide chains. These polymers have the propensity to absorb water thus swelling to become gel-like structures in solution. Upon inhalation of the resultant core formulation by a patient being treated, the gel dissolves in the body of such patient, thus releasing its drug payloads in a dissolution controlled manner. Such a polymer system forms a construct or a matrix when formed in situ with a selected medicament or medicaments whereby such medicament or medicaments forms part of the matrix or is encapsulated within the matrix. Upon such formation or encapsulation, the medicament is time- released or modulated from the site of action in the body, e.g., the lungs, the respiratory tract, ear, etc., to the surrounding environment or tissues of the subject's body. The polysaccharide polymer, e.g., an alginate salt, is typically present in the resultant controlled-release formulation in an amount ranging from about 0.000001% to about 10% by weight of the total weight of the formulation.

The therapeutic peptide is present in the inventive polymer construct in a therapeutically effective amount, that is, an amount such that the peptide can be incorporated into an aerosol formulation such as a dispersion aerosol, via oral or nasal inhalation, and cause its desired therapeutic effect, preferably with one dose, or through several doses.

Other formulations for prolonged administration are also contemplated, including use of liposomes, either multilamellar or unilamellar, the preparation of which is well known to those skilled in the art. Liposomes may be phospholipid or non-phospholipid based.

Assays Those of skill in the art will appreciate that the in vitro and in vivo assays useful for measuring the activity of the present formulation of Ac-PHSCN-NH₂ and its salts, described herein, are illustrative rather than comprehensive. These can be found, for example in Ternansky et αl, supra, and in commonly assigned co-pending applications USSN 10/074, 225 and 10/661,784), all of which are incorporated by reference in their entirety. Examples of preferred assays are set forth below.

Assay for Endothelial Cell Migration

For EC migration, transwells are coated with type I collagen (50 μg/mL) by adding 200 μL of the collagen solution per transwell, then incubating overnight at 37°C. The transwells are assembled in a 24- well plate and a chemoattractant (e.g., FGF-2) is added to the bottom chamber in a total volume of 0.8 mL media. ECs, such as human umbilical vein endothelial cells (HUVEC), which have been detached from monolayer culture using trypsin, are diluted to a final concentration of about 10⁶ cells/mL with serum-free media and 0.2 mL of this cell suspension is added to the upper chamber of each transwell. Salts of Ac-PHSCN-NH₂ may be added to both the upper and lower chambers and the migration is allowed to proceed for 5 hrs in a humidified atmosphere at 37°C. The transwells are removed from the plate stained using DiffQuik^®. Cells which did not migrate are removed from the upper chamber by scraping with a cotton swab and the membranes are detached, mounted on slides, and counted under a high-power field (40Ox) to determine the number of cells migrated.

Biological Assay of Anti-Invasive Activity

The ability of cells such as ECs or tumor cells (e.g., PC-3 human prostatic carcinoma) cells to invade through a reconstituted basement membrane (Matrigel®) in an assay known as a Matrigel® invasion assay system has been described in detail in the art (Klemman et al, Biochemistry 1986, 25: 312-318; Parish et al, 1992, Int. J. Cancer 52:378-383). Matrigel® is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin as well as transforming growth factor-β (TGFβ, urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA) and the serpin known as plasminogen activator inhibitor type 1 (PAI-I) (Chambers et al, Cane. Res. 1995, 55:1578-1585,). It is accepted in the art that results obtained in this assay for compounds which target extracellular receptors or enzymes are predictive of the efficacy of these compounds in vivo (Rabbani et al, Int. J. Cancer 1995, 63: 840-845).

Such assays employ transwell tissue culture inserts. Invasive cells are defined as cells which are able to traverse through the Matrigel® and upper aspect of a polycarbonate membrane and adhere to the bottom of the membrane. Transwells (Costar) containing polycarbonate membranes (8.0 μm pore size) are coated with Matrigel® (Collaborative Research), which has been diluted in sterile PBS to a final concentration of 75 μg/mL (60 μL of diluted Matrigel® per insert), and placed in the wells of a 24-well plate. The membranes are dried overnight in a biological safety cabinet, then rehydrated by adding 100 μL of DMEM containing antibiotics for 1 hour on a shaker table. The DMEM is removed from each insert by aspiration and 0.8 mL of DMEM/10 % FBS/antibiotics is added to each well of the 24-well plate such that it surrounds the outside of the transwell ("lower chamber"). Fresh DMEM/ antibiotics (lOOμL), human Glu-plasminogen (5 μg/mL), and any inhibitors to be tested are added to the top, inside of the transwell ("upper chamber"). The cells which are to be tested are trypsinized and resuspended in DMEM/antibiotics, then added to the top chamber of the transwell at a final concentration of 800,000 cells/mL. The final volume of the upper chamber is adjusted to 200 μL. The assembled plate is then incubated in a humid 5% CO₂ atmosphere for 72 hours. After incubation, the cells are fixed and stained using DiffQuik® (Giemsa stain) and the upper chamber is then scraped using a cotton swab to remove the Matrigel® and any cells which did not invade through the membrane. The membranes are detached from the transwell, e.g., using an X-acto^® blade, mounted on slides using Permount^® and cover-slips, then counted under a high-powered (40Ox) field. An average of the cells invaded is determined from 5-10 fields counted and plotted as a function of peptide concentration. Tube-Formation Assays of Anti- Angiogenic Activity Endothelial cells, for example, human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMVEC) which can be prepared or obtained commercially, are mixed at a concentration of 2 x 10⁵ cells/mL with fibrinogen (5mg/mL in phosphate buffered saline (PBS) in a 1:1 (v/v) ratio. Thrombin is added (5 units/ mL final concentration) and the mixture is immediately transferred to a 24-well plate (0.5 mL per well). The fibrin gel is allowed to form and then VEGF and bFGF are added to the wells (each at 5 ng/mL final concentration) along with the test compound. The cells are incubated at 37°C in 5% CO₂ for 4 days at which time the cells in each well are counted and classified as either rounded, elongated with no branches, elongated with one branch, or elongated with 2 or more branches. Results are expressed as the average of 5 different wells for each concentration of compound. Typically, in the presence of angiogenic inhibitors, cells remain either rounded or form undifferentiated tubes {e.g. 0 or 1 branch). This assay is recognized in the art to be predictive of angiogenic (or anti-angiogenic) efficacy in vivo (Min et at, Cancer Res. 1996, 56: 2428-2433,).

In an alternate assay, endothelial cell tube formation is observed when endothelial cells are cultured on Matrigel® (Schnaper et at, J. Celt Physiol. 1995, 165: 107-118). Endothelial cells (10⁴ cells/well) are transferred onto Matrigel®-coated 24-well plates and tube formation is quantitated after 48 hrs. Inhibitors are tested by adding them either at the same time as the endothelial cells or at various time points thereafter. Tube formation can also be stimulated by adding (a) angiogenic growth factors such as bFGF or VEGF, (b) differentiation stimulating agents {e.g.,. PMA) or (c) a combination of these.

While not wishing to be bound by theory, this assay models angiogenesis by presenting to the endothelial cells a particular type of basement membrane, namely the layer of matrix which migrating and differentiating endothelial cells might be expected to first encounter. In addition to bound growth factors, the matrix components found in Matrigel® (and in basement membranes in situ) or proteolytic products thereof may also be stimulatory for endothelial cell tube formation which makes this model complementary to the fibrin gel angiogenesis model previously described (Blood et al, Biochim. Biophys. Acta 1990, i 032:89-118; Odedrat al, Pharmac. Ther. 1991, 49:111-124,).

Assays for Inhibition of Proliferation

The ability of the compounds of the invention to inhibit the proliferation of EC's may be determined in a 96-well format. Type I collagen (gelatin) is used to coat the wells of the plate (0.1-1 mg/mL in PBS, 0.1 mL per well for 30 minutes at room temperature). After washing the plate (3x w/PBS), 3-6,000 cells are plated per well and allowed to attach for 4 hrs (37°C/5% CO₂) in Endothelial Growth Medium (EGM; Clonetics ) or M199 media containing 0.1-2% FBS. The media and any unattached cells are removed at the end of 4 hrs and fresh media containing bFGF (1-10 ng/mL) or VEGF (1-10 ng/rnL) is added to each well. Compounds to be tested are added last and the plate is allowed to incubate (37°C/5% CO₂) for 24-48 hrs. MTS (Promega) is added to each well and allowed to incubate from 1-4 hrs. The absorbance at 490nm, which is proportional to the cell number, is then measured to determine the differences in proliferation between control wells and those containing test compounds. A similar assay system can be set up with cultured adherent tumor cells. However, collagen may be omitted in this format. Tumor cells (e.g., 3,000-10,000/well) are plated and allowed to attach overnight. Serum free medium is then added to the wells,, and the cells are synchronized for 24 hrs. Medium containing 10% FBS is then added to each well to stimulate proliferation. Compounds to be tested are included in some of the wells. After 24 hrs, MTS is added to the plate and the assay developed and read as described above. Caspase-3 Activity

The ability of the compounds of the invention to promote apoptosis of EC's may be determined by measuring activation of caspase-3. Type I collagen (gelatin) is used to coat a PlOO plate and 5xlO⁵ ECs are seeded in EGM containing 10% FBS. After 24 hours (at 37°C in5% CO₂) the medium is replaced by EGM containing 2% FBS, 10 ng/ml bFGF and the desired test compound. The cells are harvested after 6 hours, cell lysates prepared in 1% Triton and assayed using the EnzChek®Caspase-3 Assay Kit #1 (Molecular Probes) according to the manufactures' instructions.

Corneal Angiogenesis Model The protocol used is essentially identical to that described by Volpert et al, J. Clin. Invest.

1996, 95:671-679. Briefly, female Fischer rats (120-14Og) are anesthetized and pellets (5 μl) comprised of Hydron^®, bFGF (15OnM), and the compounds to be tested are implanted into tiny incisions made in the cornea 1.0- 1.5mm from the limbus. Neovascularization is assessed at 5 and 7 days after implantation. On day 7, animals are anesthetized and infused with a dye such as colloidal carbon to stain the vessels. The animals are then euthanized, the corneas fixed with formalin, and the corneas flattened and photographed to assess the degree of neovascularization. Neovessels may be quantitated by imaging the total vessel area or length or simply by counting vessels. Matrigel® Plug Assay

This assay is performed essentially as described by Passaniti et at, 1992, Lab Invest. 67:519- 528. Ice-cold Matrigel® (e.g., 500 μL) (Collaborative Biomedical Products, Inc., Bedford, MA) is mixed with heparin (e.g., 50 μg/ml), FGF-2 (e.g., 400 ng/ml) and the compound to be tested. In some assays, bFGF may be substituted with tumor cells as the angiogenic stimulus. The Matrigel® mixture is injected subcutaneously into 4-8 week-old athymic nude mice at sites near the abdominal midline, preferably 3 injections per mouse. The injected Matrigel® forms a palpable solid gel. Injection sites are chosen such that each animal receives a positive control plug (such as FGF-2 + heparin), a negative control plug (e.g., buffer + heparin) and a plug that includes the compound being tested for its effect on angiogenesis, e.g., (FGF-2 + heparin + compound). AU treatments are preferably run in triplicate. Animals are sacrificed by cervical dislocation at about 7 days post injection or another time that may be optimal for observing angiogenesis. The mouse skin is detached along the abdominal midline, and the Matrigel® plugs are recovered and scanned immediately at high resolution. Plugs are then dispersed in water and incubated at 37°C overnight. Hemoglobin (Hb) levels are determined using Drabkin's solution (e.g., obtained from Sigma) according to the manufacturers' instructions. The amount of Hb in the plug is an indirect measure of angiogenesis as it reflects the amount of blood in the sample. In addition, or alternatively, animals may be injected prior to sacrifice with a 0.1 ml buffer (preferably PBS) containing a high molecular weight dextran to which is conjugated a fluorophore. The amount of fluorescence in the dispersed plug, determined fluorimetrically, also serves as a measure of angiogenesis in the plug. Staining with mAb anti-CD31 (CD31 is "platelet-endothelial cell adhesion molecule or PECAM") may also be used to confirm neovessel formation and microvessel density in the plugs. Chick Chorioallantoic Membrane (CAM) Angiogenesis Assay

This assay is performed essentially as described by Nguyen et at, Microvascular Res. 1994, 47:31-40. A mesh containing either angiogenic factors (bFGF) or tumor cells plus inhibitors is placed onto the CAM of an 8-day old chick embryo and the CAM observed for 3-9 days after implantation of the sample. Angiogenesis is quantitated by determining the percentage of squares in the mesh which contain blood vessels.

In Vivo Assessment of Angiogenesis Inhibition and Anti-Tumor Effects Using the Matrigel® Plug Assay with Tumor Cells

In this assay, tumor cells, for example 1-5 x 10⁶ cells of the 3LL Lewis lung carcinoma or the rat prostate cell line MatLyLu, are mixed with Matrigel® and then injected into the flank of a mouse following the protocol described in Sec. B., above. A mass of tumor cells and a powerful angiogenic response can be observed in the plugs after about 5 to 7 days. The anti-tumor and anti- angiogenic action of a compound in an actual tumor environment can be evaluated by including it in the plug. Measurement is then made of tumor weight, Hb levels or fluorescence levels (of a dextran-fluorophore conjugate injected prior to sacrifice). To measure Hb or fluorescence, the plugs are first homogenize with a tissue homogenizer.

Xenograft Model of Subcutaneous (s.c.) Tumor Growth

Nude mice are inoculated with MDA-MB-231 cells (human breast carcinoma) and Matrigel® (10⁶ cells in 0.2mL) s.c. in the right flank of the animals. The tumors are staged to 200 mm³ and then treatment with a test composition is initiated (lOOμg/animal/day given q.d. IP). Tumor volumes are obtained every other day and the animals are sacrificed after 2 weeks of treatment. The tumors are excised, weighed and paraffin embedded. Histological sections of the tumors are analyzed by H and E, anti-CD31, Ki-67, TUNEL, and CD68 staining. Xenograft Model of Metastasis The compounds of the invention are also tested for inhibition of late metastasis using an experimental metastasis model (Crowley et al, Proc. Natl. Acad. ScL USA 1993, 905021-5025). Late metastasis involves the steps of attachment and extravasation of tumor cells, local invasion, seeding, proliferation and angiogenesis. Human prostatic carcinoma cells (PC-3) transfected with a reporter gene, preferably the green fluorescent protein (GFP) gene, but as an alternative with a gene encoding the enzymes chloramphenicol acetyl-transferase (CAT), luciferase or LacZ, are inoculated into nude mice. This approach permits utilization of either of these markers (fluorescence detection of GFP or histochemical colorimetric detection of enzymatic activity) to follow the fate of these cells. Cells are injected, preferably iv, and metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human prostate cancer. For example, GFP-expressing PC-3 cells (10⁶ cells per mouse) are injected iv into the tail veins of nude (nu/nu) mice. Animals are treated with a test composition at lOOμg/animal/day given q.d. IP. Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay of the detectable label.

Inhibition of Spontaneous Metastasis In Vivo by PHSCN and Functional Derivatives The rat syngeneic breast cancer system employs Mat BUI rat breast cancer cells (Xing et al,

Int. J. Cancer 1996, 67:423-429). Tumor cells, for example, about 10⁶ suspended in 0.1 mL PBS, are inoculated into the mammary fat pads of female Fisher rats. At the time of inoculation, a 14-day Alza osmotic mini-pump is implanted intraperitoneally to dispense the test compound. The compound is dissolved in PBS (e.g., 200 mM stock), sterile filtered and placed in the minipump to achieve a release rate of about 4 mg/kg/day. Control animals receive vehicle (PBS) alone or a vehicle control peptide in the minipump. Animals are sacrificed at about day 14. In the rats treated with the compounds of the present invention, significant reductions in the size of the primary tumor and in the number of metastases in the spleen, lungs, liver, kidney and lymph nodes (enumerated as discrete foci) may be observed. Histological and immunohistochemical analysis reveal increased necrosis and signs of apoptosis in tumors in treated animals. Large necrotic areas are seen in tumor regions lacking neovascularization. Human or rabbit PHSCN and their derivatives to which ¹³¹I is conjugated (either 1 or 21 atoms per molecule of peptide) are effective radiotherapeutics and are found to be at least two-fold more potent than the unconjugated polypeptides. In contrast, treatment with control peptides fails to cause a significant change in tumor size or metastasis. 3LL Lewis Lung Carcinoma: Primary Tumor Growth

This tumor line arose spontaneously as carcinoma of the lung in a C57BL/6 mouse (Malave et al, J. Nafl. Cane. Inst. 1979, 62:83-88). It is propagated by passage in C57BL/6 mice by subcutaneous (sc) inoculation and is tested in semiallogeneic C57BL/6 x DBA/2 F₁ mice or in allogeneic C3H mice. Typically six animals per group for subcutaneously (sc) implant, or ten for intramuscular (im) implant are used. Tumor may be implanted sc as a 2-4 mm fragment, or im or sc as an inoculum of suspended cells of about 0.5-2 x 10⁶-cells. Treatment begins 24 hours after implant or is delayed until a tumor of specified size (usually approximately 400 mg) can be palpated. The test compound is administered ip daily for 11 days

Animals are followed by weighing, palpation, and measurement of tumor size. Typical tumor weight in untreated control recipients on day 12 after im inoculation is 500-2500 mg. Typical median survival time is 18-28 days. A positive control compound, for example cyclophosphamide at 20 mg/kg/injection per day on days 1-11 is used. Results computed include mean animal weight, tumor size, tumor weight, survival time. For confirmed therapeutic activity, the test composition should be tested in two multi-dose assays. 3LL Lewis Lung Carcinoma: Primary Growth and Metastasis Model

This assay is well known in the art (Gorelik et al, J. Nafl. Cane. Inst. 1980, (55:1257-1264; Gorelik et al, Rec. Results Cane. Res. 1980, 75:20-28; Isakov et al, Invasion Metas. 2:12-32 (1982); Talmadge etal, J. Nafl Cane. Inst. 1982, (59:975-980; Hilgard et al, Br. J. Cancer 1977, 35:78-86). Test mice are male C57BL/6 mice, 2-3 months old. Following sc, im, or intra-footpad implantation, this tumor produces metastases, preferentially in the lungs. With some lines of die tumor, the primary tumor exerts anti-metastatic effects and must first be excised before study of the metastatic phase (see also U.S. Patent No. 5,639,725).

Single-cell suspensions prepared from solid tumors by trypsinization are washed and suspended in PBS. Viability of 3LL cells prepared in this way is generally about 95-99%. Viable tumor cells (3 x 10⁴ - 5 x 10⁶) suspended in 50 μl PBS are injected sc, either in the dorsal region or into one hind foot pad of C57BL/6 mice. Visible tumors appear 3-4 days after dorsal sc injection of 10⁶ cells. The day of tumor appearance and the diameters of established tumors are measured every two days. The treatment is given as one to five doses of peptide or analogue per week. In another embodiment, the peptide is delivered by osmotic minipump. In experiments involving tumor excision of dorsal tumors, when tumors reach about 1500 mm³ in size, mice are randomized into two groups: (1) primary tumor is completely excised; or (2) sham surgery is performed and the tumor is left intact. Although tumors from 500-3000 mm³ inhibit growth of metastases, 1500 mm³ is the largest size primary tumor that can be safely resected with high survival and without local regrowth. The phenomenon of acceleration of metastatic growth following excision of local tumors had been repeatedly observed (see, for example, U. S. Pat. No. 5,639,725). These observations have implications for the prognosis of patients who undergo cancer surgery. After 21 days, all mice are sacrificed and autopsied. Lungs are removed, weighed and fixed in Bouin's solution and the number of visible metastases is recorded as is the diameters of the metastases. On the basis of the recorded diameters, one calculates the volume of each metastasis. To determine the total volume of metastases per lung, the mean number of visible metastases is multiplied by the mean volume.

It is also possible to measure incorporation of ¹²⁵IdUrd into lung cells (Thakur et al, J. Lab. Clin. Med. 1977, §9:217-228). Ten days following tumor amputation, 25μg of fluorodeoxyuridine is injected i.p. into tumor-bearing (and, if used, tumor-resected mice). After 30 min, mice are given lμCi of ¹²⁵IdUrd (iododeoxyuridine). One day later, lungs and spleens are removed and weighed, and the degree of ¹²⁵IdUrd incorporation is measured using a gamma counter.

In mice with footpad tumors, when tumors reach about 8-10 mm in diameter, mice are randomized into two groups: (1) legs with tumors are amputated after ligation above the knee joints; or (2) mice are left intact as nonamputated tumor-bearing controls. (Amputation of a tumor-free leg in a tumor-bearing mouse has no known effect on subsequent metastasis, ruling out possible effects of anesthesia, stress or surgery.) Mice are killed 10-14 days after amputation. Metastases are evaluated as above.

Statistics: Values representing the incidence of metastases and their growth in the lungs of tumor-bearing mice are not normally distributed. Therefore, non-parametric statistics such as the Mann- Whitney U-Test may be used for analysis.

Use of PHSCN Peptides, Analogues and Salts in Therapy

An Ac-PHSCN-NH₂ salt, or pharmaceutical compositions thereof, will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent diseases or disorders characterized by aberrant vascularization or aberrant angiogenesis, the Ac-PHSCN-NH₂ salts which may be in pharmaceutical compositions, are administered or applied in a therapeutically effective amount. The amount of a Ac-PHSCN-NH₂ salt that will be effective in the treatment of a particular disorder or condition disclosed herein will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques known in the art. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal doses or dose ranges. The amount of an Ac-PHSCN-NH₂ salt administered will, of course, be dependent on, among other factors, the subject being treated, the weight of the subject, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. For example, the dosage may be delivered in a pharmaceutical composition by a single or multiple administrations, or by controlled release. Dosing may be repeated intermittently, may be performed alone or in combination with other drugs and may be continued as long as required for effective treatment of the disease or disorder. Suitable dosage ranges for oral administration are dependent on the potency of the drug, but are generally 0.001 mg to 200 mg, preferably 0.01 mg to 50 mg, more preferably, 0.1 to 50 mg, of a compound of the invention per kilogram body weight. Dose ranges may be readily determined by methods known to those of ordinary skill in the art. Suitable dose ranges for i.v. administration are about 0.01 mg to about 100 mg per kg body weight. Suitable dosage ranges for intranasal administration are generally 0.01 mg/kg body weight to 50 mg/kg body weight or 0.10 mg/kg body weight to 10 mg/kg body weight. Suppositories generally contain about 0.01 mg to about 50 mg of the compound of the invention per kg body weight and comprise about 0.5% to about 10% by weight the active ingredient. Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual or intracerebral administration are in the range of about 0.001 mg to about 200 mg per kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well-known in the art. In a specific embodiment, the dose administered is not based on body weight, but is an absolute amount, for example, in the range of 1 mg to 1 g per dose. In another specific embodiment, the dose is 10 -750 mg per dose, e.g., 20 mg, 100 mg, or 600 mg per dose. In a specific embodiment, the dose is administered from one to several (e.g., 2, 3, 4, or 7) times per week.

The Ac-PHSCN-NH₂ salts are preferably assayed in vitro and in vivo, as described above for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays can be used to determine whether administration of an Ac-PHSCN-NH₂ salt or a combination of Ac- PHSCN-NH₂ salts is preferred for treating diseases characterized by aberrant angiogenesis or vascularization. Safety and efficacy of the Ac-PHSCN-NH₂ salts may be demonstrated using animal model systems. Preferably, a therapeutically effective dose of a Ac-PHSCN-NH₂ salt described herein will provide therapeutic benefit without causing substantial toxicity. Toxicity of Ac-PHSCN- NH₂ salts may be determined using standard pharmaceutical procedures and may be readily ascertained by the skilled artisan. In the treatment of diseases or disorders, the "therapeutic index" (ratio between a toxic and a therapeutic dose) of an Ac-PHSCN-NH₂ salt will preferably be high. The preferred dose of an Ac-PHSCN-NH₂ salt described herein will preferably result in a range of circulating concentrations of the agent that are effective but with little or no toxicity.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE I Preparation of Ac-PHSCN-NH?. hydrochloride salt

1) 20% piperidine/DMF (3 x 3 min)

2) Fmoc-Asn(trt)-OH (3 eq), HBTU (3 eq), HOBt (3 eq), NMM (6 eq), DMF, 1h

FmocHN Fmoc-His(trt)-Ser(trt)-Cys(trt)-Asn(trt) — Q

3) Repeat step 2

Rink Amide 4) Repeat steps 1 , 2 & 3 for each AM resin amino acid (Fmoc-Cys(trt)-OH, Fmoc-Ser(trt)-OH, Fmoc-His(trt)-OH) 1 ) 20% piperidine/DMF (3 x 3 min)

2) Ac-Pro-OH (3 eq), HBTU (3 eq), HOBt (3 eq), NMM (6 eq), DMF, 1h

TFA/TIS/H₂O (95:2.5:2.5), 1 h

Ac-Pro-His-Ser-Cys-Asn-NH₂ ^«TFA Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt) — φ

Amberlyst A-26 OH Resin 1 eq HCI

Ac-PHSCN-NH₂ ^«TFA Ac-PHSCN-NH₂ Ac-PHSCN-NH₂ • HCI distilled water 5 min ATN-161.030 EXAMPLE π

Preparation and Purification of Ac-Pro-His-Ser-Cys-Asn-NEk TFA salt

Rink Amide AM resin (Novabiochem) was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for three minutes with nitrogen agitation and the reaction mixture was filtered and washed once with DMF. This step was repeated an additional two times. The resin was washed three times with DMF and three times with dichloromethane. Fmoc-Asn(trt)-OH (3 eq), HBTU (3 eq), and HOBt (3 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-methylmorpholine (ΝMM) (6 eq) and the mixture was agitated for 1 hour. The reaction mixture was filtered and the resin was washed three times with DMF and three times with dichloromethane. This coupling step was repeated. The Fmoc deprotection and the coupling steps described above were sequentially used with Fmoc-Cys(trt)-OH, Fmoc-Ser(trt)-OH and Fmoc-His(trt)-OH to afford Fmoc-His(trt)-Ser(trt)-Cys(trt)-Asn(trt) bound to the resin. This resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for three minutes with nitrogen agitation and the reaction mixture was filtered and washed once with DMF. This step was repeated an additional two times. The resin was washed three times with DMF and three times with dichloromethane. Ac-Pro-OH (3 eq), HBTU (3 eq), and HOBt (3 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-methylmorpholine (ΝMM) (6 eq) and the mixture was agitated for 1 hour. The reaction mixture was filtered and the resin was washed three times with DMF and three times with dichloromethane to afford Rink Amide AM resin bound Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt). The resin was treated with

TFA/TIS/water (95:2.5:2.5, 1 mL per 100 mg of resin) and agitated with nitrogen for 2 hours. The reaction mixture was filtered, and the resin was washed once with TFA/TIS/water and three times with dichloromethane. The solvent was removed in vacuo and the resulting residue was triturated three times with ether to afford crude Ac-Pro-His-Ser-Cys-Asn-ΝH₂, TFA salt. Using this general procedure, 708 mg of crude Ac-PHSCN-NH₂, TFA salt was prepared from 2 grams of Rink Amide AM resin (loading: 0.63 mmol/g). Purification

The crude peptide, dissolved in a minimum amount of methanol and water, was purified by preparative reverse phase HPLC (Beckman) with a Phenomenex Synergi hydro-RP C18 column (250mm x 21.2 mm). The peptide was eluted using a gradient from 3-100% B over 30 min with a flow rate of 20 mL/min, where solvent A was water containing 0.1% trifluoroacetic acid and solvent B was acetonitrile containing 0.1% trifluoroacetic acid. Detection was at 220 nm. Fractions >95% pure by analytical HPLC analysis (Phenomenex hydro RP (250mm x 4.6mm) using gradient 3-100% B) (Waters) were combined, concentrated to a volume of about 2-4 ml by rotary evaporation, and lyophilized. Samples were redissolved in water and transferred to a tared 2 dram vial and lyophilized a second time. Using this method, 140 mg of pure Ac-PHSCN-NH₂, TFA salt was obtained from 338 mg of crude material: ES MS tn/z (MH-H)⁺ 598.2; HPLC: 99% pure.

EXAMPLE m Preparation of Ac-Pro-His-Ser-Cys-Asn-NHg

Ac-Pro-His-Ser-Cys-Asn-NH₂, TFA salt (140 mg, 0.197 mmol) was dissolved in 2 mL of distilled water and Amberlyst A-26 (OH) resin (4.2 meq/g, 273 mg, 5.8 eq) was added. The reaction mixture was stirred at room temperature for 5 minutes. The aqueous solution was decanted, the resin was washed twice with distilled water, and the combined aqueous layers were lyophilized to afford 81 mg (69%) Of Ac-PHSCN-NH₂ as a fluffy, white solid: ES MS m/z (M+H)⁺ 598.2; HPLC: 94% monomer, 6% dimer.

EXAMPLE IV

Preparation of Ac-Pro-His-Ser-Cys-Asn-NEh_' hydrochloride salt

Ac-Pro-His-Ser-Cys-Asn-NH₂ (77 mg, 0.13 mmol) was dissolved in 3 mL of distilled water at room temperature and 1 M hydrochloric acid (0.13 mL, 0.13 mmol) was added immediately. The mixture was swirled once and then frozen and lyophilized to afford Ac-PHSCN-NH₂, hydrochloride salt as a fluffy white solid: ¹H NMR (300 MHz, DMSO-d6) δ 9.00 (s, IH), 8.57-8.26 (m, 2H), 8.21- 8.03 (m, 2H), 7.45-7.36 (m, 2H), 7.13 (s, IH), 7.09 (s, IH), 6.94 (s, IH), 4.79-4.59 (m, IH), 4.50- 4.25 (m, 4H), 3.74-3.56 (m, 3H, overlapping with water peak), 3.25-3.15 (m, 2H, overlapping with water peak), 3.11-2.98 (m, IH), 2.88-2.72 (m, 2H), 2.57-2.38 (m, 2H, overlapping with DMSO peak), 2.02 (s, 3H), 1.92-1.65 (m, 4H); HPLC: 93% monomer, 7% dimer. EXAMPLE V

Materials and Methods in Formulating and Testing

Formulations

Ac-PHSCN-NH₂, 50 mg/ml, was formulated in solutions that included the 5OmM Citrate and the ingredients shown in Table 1, below. The solutions were lyophilized and dispensed into vials for reconstitution in 1 mL water. TABLE 1

Testing of Stability

Accelerated Stability was tested by storage of the solution for 4 day at 40°C and 34 days at 55°C. Accelerated stability experiments are performed using extreme conditions of temperature and/or humidity to force degradation and to quickly test the relative stability of a particular formulation (typical storage conditions are 22°, 4°, or -20°C for most drugs). This data then provides the ability to select a formulation that will most likely be the most stable from a number of test formulations. After reconstitution with water each formulation was examined by reverse phase HPLC using a standard mobile phases such as water/methanol or water/acetonitrile. This methodology allows the separation of Ac-PHSCN-NH₂ from Ac-PHSCN-NH₂ degradation products such as fragments of Ac-PHSCN-NH₂ or the disulfide-bonded dimer.

Calculation of Potency

Potency was calculated as the area under the Ac-PHSCN-NH₂ peak in the HPLC chromatogram vs. a standard curve. Calculation of Purity

Purity was calculated as the area under the Ac-PHSCN-NH₂ peak as a percentage of the integrated area of entire chromatogram.

Calculation of % Dimer

The relative amount OfAc-PHSCN-NH₂ dimer was calculated as the area under the dimer peak as a percentage of the integrated area of entire chromatogram.

EXAMPLE VI Stability of Various Formulations of Ac-PHSCN-NH;

The stability of the peptide formulations were expressed in three ways: Potency (mg/mL) Purity (% monomer and % dimer). The results are shown in Table 2 and Table 3 below. Table 3 shows the potency normalized to that of the solution at t=0; the columns indicated with an N show the normalized values.

TABLE 2

I ³OTENCY mg/mL

Solution # Prior to Lyo^* t(0) 1 day 5 days 9 days 17 days

1 50 50.4 48.9 49.5 48.3 48.5

2 50 49.9 48.7 49.7 50.8 47.0

3 50 49.8 50.0 49.8 49.8 46.6

4 50 50.8 50.7 50.1 49.9 45.6

5 50 50.4 50.9 50.4 50.1 47.4

6 50 50.2 50.2 49.9 49.7 46.2

PURITY %Monomer

Solution # Prior to Lyo* t(0) 1 day 5 days 9 days 17 days

1 95 95.2 94.6 94.5 94.2 94.1

2 95 95.1 95.0 95.0 94.5 94.5

3 95 94.8 94.9 94.7 95.0 94.6

4 95 95.0 94.9 95.2 94.7 94.5

5 95 95.0 95.0 95.2 94.6 94.3

6 95 95.3 94.8 94.8 94.6 93.2

PURITY %Dimer

Solution # Prior to Lyo* t(0) 1 day 5 days 9 days 17 days

1 0.9 0.7 0.8 0.9 1.1 1.3

2 0.9 0.9 0.7 0.7 1.0 1.2

3 0.9 1.0 0.8 0.8 0.8 1.1

4 0.9 0.9 0.8 0.7 1.0 1.4

5 0.9 0.8 0.8 0.8 1.0 1.3

6 0.9 0.7 0.8 0.7 1.0 1.8

* Lyo=lyophilization

TABLE 3

Formulation/Solution #

Time

(days) 1 1 N* 2 2N 3 3N 4 4N 5 5N 6 6N

0 50.4 100.0 49.9 100.0 49.8 100.0 50.8 100.0 50.4 100.0 50.2 100.0

1 48.9 97.0 48.7 97.4 50.0 100.4 50.7 99.9 50.9 101.0 50.2 99.9

5 49.5 98.1 49.7 99.5 49.8 100.1 50.1 98.6 50.4 99.9 49.9 99.3

9 48.3 95.9 50.8 101.7 49.8 99.9 49.9 98.1 50.1 99.4 49.7 98.8

17 48.5 96.2 47.0 94.0 46.6 93.6 45.6 89.8 47.4 94.1 46.2 91.9 ^r = N=normalized EXAMPLE Vn

Selection of Preferred Formulation(s)

The following summarizes the characteristics of the peptide formulations

A. The formulation in Solutions 1-4 were characterized as follows: 1. Inconsistent Cake shape, a majority collapsed in the lyophilizer

2. Loss of cake volume during accelerated stability.

3. Unreasonable reconstitution times for collapsed cakes.

B. The formulation in Solution 6 was characterized as follows:

Color changed to brown during accelerated stability most likely due to a reaction between glucose and glycine.

C. The formulation in Solution 5 was characterized as follows:

1. Good quality cake, white in color, rapid reconstitution and low moisture content.

The composition of solution 5 is 50mg/mL of the peptide, 50mg/mL glycine, 5OmM citrate, pH 5.0 , lyophilized (ImL). 2. Robust performance in the lyophilizer, uniform appearance with no collapse in any sample.

3. Maintained appearance and reconstitution ability during accelerated stability.

4. Maintained stability that was equal to (+2%) or better than other formulations.

EXAMPLE Viπ Real time stability data indicated that the preferred intravenous formulation (100 mg

Ac-PHSCN-NH₂ (=ATN-161) 50 niM citrate 50 mg/mL glycine, pH 5.0, lyophilized from a 2 mL solution of 50 mg/mL ATN-161) remained within specifications (i.e., >90% ATN- 161) for 3 years when stored at -20°C or at 2-8°C. It was decided based on these results that a low pH formulation with sufficient bulking agent (about 50 mg/ml glycine) and low moisture content in the vial after lyophilization would yield the desired quality and stability for the ATN-161 drug product. Moisture content can be varied by optimizing the lyophilization cycle using methods well-known in the art. Similar parameters would be followed for developing additional formulations for ATN-161 or any of its acid addition salts or analogues. All the references cited above are incorporated herein by reference in their entirety, whether specifically incorporated or not. Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising

(a) a peptide Pro-His-Ser-Cys-Asn, an analogue thereof, or a salt of the peptide or of the analogue, which is formulated with (b) at least one additional compound that stabilizes the peptide, analogue or salt against spontaneous tandem dimerization or higher oligomerization.

2. The composition of claim 1 wherein the peptide is capped at its N- and C-termini with an N-terminal cap and a C-terminal cap, respectively.

3. The composition of claim 2 wherein the N-terminal cap is an acyl group and the C terminal cap is an amide group.

4. The composition of claim 3 wherein the N-terminal cap is an acetyl group.

5. The composition of any of claims 1-4 wherein the additional compound inhibits, prevents or reverses disulfide bond formation between sulfhydryl groups of Cys residues.

6. The composition of any of claims 1-4 wherein the additional compound is a biocompatible acid buffer with a pK of about 5.

7. The composition of claim 3 wherein in the presence of the buffer, the pH of the solution is greater than 3.0 and less than, or equal to, 7.5.

8. The composition of claim 6, wherein the acid buffer is citrate, acetate or 2-(N- rnorpholino)ethanesulfonic acid (MES).

9. The composition of claim 8 wherein the acid buffer is citrate at a concentration of about 25 mM.

10. The composition of any of claims 6-9, wherein the buffer is supplemented with glycine as an excipient and bulking agent.

11. The composition of claim 10 wherein the concentration of glycine is about 50 mg/ml.

12. The composition of any of claims 6-9, wherein the buffer comprises citrate and acetate.

13. The composition of any of claims 6-8 wherein the buffer also comprises Tris.

14. The composition of any of claims 1-13 that comprises (i) the peptide or salt of the peptide or analogue, (ii) about 50 mM citrate, and (iii) about 50 mg/ml glycine.

15. The composition of claim 14, wherein the composition is in a container or vial in lyophilized form having 100 mg peptide or salt of the peptide or analogue, 50 mM citrate, 50 mg/ml glycine lyophilized from 2 mL of a pH 5.0 solution.

16. The composition of any of claims 1-15 further comprising one or more reducing agents.

17. The composition of claim 16 wherein said reducing agents comprise dithiothreitol, β-mercaptoethanol or glutathione.

18. The composition of claim 17 wherein the concentration of the reducing agent or agents does not exceed about 10 mM.

19. The composition of any of claims 16-18 further comprising a non- thiol biocompatible anti-oxidant.

20. The composition of any of claims 1-19 that comprises a lyoprotectant present in an lyoprotecting amount.

21. The composition of claim 20 wherein the molar ratio of lyoprotectant to peptide is about 50-600 mole lyoprotectant to 1 mole peptide.

22. The composition of claim 20 or 21, wherein the lyoprotectant is one or more sugars, one or more amino acids, one or more rnethylamines, one or more lyotropic salts, and/or one or more polyols.

23. The composition of any of claims 20-22, wherein the lyoprotectant is sucrose or trehalose; monosodium glutamate or histidine; betaine; magnesium sulfate; or a trihydric or higher sugar alcohol.

24. The composition of claim 22 or 23 comprising one or more polyols selected from the group consisting of glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, , polyethylene glycol, and a combination thereof.

25. The composition of claim 22 wherein the lyoprotectant is a non-reducing sugar.

26. The composition of claim 25 wherein the sugar is trehalose or sucrose.

27. The composition of any of claims 1-26 which is sterile and formulated for in vivo administration.

28. An article of manufacture or kit comprising

(a) a first container which contains a composition according to any of claim 1-27 in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstituting solution for the lyophilized composition; and

(c) optionally, instructions for (i) use of the solution or (ii) reconstitution and/or use of the lyophilized composition.

29. The article or kit of claim 28, further comprising one or more of (i) another buffer, (ii) a diluent, (iii) a filter, (iv) a needle, or (v) a syringe.

30. The article or kit of claim 28 wherein the first and optional second container is a bottle, a vial, a syringe or test tube.

31. The article or kit of claim 28 wherein the first and optional second container is a multi-use container.

32. The article or kit of any of claims 28-31 wherein the composition is in lyophilized form.

33. A method of inhibiting angiogenesis in a subject, comprising administering to the subject the composition of any of claims 1-27, wherein the peptide or analogue is administered in an anti-angiogenic effective amount.

34. A method for treating cancer in a subject by inhibiting angiogenesis, comprising administering to the subject the composition of any of claims 1-27, wherein the peptide or analogue is administered in a cancer-therapeutic effective amount.

35. A method for treating Crohn's disease in a subject by inhibiting angiogenesis, comprising administering to the subject the composition of any of claims 1-27, wherein the peptide or analogue is administered in a Crohn's disease- therapeutic effective amount.

36. Use of a composition according to any of claims 1-27 in a medicament for administration to a subject to inhibit undesired angiogenesis.

37. Use according to claim 36 for administration to a subject with cancer to treat said cancer.

38. Use according to claim 36 for administration to a subject with Crohn's disease to treat said disease.

39. Use according to any of claims 36-38, wherein the peptide, analogue, or salt of the peptide or analogue is administered in an anti-angiogenic effective amount.

40. Use of a composition according to any of claims 1-27 in the manufacture of a medicament for administration to a subject to inhibit undesired angiogenesis.

41. Use according to claim 41 in the manufacture of a medicament for administration to a subject with cancer to treat said cancer.

42. Use according to claim 41 in the manufacture of a medicament for administration to a subject with Crohn's disease to treat said disease.

43. Use according to any of claims 40-42 wherein the peptide, analogue, or salt of the peptide or analogue is administered in an anti-angiogenic effective amount.