JP2008528630A - Composition comprising anti-angiogenic PHSCN peptide - Google Patents

Abstract

Cys-containing anti-angiogenic peptide Pro-His-Ser-Cys-Asn (preferably capped as Ac-PHSCN-NH ₂ ) or an acid addition salt or analog thereof, and the peptide or analog spontaneously Disclosed are compositions / formulations containing at least one additional compound that stabilizes from stable tandem dimerization or higher oligomerization. A preferred formulation comprises an acidic buffer such as citric acid, an excipient and glycine as a bulking agent. Additional optional ingredients of the formulation include reducing agents, biocompatible non-thiol antioxidants, cryoprotectants (typically one or more sugars, one or more amino acids, one or more methylamines, one A lyotropic salt and / or one or more polyols). Further provided is a product or kit comprising the formulation in solution or lyophilized form. Also disclosed is a method for inhibiting angiogenesis in a subject comprising administering the peptide as a formulation to the subject.

Description

  The present invention relates to a composition comprising an improved formulation of the peptide that prevents degradation and spontaneous oxidative dimerization or oligomerization of the Cys-containing anti-angiogenic peptide Pro-His-Ser-Cys-Asn .

  Most forms of cancer appear as solid tumors or are derived from solid tumors (Shockley et al., Ann. N.Y. Acad. Sci. 1991, 617: 367-82). This type of tumor is generally known to be clinically resistant to biological therapeutic agents such as monoclonal antibodies and immunotoxins. Anti-angiogenic therapies to treat cancer were developed from the recognition that solid tumors require angiogenesis (ie, the formation of new blood vessels) for sustained growth (Folkman, Ann. Surg. 1972, 175: 409-16; Folkman, Mol. Med. 1995, 1: 120-22; Folkman, Breast Cancer Res. Treat. 1995, 36: 109-18; Hanahan et al., Cell 1996, 86: 353-64). The efficacy of anti-angiogenic therapy in animal models has been demonstrated in many studies, for example, Milluerer et al., 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 Ther. 2002, 1: 327-37; van Golen KL et al., Neoplasia 2002, 4: 373-9; Cox C et al., Arch See Otolaryngol Head Neck Surg. 2003, 129: 781-5. Without angiogenesis, the solid cell inner cell layer does not grow well. Furthermore, angiogenesis (ie, abnormal neovascularization) has now been shown to be required for the growth of non-solid hematological tumors, and many other diseases (ocular neovascularization) Disease, macular degeneration, rheumatoid arthritis, etc.).

  In contrast, normal tissue does not require angiogenesis except for angiogenesis under special circumstances such as wound repair and proliferation of the uterine lining during the menstrual cycle. Thus, the need 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 the differences in cell replication and cell death. The latter difference is commonly used to treat cancer.

  Tumor angiogenesis is a cytokine such as vascular endothelial growth factor (VEGF) and / or fibroblast growth factor (FGF), which binds to specific receptors on endothelial cells (EC) of the local vasculature Can be initiated under hypoxic conditions. Activated EC secretes enzymes that remodel the associated tissue matrix and regulate the expression of adhesion molecules such as integrins. After matrix degradation, the EC grows and migrates towards the tumor, resulting in the formation and maturation of new blood vessels.

  Interestingly, protein fragments such as endostatin, kringle 5, and PEX that inhibit angiogenesis are produced by degradation of matrix proteins (O'Reilly et al., Cell 1997, 88: 277-85; O'Reilly et al., Cell, 1994, 79: 315-28; Brooks et al., Cell, 1998, 92: 391-400). Thus, these protein fragments may inhibit new angiogenesis and thus prevent tumor growth and metastasis. However, these protein fragments have significant difficulties associated with their use. They are difficult and expensive to produce in large quantities, have weak pharmacological properties, and are susceptible to degradation. One approach was to identify short peptide fragments of these larger proteins, or longer fragments or subunits that retain most of the anti-angiogenic activity of the parent protein.

Although the search for peptides that inhibit angiogenesis has provided compounds that have a considerable effect in preventing the growth of new blood vessels, molecules with excellent activity profiles are still needed. Thus, new peptides need to fully explore the peptide's potential in preventing angiogenesis and detecting abnormal neovascularization. The new peptide has a longer plasma half-life, is more resistant to degradation and has an improved biologic compared to peptides disclosed in the art (Livant, U.S. Patent Nos. 6,001,965 and 6,472,369). May have availability, higher affinity, greater selectivity, etc. Such novel peptides can be effective in inhibiting cell migration, invasion, proliferation, and in treating or preventing various diseases associated with undesirable angiogenesis and abnormal neovascularization. As an example of such a peptide, the derivative of the capped pentapeptide Ac-PHSCN-NH ₂ (also referred to herein as ATN-161) was assigned to the same assignee as the present application on November 25, 2003. US patent application Ser. No. 10 / 723,144 (published as Allan et al., US 20040162239A1) and US Ser. No. 10 / 722,843 (published as Ternansky et al., US 2005002081OAl) on Nov. 25, 2003, all of which are incorporated herein by reference. Is incorporated herein by reference.

What is needed in the art is a method of preventing degradation of Ac-PHSCN-NH ₂ in both the liquid phase and solid phase. One approach to improving the activity profile of anti-angiogenic peptides is to develop their formulation methods as a means of extending shelf life and increasing resistance to degradation. In the case of peptides containing Cys, it is also important to prevent spontaneous oxidative dimerization or higher oligomerization or polymerization through disulfide bond formation. The present invention relates to such improved formulations for the anti-angiogenic peptide Ac-PHSCN-NH ₂ and its anti-angiogenic derivatives.

  We conserve the biological / biochemical potency of Cys-containing peptides (especially by inhibiting the formation of disulfide bonds that lead to undesired dimerization) of Cys-containing peptides and their salts. A composition comprising an improved formulation was developed.

Ac-PHSCN-NH ₂ acid addition salts useful in the formulations of the present invention are described in a co-pending patent application by Trenansky et al. These applications are US Provisional Application No. 60 / 649,308 dated February 1, 2005 entitled “Ac-PHSCN-NH ₂ Acid Addition Salt” and International Application (No TBD) dated February 1, 2006 (Attorney) No. 9715-022-228), the entirety of which is incorporated herein by reference. The above application, Ac-PHSCN-NH ₂ an acid addition salt, the acid addition process for the preparation of salts of Ac-PHSCN-NH _2, a pharmaceutical composition comprising an acid addition salt of Ac-PHSCN-NH _2, angiogenesis and abnormal A method for using an acid addition salt of Ac-PHSCN-NH ₂ and a pharmaceutical composition thereof for the treatment of diseases associated with new neovascularization, and a method for preventing degradation of Ac-PHSCN-NH ₂ by salt formation Disclosure. Therefore, the novel formulations of the present invention include formulations containing salts described by Trenansky et al. (Supra).

  Thus, the present invention relates to the peptide Pro-His-Ser-Cys-Asn (PHSCN), its analogs, or salts of the peptide or analog, and the peptide or analog spontaneously tandem dimerization or It relates to a formulation comprising at least one further compound that stabilizes against a higher degree of oligomerization. Preferably, the peptide is capped at both ends, capped by an acetyl group at the N-terminus and by an amide group at the C-terminus. Further compounds are those that inhibit, prevent or reverse the formation of disulfide bonds between sulfhydryl groups of Cys residues.

  Also provided is a formulation as described above wherein the additional compound is a biocompatible acid buffer having a pK of about 5.

  Preferably, in the presence of the buffer, the pH of the solution is greater than about 3.0 and equal to or less than 7.5. A preferred acid buffer is citric acid, acetic acid, or 2- (N-morpholino) ethanesulfonic acid (MES).

  In the above formulation, the acid buffer is preferably citric acid at a concentration of about 25 mM. The buffer may contain glycine as an excipient and bulking agent at a concentration of about 50 mg / ml. The buffer may include citric acid and acetic acid, and may include Tris.

  In a preferred embodiment, the formulation comprises (i) a peptide, a capped peptide or analog, (ii) about 50 mM citric acid, and (iii) about 50 mg / ml glycine.

  The formulation of the preferred embodiment is lyophilized from 2 mL of pH 5.0 solution in a lyophilized form with 100 mg peptide, capped peptide, salt or analog, 50 mM citric acid, 50 mg / ml glycine. Or present in a vial.

  The above formulation can further comprise one or more reducing agents, suitable reducing agents are dithiothreitol, β-mercaptoethanol, or glutathione. Preferably, the concentration of the one or more reducing agents does not exceed about 10 mM.

  The above preparation may contain a biocompatible non-thiol antioxidant.

  The formulation can include a lyoprotectant in a cryoprotective amount, for example, in an amount of about 50-600 moles of cryoprotectant per mole of peptide. Cryoprotectants include one or more saccharides, one or more amino acids, one or more methylamines, one or more lyotropic salts, and / or one or more polyols. In the preferred formulations described above, (a) the sugar is sucrose or trehalose, (b) the amino acid is monosodium glutamate or histidine, (c) methylamine is betaine, and (d) lyotropic. The salt is magnesium sulfate, and (e) the polyol is a trihydric or higher sugar alcohol.

  The above formulation may comprise one or more polyols selected from the group consisting of glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, propylene glycol, and combinations thereof. In some formulations, the cryoprotectant is a non-reducing sugar and trehalose or sucrose is preferred.

In all the above formulations, the peptide is preferably capped at its N-terminus and C-terminus, the N-terminal cap is an acyl group, the C-terminal cap is an amide group, and the peptide is CO-NH Most preferably it ends with ₂ .

  The above formulations are preferably sterile and are formulated for in vivo administration.

The present invention
(a) a container containing the above formulation in solution or lyophilized form,
(b) optionally a second container containing a diluent or reconstitution liquid for the lyophilized formulation, and
(c) optionally (i) use of the solution, or (ii) instructions for re-preparation and / or use of the lyophilized formulation,
A product or kit comprising

  The product 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, vial, syringe, or test tube. It may be a multi-use container and the formulation is preferably lyophilized.

  The present invention relates to a method for inhibiting angiogenesis in a subject, comprising administering the above-mentioned preparation to the subject, wherein the peptide, analog or salt is administered in an amount effective to inhibit angiogenesis. Is done. Such inhibition of angiogenesis has been utilized in methods of treating cancer and / or Crohn's disease. The present invention provides the use of the above composition in a medicament to be administered to a subject to suppress unwanted angiogenesis, such as the treatment of cancer or Crohn's disease. Further included is the use of the above composition in the manufacture of a medicament to be administered to a subject to inhibit unwanted angiogenesis and thereby treat cancer or Crohn's disease.

We have the Cys-containing peptide PHSCN, most preferably acetyl-PHSCN-NH ₂ , which is a terminal-capped pentapeptide, and various salts thereof, and / or capped or uncapped PHSCN. Improved formulations for analogs and salts of the derivatives and analogs have been developed. Various other capping groups are described below. An “analog” of PHSCN refers to a natural or non-natural molecule that is substantially structurally and / or functionally similar to PHSCN. Examples include conservative amino acid substitution variants, addition variants of up to about 20 residues (excluding the natural polypeptide or its structural domain), and chemically modified peptides. Other analogs include peptidomimetics and aptamers.

  The peptide to be formulated is preferably pure or essentially pure, and desirably essentially homogeneous (ie, free from contaminating peptides, proteins, etc.). “Essentially pure” means a peptide preparation in which 90% by weight or more (preferably 95% by weight or more) is the peptide based on the total weight of the preparation. By “essentially homogeneous” preparation is meant a peptide preparation comprising 99% or more by weight of peptide, based on the total weight of the peptide in the preparation.

As described above, the PHSCN peptide preferably has its N-terminal and C-terminal capped with an acyl (abbreviated as “Ac”) group and an amide (abbreviated as “Am”) group, For example, the N-terminus is capped with an acetyl (CH ₃ CO—) group and the C-terminus is capped with an amide (CO—NH ₂ ) group.

N-terminal capping groups A wide range of N-terminal capping groups (preferably attached to the terminal amino group) are envisioned, for example:
Formyl;
C _1-10 alkanoyl, such as acetyl, propionyl, butyryl;
C _1-1O alkenoyl, such as hexa-3-enoyl;
C _1-10 alkinoyl, those having _1-10 carbon atoms, such as hexa-5-inoyl;
Aroyl, such as benzoyl or 1-naphthoyl;
Heteroaroyl, such as 3-pyroyl 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 _1-10 substituted alkenoyl, such as 6-hydroxy-hex-3-enoyl;
C _1-10 substituted alkinoyl, such as 3-hydroxy-hex-5-inoyl;
Substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphth-2-oil;
Substituted heteroaroyl such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazoline-6-oil;
Substituted alkylsulfonyl, such as 2-aminoethanesulfonyl;
Substituted arylsulfonyl, such as 5-dimethylamino-1-naphthalenesulfonyl;
Substituted heteroarylsulfonyl, such as l-methoxy-6-isoquinolinesulfonyl;
Carbamoyl or thiocarbamoyl;
Substituted carbamoyl (R'-NH-CO) or substituted thiocarbamoyl (R'-NH-CS), where 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), where R 'is alkanoyl, alkenoyl, alkinoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkinoyl, substituted aroyl, Or substituted heteroaroyl.

C-terminal capping group
The C-terminal capping group is either amide linked to the terminal carboxyl or ester linked. A capping group that provides an amide bond is described as NR ¹ R ² , where R ¹ and R ² are independently selected from the following groups:
hydrogen;
C _1-10 alkyl, such as methyl, ethyl, isopropyl;
C _1-10 alkenyl, such as prop-2-enyl;
C _1-10 alkynyl, such as prop-2-ynyl;
C _1-10 substituted alkyl such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, halogenalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl;
C _1-10 substituted alkenyl such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, halogen alkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl;
C _1-10 substituted alkynyl, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, halogen alkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, carboxyalkynyl, carbamoylalkynyl;
C _1-10 aroylalkyl, such as phenacyl or 2-benzoylethyl;
Aryl, such as phenyl or 1-naphthyl;
Heteroaryl, such as 4-quinolyl;
C _1-10 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 SO ₂ —R ′ ″ or SO—R ″. Where R ′ ″ is a substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl.

  A capping group that provides an ester linkage is described as OR, where R can be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted aryloxy; Substituted aralkyloxy; or substituted heteroaralkyloxy.

  Either or both of the N-terminal or C-terminal capping groups may be of a structure such that the capped molecule functions as a prodrug (a physiologically inactive derivative of the parent drug molecule). Prodrugs undergo spontaneous or enzymatic transformations in the body to release the active drug and have improved delivery characteristics compared to the parent drug molecule (Bundgaard H: Design of Prodrugs, Elsevier, Amsterdam, 1985).

  It is possible to add other active molecules to the peptide by appropriate selection of the capping group. For example, the presence of a sulfhydryl group linked to an N- or C-terminal cap will allow conjugation of the derivatized peptide with other molecules.

  A “lyoprotectant”, 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. It is a molecule. Exemplary cryoprotectants include: sugars such as sucrose or trehalose; amino acids such as monosodium glutamate or histidine; methylamines such as betaine; lyotropic salts such as magnesium sulfate; Polyols, such as trihydric or higher sugar alcohols, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; pluronic; and combinations thereof. A preferred cryoprotectant is a non-reducing sugar such as trehalose or sucrose. The cryoprotectant is added in a “cryoprotective amount” to the preparation prior to lyophilization, which is the amount of the peptide that has been lyophilized in the presence of a cryoprotective amount of the cryoprotectant after the peptide has been lyophilized. The amount that essentially retains physical and chemical stability and integrity during lyophilization and storage.

  A “diluent” is one that is pharmaceutically acceptable (safe and non-toxic for human administration) and is useful for preparing a formulation prepared at the time of use. Typical diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. Is mentioned.

  A “preservative” is a compound that is added to a diluent to essentially reduce bacterial activity in the reconstituted formulation, and thus, for example, the production of a reconstituted formulation for multiple use Becomes easier. Examples of preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols (eg phenol), butyl and benzyl alcohol, alkyl parabens (eg methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. included. The optimal preservative in the present invention is benzyl alcohol.

  A “bulking agent” adds mass to the lyophilized mixture to contribute to the physical structure of the lyophilized cake (eg, facilitates the production of an essentially homogeneous lyophilized cake that maintains an open pore structure). It is a compound. Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol.

  One important objective of the formulation is to stabilize these peptides or analogs against spontaneous dimerization. To accomplish this goal, the peptide is preferably formulated at a low pH that prevents the formation of disulfide bonds between the two Cys residues. Such acid buffer is preferably biocompatible. Examples include citric acid, acetic acid, 2- (N-morpholino) ethanesulfonic acid (MES), or similar buffer at about pK5, and in the presence of such buffer, the pH of the solution will be 7.5 or lower. Is preferably not lower than 3.0. A suitable acidic formulation comprises citric acid, preferably about 25 mM. It is preferable to add glycine (Gly) as an excipient and a bulking agent to the buffer. Another advantage of Gly is that it is an approved excipient suitable for intravenous infusion or injection into humans.

  Other amino acids or compounds can be used in place of Gly. Examples of desirable acids or combinations thereof are citric acid + acetic acid, acetic acid and Tris, and the like. The ultimate 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 encompasses stabilized liquid pharmaceutical compositions containing, in addition to lyophilized compositions, the peptides described herein (preferably those capped), but usually the peptides The effectiveness of as a therapeutically active ingredient decreases during storage as a liquid formulation as a result of dimerization or higher oligomerization, aggregation, and the like. A stabilized liquid pharmaceutical composition includes an amount of an amino acid base sufficient to reduce the formation of aggregates during storage, wherein such amino acid base is a single amino acid or combination of amino acids, A given amino acid exists in either its free form or its salt form. It is understood that the composition includes a buffer that maintains the pH of the liquid composition within an acceptable range for peptide stability, in which case the buffer is a substantially salt-form free acid, A salt form of an acid or a mixture of an acid and its salt form. The amino acid base helps to stabilize the peptide against aggregate formation during storage of the liquid pharmaceutical composition, while the use of an acid buffer (in either form) is nearly isotonic. This results in a liquid composition having osmotic pressure. Other stabilizers (especially methionine, nonionic surfactants such as polysorbate 80, and EDTA) may additionally be added to the liquid pharmaceutical composition to further enhance the stability of the peptide. It can be said that such a liquid pharmaceutical composition is stabilized. This is because adding a substantially salt-form-free acid, a salt-form acid, or a mixture of an acid and its salt form in combination with an amino acid base can be formulated without using a combination of these two components This is because the storage stability is improved as compared with the liquid composition. Methods for increasing the stability of peptides in liquid pharmaceutical compositions (and also for increasing the storage stability of such compositions) are sufficient to reduce aggregate formation of polypeptides during storage. Adding an amount of an amino acid base and a buffer that is a substantially salt-form free acid, a salt form acid, or a mixture of an acid and its salt form to a liquid composition.

Ac-PHSCN-NH ₂ acid addition salts (see Ternansky et al., Supra) can be formed from both organic and inorganic acids. Typical organic acids generally include carboxylic acids and sulfonic acids, such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic 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-disulfone 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, laurylsulfuric acid, gluco Acid, glutamic acid, hydroxy Na futon acid, salicylic acid, stearic acid, and muconic acid. Other organic acids are known to those skilled in the art. In some embodiments, the acid addition salt of Ac-PHSCN-NH ₂ is formed from methanesulfonic acid, acetic acid, glycolic acid, (+) camphorsulfonic acid, mandelic acid, salicylic acid, succinic acid, or combinations thereof .

Typical 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, hypophosphorous acid, Examples include nitric acid, cyanic acid, chromic acid, sulfurous acid, and hydrazoic acid. Other inorganic acids are also known to those skilled in the art. In some embodiments, the acid addition salt of Ac—PHSCN—NH ₂ is formed from hydrobromic acid, nitric acid, hydrochloric acid, phosphoric acid, or combinations thereof. In other embodiments, the acid addition salt of Ac—PHSCN—NH ₂ is formed from hydrochloric acid.

In general, the acid addition salt of Ac—PHSCN—NH ₂ may be prepared by any conventional method known to those skilled in the art. Such methods include saturating the Ac—PHSCN—NH ₂ solution with a gaseous acid, adding the acid solution to the Ac—PHSCN—NH ₂ solution, and the like. In some embodiments, an acid addition salt of Ac-PHSCN-NH ₂ is added to a solution of Ac-PHSCN-NH ₂ dissolved in distilled water by adding slightly more than 1 equivalent (eg, 1.05 equivalents) of acid. To prepare. Acid addition salts are typically isolated as solids from aqueous mixtures.

Ac-PHSCN-NH ₂ acid addition salts are generally much more stable than free bases in both solid and solution phases. Acid addition salts are believed to prevent oxidative dimerization of Ac-PHSCN-NH ₂ in which cysteine is interposed. Acid addition salts that prevent decomposition of Ac-PHSCN-NH ₂ include, for example, methanesulfonic acid, acetic acid, glycolic acid, sulfuric acid, (+) camphorsulfonic acid, mandelic acid, salicylic acid, succinic acid, hydrobromic acid, hydrochloric acid , Nitric acid, and phosphoric acid.

  The formulations of the present invention preferably contain one or more reducing agents, such as low concentrations (preferably not exceeding about 10 mM) of dithiothreitol (DTT), β-mercaptoethanol, glutathione (GSH), or Contains other Cys-containing reducing agents. Other non-thiol-containing antioxidants that are biocompatible, such as metal binding compounds (EDTA, EGTA) can also be used.

Another embodiment of the invention is directed to a formulation that increases the half-life of the peptide after parenteral injection. Examples of these are the formulation of Ac-PHSCN-NH ₂ with cyclodextrins, cremaphor, or liposome formulations.

“Cyclodextrin” refers to a cyclic molecule in which 6 or more α-D-glucopyranose units are linked at the 1,4-position by α-bond like amylose. β-cyclodextrin or cycloheptaamylose contains 7 α-D-glucopyranose units. As used herein, the term “cyclodextrin” includes cyclodextrin derivatives such as hydroxypropyl and sulfobutyl ether cyclodextrin. Such derivatives are described, for example, in US Pat. Nos. 4,727,064 and 5,376,645. One preferred cyclodextrin is hydroxypropyl β-cyclodextrin (HβCD) having a degree of substitution of about 4.1 to 5.1 as measured by Fourier transform infrared spectroscopy. This type of cyclodextrin is available under the name Cavitron 82003 ^™ from Cerestar (Hammond, Ind., USA).

In certain preferred embodiments, Ac-PHSCN-NH ₂ or a derivative thereof is formulated in an aqueous solution containing cyclodextrin. In another embodiment, the peptides of the invention are formulated as a lyophilized powder containing cyclodextrin or as a sterile powder containing cyclodextrin. Preferably, the cyclodextrin is HβCD or sulfobutyl ether β-cyclodextrin, more preferably the cyclodextrin is hydroxypropyl-β-cyclodextrin. In injectables, cyclodextrins will generally comprise about 1-25% (w / w) of the formulation, preferably about 2-10%, more preferably about 4-6%. Further, the weight ratio of cyclodextrin to peptide is from about 1: 1 to about 10: 1.

In another embodiment of the invention, Ac-PHSCN-NH ₂ or a derivative thereof is formulated to make the peptide available orally or intranasally.

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

  Formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes before or after lyophilization and in-use preparation. Alternatively, sterility of the entire mixture is achieved by autoclaving the components prior to addition of the peptide, for example at about 120 ° C. for about 30 minutes.

The formulation is lyophilized after the peptide, cryoprotectant, and other optional ingredients are mixed together, but many different lyophilizers are available for that purpose. For example, a Hull 50 ^™ (Hull, USA) or GT20 ^™ (Leybold-Heraeus, Germany) freeze dryer. Freeze-drying is performed by sublimating ice from the frozen contents at a temperature suitable for primary drying after freezing the formulation. Under this condition, the product temperature is below the eutectic or disintegration temperature of the formulation. Typically, the primary drying shelf temperature is about -30-25 ° C. at a suitable pressure (typically in the range of about 50-250 mTorr), but the product remains frozen during the primary drying. ) The time required for drying mainly depends on the formulation itself, the size and type of the container holding the sample (eg glass vial), the volume of the liquid, and ranges from several hours to several days (eg 40-60 hours). It is possible. The secondary drying step can be performed at about 0-40 ° C., depending mainly on the type and size of the container and the nature of the peptide. For example, the shelf temperature during the total water removal stage of lyophilization can be about 15-30 ° C. (eg, about 20 ° C.). The time and pressure required for secondary drying is the time and pressure that results in a suitable lyophilized cake and depends on, for example, temperature and other parameters. The secondary drying time depends on the desired residual moisture in the product and will typically take at least about 5 hours (eg, 10-15 hours). The pressure may be the same as that used in the primary drying stage. Freeze-drying conditions can vary depending on the formulation and vial size.

  In some cases, it may be desirable to lyophilize the peptide formulation in a container in which the peptide will be prepared at the time of use to avoid the peptide transfer step. The container in this case may be, for example, a 3, 5, 10, 20, 50 or 100 cc vial.

  As a general suggestion, lyophilization will result in a lyophilized formulation having a moisture content of about 5% or less, preferably about 3% or less.

Preparation at the time of use of the lyophilized formulation At the desired stage, typically when administering the peptide to a patient, the lyophilized formulation is prepared at the time of use with a diluent, so that the concentration of the peptide in the ready-to-use formulation is at least mg / mL, such as about 10 mg / mL to about 1000 mg / mL, more preferably about 50 mg / mL to about 500 mg / mL, most preferably about 100 mg / mL to about 500 mg / mL. To do. Such high peptide concentrations in the ready-to-use formulation are believed to be particularly useful when attempting to deliver a ready-to-use formulation subcutaneously. However, for other routes of administration such as intravenous (iv) administration, lower peptide concentrations in the ready-to-use formulation (eg, about 1-100 mg / mL in the ready-to-use formulation, or about 5-50 mg / mL peptide) may be desirable. In certain embodiments, the peptide concentration in the ready-to-use formulation is significantly higher than that in the formulation prior to lyophilization. For example, the peptide concentration in the ready-to-use formulation is about 2 to 40 times, preferably 3 to 10 times, most preferably 3 to 6 times (e.g. at least 3 times or at least the concentration in the formulation prior to lyophilization). 4 times).

  The on-use preparation is performed at a temperature of about 25 ° C. to ensure complete hydration, but may be performed at other temperatures as desired. The time required for in-use preparation will vary depending on, for example, the type of diluent, the amount of excipients, and the peptide. Typical diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (eg, phosphate buffered saline, PBS), sterile saline, Ringer's solution, or dextrose solution. . The diluent may optionally contain a preservative. Although typical preservatives have been described above, aromatic alcohols such as benzyl alcohol and phenol alcohol are preferred preservatives. The amount of preservative used is determined by evaluating various preservative concentrations for peptide compatibility and preservative efficacy testing. For example, when the preservative is an aromatic alcohol (eg, benzyl alcohol), it may be present in an amount of about 0.1-2.0%, preferably about 0.5-1.5%, most preferably about 1.0-1.2%.

Preferably, the ready-to-use formulation has less than 6000 particles (size > 10 μm) per vial.

In another embodiment of the present invention, a product is provided that includes a lyophilized formulation of the present invention and is accompanied by instructions for its in-use preparation and / or use. This product is in a container. Suitable containers include, for example, bottles, vials (eg, two-chamber vials), syringes (eg, two-chamber syringes), and test tubes. Containers can be made from a variety of materials such as glass and plastic. The container contains the lyophilized formulation and a label affixed to or associated with the container may provide instructions for use and / or use. The label indicates, for example, that the lyophilized formulation is prepared at the time of use to the peptide concentration as described above. The label may further indicate that the formulation is useful for subcutaneous administration or intended for subcutaneous administration. The container into which the formulation is placed may be a multi-use container, which allows repeated administration (eg, 2-6 administrations) of the ready-to-use formulation. The product can further include a second container with a suitable diluent (eg, BWFI). When mixing the diluent and lyophilized formulation, the final protein concentration in the ready-to-use formulation should generally be at least 50 mg / mL. The product may contain other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, in-package prints containing instructions for use.

The therapeutic kit is a single container comprising a formulation of Ac-PHSCN-NH ₂ pharmaceutical composition, with or without other components (eg, other compounds or pharmaceutical compositions of these other compounds) Or have a separate container for each component. Preferably, the therapeutic kit of the present invention comprises a second compound (eg, chemotherapeutic agent, natural product, hormone or antagonist, anti-angiogenic agent or inhibitor, apoptosis inducer or chelator) or a pharmaceutical composition thereof. A formulation of Ac-PHSCN-NH ₂ or an acid addition salt thereof as described herein packaged together for co-administration with a product. The components of the kit may be pre-combined or each component may be placed in a separate container prior to administration to the patient. The components of the kit may be provided as one or more solutions, preferably an aqueous solution, more preferably a sterile aqueous solution. The components of the kit can also be provided as a solid, which is converted to a liquid by adding a suitable solvent (preferably provided in a separate container).

  The container of the treatment kit can be a vial, test tube, flask, bottle, syringe, or any other means of containing a solid or liquid. Usually, if more than two components are present, the kit includes a second vial or other container that allows separate dosing. The kit may also include a separate container for pharmaceutically acceptable liquids. Preferably, the therapeutic kit will include devices (eg, one or more needles, syringes, eye drops, pipettes, etc.) that allow administration of the agents of the invention that are components of the kit.

  The preparation of the present invention is suitable for administering a peptide by an acceptable route such as oral (intestinal), subcutaneous, intramuscular, intravenous and transdermal. Administration may be performed with an infusion pump. It is understood that any mode of administration and route disclosed by Ternansky et al. (Supra) are useful in the present formulation, all of which are incorporated herein by reference in their entirety.

  The present invention is also directed to the formulation of peptides suitable for administration by inhalation. Many drugs currently administered by inhalation are primarily administered as inhalable liquid or solid aerosol particles. In the case of biologically derived therapeutics, this can pose certain problems. This is because many of these pharmaceuticals are unstable in an aqueous environment for extended periods of time and are rapidly denatured when pulverized with high shear or other grinding methods when provided as a dry powder. Because. Moreover, some of these medications do not remain long enough in the lung because they are quickly extracted from the pulmonary environment after being administered as an inhaled aerosol. Also, as a result of the drug's reactivity with the surface of the device or container, or during aerosol application (especially in high shear, energy intensive spray systems), the drug is rendered inactive (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-glycolide), which are biotherapeutic proteins and Developed for peptides (Liu, R. et al., Biotechnol. Bioeng., 57: 177-184 (1991)). Most therapeutic peptides are poorly absorbed from biological membranes, even when formulated with penetration enhancers. Many therapies increase drug doses by several orders of magnitude to achieve the minimum systemic concentration required for efficacy. In other cases, drugs are formulated with exogenous absorption enhancers to improve permeability through absorption barriers, which often results in toxicity. The mode of drug administration to living organisms is also gradually spreading from oral and parenteral to transdermal, rectal and pulmonary routes (ie, nasal cavity and lung). Some of these drug delivery methods are successful and others are unsuccessful, which treats various diseases such as infections, malignancies, cardiovascular diseases, endocrine diseases, neurological diseases, and various immunocompromised diseases This is because of the lack of acceptance of relatively new and complex molecules that must be used.

  Thus, although the present invention takes advantage of the presence of a fluid propellant formulation system comprising a peptide (drug), the peptide is stable, protected by a rate limiting carrier, easily manufactured, and as fluid diffusing particles in the patient's lungs It is therapeutically effective when administered. As stated above, the present invention encompasses peptide formulations for inhalation through the mouth or nose. Such formulations include, but are not limited to, modified release aerosol particles and polysaccharide vesicles (associated with a given drug, here a peptide) to form part of a construct with the drug, or Medical respirable aerosol particles, which contain the drug and provide its sustained release (disclosed in US Pat. No. 6,551,578, which is incorporated by reference in its entirety). In this method, the peptide is formulated so as to be suitable for administration by inhalation through the mouth or nose. Stable colloidal dispersion of peptides in fluids such as air, hydrocarbon gases, chlorofluorocarbon (CFC) propellants, or non-CFC propellants such as tetrafluoroethane (HFA-134a) or heptafluoropropane (HFA-227) The body is considered.

  In a formulation of the invention intended for inhalation into the lung, the peptide is associated with a natural polysaccharide polymer (to which the peptide is to be bound). In this context, “association” refers to whether the peptide is present with the polysaccharide polymer as a matrix or as part of a polymer construct, encapsulated in the polysaccharide polymer or in the polysaccharide polymer construct particles, or Meaning that the amount or percentage (eg, 95% or more) of the peptide that is present on the surface of such particles is therapeutically effective for the peptide is particulate. Typically, the construct particles have a particle size of less than about 10 μm, preferably less than about 5 μm, such that the particles are inhaled into the respiratory tract and / or lungs of the patient to be treated.

  Suitable polymer constructs incorporate a given peptide therein, i.e. encapsulate, from there to the site of action or application of the patient's body (e.g. from the patient's lungs to the surrounding local environment). Providing controlled or modified release of the medicament.

Suitable polysaccharides are polymers selected from the group of alginate (in which the cations are for example Li ⁺ , Na ⁺ , K ⁺ , Ca ⁺⁺ , NH ³⁺ , NH ⁴⁺ etc.), for example Sodium alginate, calcium alginate, sodium alginate-calcium, ammonium alginate, sodium alginate-ammonium alginate, or calcium alginate-ammonium alginate. A preferred alginate modified release agent is ammonium-calcium alginate. These materials are commonly used in injectable implants and microsphere formulations for controlled release. A commercial form of ammonium-calcium alginate is Keltose ^™ manufactured and sold by International Specialty Products (Wayne, NJ). As used herein, “alginic acid” refers to either alginic acid or a salt thereof; or other polymers based on natural polysaccharides or carbohydrates such as gum arabic, pectin, galacturonic acid, karaya gum; benjamin gum, plantago ovata gum Meaning agar; carrageenan; cellulose; gelatin; or a mixture of any of the aforementioned polymers. Alginic acid is a pharmaceutical excipient that is generally considered safe and is used to produce a variety of well-documented pharmaceutical systems (US Pat. Nos. 6,166,084; 6,166,043; 6,166,042; 6,166,004) ; And No. 6,165,615). Alginic acid is a naturally occurring polymer containing polysaccharide chains. These polymers have the property of absorbing water and swelling to form a gel-like structure in solution. When the resulting core formulation is inhaled by the patient to be treated, the gel dissolves in the patient's body and releases its drug load in a dissolution controlled manner. Such a polymer system, when formulated in situ with a given medicament, forms a construct or matrix whereby the medicament forms part of the matrix or is encapsulated within the matrix. Once formed or encapsulated in this way, the drug is time released or adjusted from the site of action within the body (eg, lungs, airways, ears, etc.) to the surrounding environment or tissue of the patient's body. The polysaccharide polymer (eg, alginate) is present in the resulting controlled release formulation, typically in an amount of about 0.000001 to 10% by weight, based on the total weight of the formulation.

  The therapeutic peptide is present in the polymer construct of the present invention in a therapeutically effective amount, i.e., the peptide is incorporated into an aerosol formulation, such as a dispersion aerosol by oral or nasal inhalation, preferably in a single dose or multiple doses. The dose is present in an amount that produces its desired therapeutic effect.

  Other formulations for continuous administration are also conceivable, for example the use of multilamellar or single membrane liposomes. The production of liposomes is well known to those skilled in the art, and the liposomes may be phospholipid-based or non-phospholipid-based.

The assay useful in vitro and in vivo assays to measure the activity of the preparation containing Ac-PHSCN-NH ₂ or a salt thereof described herein are exemplary rather than inclusive, Those skilled in the art will understand. These can be found, for example, in Ternansky et al., Supra, and co-pending US patent applications USSN 10 / 074,225 and 10 / 661,784, assigned to the same assignee as the present application, all of which are incorporated by reference in their entirety. It is incorporated herein. Examples of suitable assays are described below.

Endothelial Cell Migration Assay For endothelial cell (EC) migration, add 200 μL collagen solution per transwell and incubate overnight at 37 ° C. to coat the transwell with type I collagen (50 μg / mL) . The transwell is assembled into a 24-well plate and an attractant (eg, FGF-2) is added to the lower chamber with a total volume of 0.8 mL of medium. ECs such as human umbilical vein endothelial cells (HUVEC) that had been detached from the monolayer culture with trypsin were diluted with serum-free medium to a final concentration of about 10 ⁶ cells / mL. Add 0.2 mL of the solution to the upper chamber of each transwell. Ac-PHSCN-NH ₂ salt is added to both the upper and lower chambers and allowed to move for 5 hours at 37 ° C. in a humidified atmosphere. The transwell is removed from the plate and stained using DiffQuik®. Unmigrated cells are removed from the upper chamber by rubbing with a cotton swab, the membrane is removed and placed on a slide and counted in a high power field (400 ×) to determine the number of migrated cells.

Biological Assays matrigel anti intrusion activity (Matrigel: registered trademark) penetrates through the reconstituted basement membrane (Matrigel) in an assay known as invasion assay system, EC or tumor cells (e.g., PC-3 human prostate cancer The ability of cells such as) has been described in detail in the art (Kleinman et al., Biochemistry 1986, 25: 312-318; Parish et al., 1992, Int. J. Cancer 52: 378-383). Matrigel is type IV collagen, laminin, heparan sulfate proteoglycan (eg perlecan; binds and localizes bFGF), vitronectin, and transforming growth factor-β (TGFβ), urokinase-type plasminogen activator (uPA) is a reconstituted basement membrane containing serpin known as tissue plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) (Chambers et al., Canc. Res. 1995, 55: 1578-1585). It is recognized in the art that the results obtained in this assay for compounds targeting extracellular receptors or enzymes are predictive of the in vivo efficacy of these compounds (Rabbani et al., Int. J. Cancer 1995, 63: 840-845).

Such assays use transwell tissue culture inserts. Invasive cells are defined as cells that can pass across the top surface of the matrigel and polycarbonate membrane and adhere to the bottom surface of the membrane. Coat Matrigel (Collaborative Research) diluted to a final concentration of 75 μg / mL with sterile PBS (Costar) containing polycarbonate membrane (pore size 8.0 μm) (60 μL diluted Matrigel per insert), 24 wells Place in the well of the plate. The membrane is dried overnight in a biological safety cabinet, after which 100 μL of antibiotic-containing DMEM is added and rehydrated for 1 hour on a shaker. Remove DMEM by aspiration from each insert and add 0.8 mL DMEM / 10% FBS / antibiotic to each well of a 24-well plate so that it surrounds the outside of the transwell (the “lower chamber”). Fresh DMEM / antibiotic (100 μL), human Glu-plasminogen (5 μg / mL), and the inhibitor to be tested are added to the upper inside of the transwell (“upper chamber”). The cells to be tested are trypsinized, resuspended in DMEM / antibiotic, and then added to the upper chamber of the transwell at a final concentration of 800,000 cells / mL. Adjust the final volume of the upper chamber to 200 μL. The assembled plate is then incubated for 72 hours in a humidified 5% CO ₂ atmosphere. After incubation, the cells are fixed and stained with Diff-Quick (Giemsa staining). The upper chamber is then rubbed with a cotton swab to remove Matrigel and cells that did not enter the membrane. The membrane is removed from the transwell using, for example, an X-acto® blade, placed on a slide using Permount® and a cover glass, and counted in a high power field (400 ×). Average the number of invading cells from the counted 5-10 fields and plot as a function of peptide concentration.

Tube formation assay of anti-angiogenic activity 2 × 10 ⁵ endothelial cells, for example human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMVEC), which can be prepared or commercially available Mix in a ratio of 1: 1 (v / v) with fibrinogen (5 mg / mL in phosphate buffered saline (PBS)) at a concentration of mL. Thrombin is added (final concentration 5 units / mL) and the mixture is immediately transferred to a 24-well plate (0.5 mL / well). A fibrin gel is formed, after which VEGF and bFGF (final concentrations of 5 ng / mL each) are added to the wells along with the test compound. Cells are incubated for 4 days at 37 ° C. with 5% CO ₂ , at which point the cells in each well are counted. The cells are then classified as either round shapes, elongated shapes without branches, elongated shapes with one branch, or elongated shapes with two or more branches. Results are expressed as an average of 5 separate wells for each compound concentration. Generally, in the presence of an angiogenesis inhibitor, the cells remain round or form undifferentiated tubes (eg, 0 or 1 branch). This assay is recognized in the art as predicting angiogenic (or anti-angiogenic) efficacy in vivo (Min et al., Cancer Res. 1996, 56: 2428-2433).

In another assay, endothelial cell tube formation is observed when endothelial cells are cultured on Matrigel (Schnaper et al., J. Cell Physiol. 1995, 165: 107-118). Transfer endothelial cells (10 ⁴ cells / well) to a matrigel-coated 24-well plate and examine tube formation 48 hours later. The inhibitor is tested by adding it at the same time as the endothelial cells or at various time points thereafter. Tube formation can also be stimulated by adding (a) an angiogenic growth factor such as bFGF or VEGF, (b) a differentiation promoting agent such as PMA, or (c) a combination thereof.

  While not wishing to be bound by theory, this assay presents endothelial cells with a specific type of basement membrane (ie, the layer of matrix that is expected to encounter the endothelial cells that migrate and differentiate first). To model angiogenesis. In addition to the bound growth factors, matrix components present in Matrigel (in situ basement membrane) or its proteolytic products are also stimulating to tube formation of endothelial cells, which makes this model previously described Complementing the developed fibrin gel angiogenesis model (Blood et al., Biochim. Biophys. Acta 1990, 1032: 89-118; Odedrat et al., Pharmac. Ther. 1991, 49: 111-124).

Growth inhibition assay
The ability of the compounds of the invention to inhibit EC proliferation can be examined in a 96 well format. Coat plate wells with type I collagen (gelatin) (0.1-1 mg / mL in PBS, 0.1 mL / well for 30 min at room temperature). After washing the plate (3x w / PBS), seed 3 to 6,000 cells per well and in endothelial growth medium (EGM; Clonetics) or M199 medium with 0.1 to 2% FBS for 4 hours (37 ° C / 5% CO ₂ ) Incubate to attach. After 4 hours, all media and non-adherent cells are removed and fresh media containing bFGF (1-10 ng / mL) or VEGF (1-10 ng / mL) is added to each well. The compound to be tested is added last and the plate is incubated for 24-48 hours (37 ° C./5% CO ₂ ). MTS (Promega) is added to each well and incubated for 1-4 hours. The absorbance at 490 nm (proportional to cell number) is then measured to determine the growth difference between control wells and test compound containing wells.

  A similar assay system can be set up with adherent cultured tumor cells. However, this format may exclude collagen. Tumor cells (eg, 3,000-10,000 cells / well) are seeded and cultured overnight to attach. Next, serum-free medium is added to the wells and the cells are cultured in synchrony for 24 hours. Medium containing 10% FBS is added to each well to stimulate growth. Add test compound to some wells. After 24 hours, MTS is added to the plate and the assay is developed and read as above.

Caspase-3 activity
The ability of the compounds of the invention to promote EC apoptosis can be examined by measuring caspase-3 activation. P100 plates are coated with type I collagen (gelatin) and 5 × 10 ⁵ ECs are seeded in EGM containing 10% FBS. After 24 hours (37 ° C./5% CO ₂ ), the medium is replaced with EGM containing 2% FBS, 10 ng / ml bFGF, and the desired test compound. Cells are harvested after 6 hours, cell lysates are prepared in 1% Triton and assayed using EnzChek® caspase-3 assay kit # 1 (Molecular Probes) according to the manufacturer's instructions.

The protocol used for the corneal neovascularization model is essentially the same as that described in Volpert et al., J. Clin. Invest. 1996, 98: 671-679. Briefly, female Fischer rats (120-14Og) are anesthetized and a pellet (5 μl) containing hydrone (Hydron®), bFGF (15OnM) and test compound is placed 1.0-1.5 mm from the limbus. Implant a small incision made there. Evaluate neovascularization 5 and 7 days after implantation. On day 7, the animal is anesthetized and a pigment such as colloidal carbon is poured to stain the blood vessels. The animals are then euthanized, the cornea is fixed in formalin, the cornea is flattened and a picture is taken to assess the extent of new blood vessel formation. New blood vessels can be quantified by imaging the total vessel area or length or simply counting the number of vessels.

Matrigel plug assay This assay is performed essentially as described in Passaniti et al., 1992, Lab Invest. 67: 519-528. Ice-cold Matrigel (eg, 500 μL) (Collaborative Biomedical Products, Inc., Bedford, Mass.) Is mixed with heparin (eg, 50 μg / ml), FGF-2 (eg, 400 ng / ml), and test compound. In some assays, bFGF is replaced with tumor cells as angiogenic stimuli. The Matrigel mixture is injected subcutaneously in the vicinity of the ventral midline gland of 4-8 week old athymic nude mice, preferably 3 sites per mouse. The injected Matrigel forms a tactile solid gel. The injection site is a plug (eg, FGF-2 + heparin) where each animal has a positive control plug (eg, FGF-2 + heparin), a negative control plug (eg, buffer + heparin), and a compound to be tested for effects on angiogenesis. + Compound) is selected. Preferably, all treatments are repeated 3 times. Animals are sacrificed by cervical dislocation about 7 days after injection or on another day optimal for observing angiogenesis. The skin of the mouse is peeled along the ventral midline and the Matrigel plug is collected and immediately scanned at high resolution. The plug is then dispersed in water and incubated overnight at 37 ° C. Hemoglobin (Hb) levels are measured using Drabkin's solution (eg, obtained from Sigma) according to the manufacturer's instructions. The amount of Hb in the plug is an indirect measure of angiogenesis because it reflects the amount of blood contained in the sample. In addition or alternatively, the animal may be injected with a buffer (preferably PBS) containing a high molecular weight dextran conjugated with a fluorophore prior to sacrificing the animal. The amount of fluorescence in the dispersion plug, measured fluorometrically, also serves as a measure of angiogenesis at the plug. Staining with mAb anti-CD31 (CD31 is “platelet-endothelial cell adhesion molecule or PECAM”) can also be used to confirm the formation of new blood vessels and microvessel density in the plug.

Chicken embryo chorioallantoic membrane (CAM) angiogenesis assay This assay is performed essentially as described in Nguyen et al., Microvascular Res. 1994, 47: 31-40. Meshes containing either angiogenic factor (bFGF) or tumor cells and inhibitors are placed on CAMs of 8-day-old chicken embryos and the CAMs are observed for 3-9 days after sample implantation. Angiogenesis is quantified by determining the percentage of squares in the mesh containing the blood vessels.

In vivo evaluation of angiogenesis inhibition and anti-tumor effects using Matrigel plug assay with tumor cells In this assay, tumor cells, e.g. 1-5 x 10 ⁶ 3LL Lewis lung cancer or rat prostate cell line MatLyLu, were mixed with Matrigel. The mice are then injected into the flank according to the protocol described in section B above. Tumor cell mass and a strong angiogenic response are observed in the plugs after about 5-7 days, and the compound's anti-tumor and anti-angiogenic effects in the actual tumor environment can be assessed by including the compound in the plug. Tumor weight, Hb levels, or fluorescence levels (fluorescence levels of dextran-fluorophore conjugate injected prior to sacrifice) are then measured. To measure Hb or fluorescence, first homogenize the plug using a tissue homogenizer.

Subcutaneous (sc) tumor-grown xenograft model nude mice are inoculated subcutaneously with MDA-MB-231 cells (human breast cancer) and Matrigel (10 ⁶ cells in 0.2 mL) on the right flank. Tumors are grown to 200 mm ³ before treatment with the test composition is started (100 μg / animal / day administered intraperitoneally daily). Tumor volume is measured every other day and the animals are sacrificed after 2 weeks of treatment. The tumor is excised, weighed and embedded in paraffin. Histological sections of the tumor are analyzed with H & E, anti-CD31, Ki-67, TUNEL, and CD68 staining.

Xenograft Model of Metastasis Compounds of the invention are also tested for the prevention of late metastasis using an experimental metastasis model (Crowley et al., Proc. Natl. Acad. Sci. USA 1993, 90 5021-5025). Late metastases include tumor cell attachment and extravasation, local invasion, seeding, proliferation and angiogenesis steps. Human prostate cancer cells (PC-3) are inoculated into nude mice that are transfected with a reporter gene, preferably a green fluorescent protein (GFP) gene (but as an alternative gene, the enzyme chloramphenicol) A gene encoding acetyltransferase (CAT), luciferase or LacZ may be used). This approach makes it possible to track the fate of these cells using either of these markers (fluorescence detection of GFP or histochemical colorimetric detection of enzyme activity). Cells are preferably injected intravenously and metastases are confirmed after about 14 days, particularly in the lungs (but also in the regional lymph nodes, femur and brain). This mimics the organ orientation of metastasis that naturally occurs in human prostate cancer. For example, PC-3 cells expressing GFP (10 ⁶ per mouse) are injected into the tail vein of nude (nu / nu) mice. Animals are treated with the test composition (100 μg / animal / day administered intraperitoneally daily). Metastatic single cells and lesions are visualized and quantified by histochemistry with a fluorescence or light microscope, or by disrupting the tissue and performing a quantitative colorimetric assay of the detectable label.

Suppression of spontaneous metastasis in vivo by PHSCN and functional derivatives Rat syngeneic breast cancer lines utilize Mat BIII rat breast cancer cells (Xing et al., Int. J. Cancer 1996, 67: 423-429). Tumor cells (eg, about 10 ⁶ suspended in 0.1 mL PBS) are inoculated into the fat pad of a female Fisher rat's breast. At the time of inoculation, a 14-day Alza osmotic minipump is implanted intraperitoneally to dispense the test compound. The compound is dissolved in PBS (eg, 200 mM stock), sterile filtered and placed in a minipump to achieve a release rate of about 4 mg / kg / day. Control animals receive vehicle (PBS) alone or vehicle control peptide by minipump. Sacrifice animals around day 14. In rats treated with the compounds of the present invention, there was a significant reduction in primary tumor size and a significant reduction in the number of metastases to the spleen, lung, liver, kidney and lymph nodes (counted as individual lesions). Can be observed. Histological and immunohistochemical analysis indicates that signs of tumor necrosis and apoptosis increased in treated animals. A large necrotic area is seen in the tumor area where no neovascularization is present. Human or rabbit PHSCN conjugated with ¹³¹ I (1 or 2 I atoms per peptide molecule) and derivatives thereof are effective radiotherapeutic agents and are at least more potent than unconjugated polypeptides. I know it is twice as expensive. In contrast, treatment with the control peptide cannot cause a significant change in tumor size or metastasis.

3LL Lewis Lung Cancer: Primary Tumor Growth This tumor line naturally developed as a lung carcinoma in C57BL / 6 mice (Malave et al., J. Natl. Canc. Inst. 1979, 62: 83-88). This is subcultured in C57BL / 6 mice by subcutaneous inoculation and tested in semi-allelic C57BL / 6 x DBA / 2 F ₁ mice or allogeneic C3H mice. Typically, a group of 6 animals is used for subcutaneous (sc) implantation, or 10 animals are used for intramuscular (im) implantation. Tumors may be sc transplanted as 2-4 mm fragments, or im or sc transplanted as an inoculum of about 0.5-2 × 10 ⁶ suspension cells. Treatment begins 24 hours after transplantation or when the tumor becomes palpable to a specific size (usually around 400 mg). Test compounds are administered intraperitoneally daily for 11 days.

  Subsequently, the animals are weighed, palpated and the size of the tumor is measured. Tumor weight in untreated control recipients 12 days after im inoculation is generally 500-2500 mg. Typical median survival is 18-28 days. As a positive control compound, for example, 20 mg / kg / injection cyclophosphamide per day is used from day 1 to day 11. Results to be measured include average animal weight, tumor size, and survival time. When confirming therapeutic activity, the test composition should be tested in two multiple dose assays.

3LL Lewis Lung Cancer: Primary Tumor Growth and Metastasis Model This assay is well known in the art (Gorelik et al., J. Natl. Canc. Inst. 1980, 65: 1257-1264; Gorelik et al., Rec. Results Canc 1980, 75: 20-28; Isakov et al., Invasion Metas. 2: 12-32 (1982); Talmadge et al., J. Natl Canc. Inst. 1982, 69: 975-980; Hilgard et al., Br. J. Cancer 1977, 35: 78-86). Test mice are male C57BL / 6 mice 2-3 months old. After sc, im, or footpad implantation, the tumor preferentially metastasizes to the lungs. In some tumor lines, the primary tumor exerts an anti-metastatic effect and must be excised first before conducting metastatic studies (see also US Pat. No. 5,639,725).

Single cell suspensions prepared from solid tumors by trypsinization are washed and suspended in PBS. The viability of 3LL cells prepared in this way is generally about 95-99%. Viable tumor cells (3 × 10 ^{4 to} 5 × 10 ⁶ ) suspended in 50 μl of PBS are injected sc into either the dorsal region of C57BL / 6 mice or one posterior footpad. Visible tumors appear 3-4 days after sc injection of 10 ⁶ cells dorsally. The day the tumor appears and the diameter of the established tumor are measured every 2 days. Treated by administering 1-5 doses of peptide or analog per week. In another embodiment, the peptide is delivered with an osmotic minipump.

In experiments that require resection of the dorsal tumor, mice are randomly divided into two groups when the tumor reaches a size of approximately 1500 mm ³ . (1) Completely remove the primary tumor; or (2) Perform a sham operation to leave the tumor intact. Tumor 500 to 3,000 mm ³ inhibit growth of metastases, with high viability, but without local regrowth, the maximum size of the primary tumor that can be safely resected is 1500 mm ^3. The acceleration phenomenon of metastatic growth after resection of a local tumor has been repeatedly observed (see, eg, US Pat. No. 5,639,725). These observations affect the prognosis of patients undergoing cancer surgery. After 21 days, necropsy is performed at the expense of all mice. The lungs are removed and weighed, fixed in Bouin's solution, the number of visible metastases recorded, and the diameter of the metastasis recorded as well. Based on the recorded diameter, determine the volume of each transition. To determine the total metastatic volume per lung, the average number of visible metastases is multiplied by the average volume.

It is also possible to measure the uptake of ¹²⁵ IdUrd into lung cells (Thakur et al., J. Lab. Clin. Med. 1977, 89: 217-228). Ten days after tumor transection, 25 μg of fluorodeoxyuridine is injected intraperitoneally into tumor-bearing mice (and tumor excised mice, if used). Thirty minutes later, mice are administered 1 μCi of ¹²⁵ IdUrd (iododeoxyuridine). One day later, the lungs and spleen are removed and weighed and the extent of ¹²⁵ IdUrd uptake measured using a γ counter.

  For mice with tumors on the footpad, when the tumor reaches 8-10 mm in diameter, the mice are randomly divided into two groups. That is, (1) the tumor-bearing leg is ligated above the knee joint and then cut; or (2) the mouse is left intact as an uncut tumor-bearing control. (Tumor-free amputation of tumor-bearing mice has no known effect on subsequent metastases, except that anesthesia, stress or surgery may have an effect.) 10-14 days after amputation Kill the mouse. Metastasis is assessed as described above.

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

Use of PHSCN peptides, analogs and salts in therapy
Ac-PHSCN-NH ₂ salt or a pharmaceutical composition thereof is generally used in an amount effective to achieve the intended purpose. Ac-PHSCN-NH ₂ salt (which may be in the form of a pharmaceutical composition) is therapeutically effective for use in the treatment or prevention of diseases or disorders characterized by abnormal angiogenesis or abnormal angiogenesis Administer or apply by volume. The amount of Ac-PHSCN-NH ₂ salt that may be effective in the treatment of a particular disorder or symptom disclosed herein will vary depending on the nature of the disorder or symptom, but standard clinical methods known in the art Can be determined. In addition, in vitro or in vivo assays may be utilized to help identify optimal dosages or dosage ranges. The amount of Ac-PHSCN-NH ₂ salt administered will, of course, depend on, inter alia, the subject being treated, the subject's weight, the severity of the disease, the mode of administration, and the judgment of the physician. For example, the dosage is delivered as a pharmaceutical composition by one or more administrations or by controlled release. Dosing may be repeated intermittently, performed alone or in combination with other drugs, and may continue for as long as required for effective treatment of the disease or disorder. Suitable dosage ranges for oral administration vary with 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 kg body weight. The dose range can be easily determined by methods known to those skilled in the art. Suitable dosage ranges for intravenous administration are from about 0.01 mg / kg to about 100 mg / kg body weight. Suitable dosage ranges for intranasal administration are generally 0.01 mg to 50 mg or 0.10 mg to 10 mg per kg body weight. Suppositories generally contain about 0.01 mg to about 50 mg of a compound of the present invention per kg body weight and contain about 0.5 to 10% by weight of the active ingredient. Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, or intracerebral administration range from about 0.001 mg / kg to about 200 mg / kg body weight. Effective doses can 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 certain embodiments, the dose administered is not based on body weight and is in an absolute amount, for example, ranging from 1 mg to 1 g at a time. In another specific embodiment, the dose is 10-750 mg at a time, for example 20 mg, 100 mg, or 600 mg at a time. In certain embodiments, the dose is administered once to several times per week (eg, 2, 3, 4, or 7 times).

Ac-PHSCN-NH ₂ salts are preferably assayed in vitro and in vivo prior to human use, as described above for the desired therapeutic or prophylactic activity. For example, an in vitro assay is performed to determine whether administration of Ac-PHSCN-NH ₂ salt or a combination of Ac-PHSCN-NH ₂ salts is suitable for the treatment of diseases characterized by abnormal angiogenesis or angiogenesis can do. The safety and efficacy of Ac-PHSCN-NH ₂ salt can be demonstrated using animal model systems. Preferably, the therapeutically effective amount of Ac-PHSCN-NH ₂ salts described herein, without causing substantial toxicity and gives a therapeutic effect. The toxicity of Ac-PHSCN-NH ₂ salt can be examined using standard pharmaceutical methods and is readily ascertainable by one skilled in the art. In the treatment of a disease or disorder, it is preferred that the “therapeutic index” (ratio of toxic dose to therapeutic dose) of Ac—PHSCN—NH ₂ salt is high. Suitable doses of Ac-PHSCN-NH ₂ salts described herein are preferably are effective, the dose resulting in a circulating concentration range of little or no drug toxicity.

  Although the present invention has been generally described above, the present invention will be more readily understood by reference to the following examples. These examples are provided for purposes of illustration and are not intended to limit the invention unless otherwise indicated.

Example I
Preparation of Ac-PHSCN-NH ₂ hydrochloride

Example II
Preparation and purification of Ac-Pro-His-Ser-Cys-Asn-NH ₂ TFA salt
Rink Amide AM resin (Novabiochem) was treated with 20% piperidine in DMF (1 mL per 100 mg resin) for 3 minutes under nitrogen stirring and the reaction mixture was filtered and washed once with DMF. This process was repeated two more times. The resin was washed 3 times with DMF and 3 times with dichloromethane. Fmoc-Asn (trt) -OH (3 eq), HBTU (3 eq), and HOBt (3 eq) are dissolved in DMF (1 mL per 100 mg resin) and added to the resin followed by N-methyl Morpholine (NMM) (6 eq) was added and the mixture was stirred for 1 hour. The reaction mixture was filtered and the resin was washed 3 times with DMF and 3 times with dichloromethane. This coupling process was repeated. The Fmoc deprotection and the above coupling steps were performed sequentially using Fmoc-Cys (trt) -OH, Fmoc-Ser (trt) -OH and Fmoc-His (trt) -OH to obtain Fmoc bound to the resin. -His (trt) -Ser (trt) -Cys (trt) -Asn (trt) was obtained. The resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for 3 minutes under nitrogen stirring and the reaction mixture was filtered and washed once with DMF. This process was repeated two more times. The resin was washed 3 times with DMF and 3 times with dichloromethane. Ac-Pro-OH (3 eq), HBTU (3 eq), and HOBt (3 eq) are dissolved in DMF (1 mL per 100 mg resin) and added to the resin followed by N-methylmorpholine (NMM ) (6 eq) was added and the mixture was stirred for 1 h. The reaction mixture was filtered and the resin was washed 3 times with DMF and 3 times with dichloromethane to give Ac-Pro-His (trt) -Ser (trt) -Cys (trt)-conjugated to Rink Amide AM resin. Asn (trt) was obtained. The resin was treated with TFA / TIS / water (95: 2.5: 2.5, 1 mL per 100 mg resin) and stirred under 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 with ether to give the crude Ac-Pro-His-Ser-Cys-Asn-NH ₂ TFA salt.

Using this general procedure, 708 mg of crude Ac-PHSCN-NH ₂ TFA salt was prepared from 2 grams of Rink Amide AM resin (loading: 0.63 mmol / g).

The crude peptide dissolved in the minimum amount of methanol and water was purified by reverse phase HPLC (Beckman) for separation using a Phenomenex Synergi hydro-RP C18 column (250 mm × 21.2 mm). Peptides were eluted over 30 minutes at a flow rate of 20 mL / min using a gradient from 3% to 100% B, where solvent A is water containing 0.1% trifluoroacetic acid and solvent B is 0.1% triflic acid. Acetonitrile containing fluoroacetic acid. Detection was at 220 nm. Analytical HPLC analysis (Phenomenex hydro RP (250mm x 4.6mm) using a gradient from 3% to 100% B) (Waters), combined fractions> 95% pure and volume about 2-4ml on a rotary evaporator Concentrated to lyophilized. The sample was redissolved in water, transferred to a tared 2 drum vial and second lyophilized.

Using this method, 140 mg of pure Ac-PHSCN-NH ₂ TFA salt was obtained from 338 mg of crude material: ES MS m / z (M + H) ⁺ 598.2; HPLC: 99% purity.

Example III
Preparation of Ac-Pro-His-Ser-Cys-Asn-NH ₂
Ac-Pro-His-Ser-Cys-Asn-NH ₂ TFA salt (140 mg, 0.197 mmol) is dissolved in 2 mL of distilled water and Amberlyst A-26 (OH) resin (4.2 meq / g, 273 mg, 5.8 equivalents) Was added. The reaction mixture was stirred at room temperature for 5 minutes. The aqueous liquor was decanted, the resin was washed twice with distilled water, and the combined aqueous phases were lyophilized to give 81 mg (69%) of Ac-PHSCN-NH ₂ 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-NH 2 hydrochloride
Ac-Pro-His-Ser-Cys-Asn-NH ₂ (77 mg, 0.13 mmol) was dissolved in 3 mL of distilled water at room temperature, and 1M hydrochloric acid (0.13 mL, 0.13 mmol) was immediately added. The mixture was stirred once, then frozen and lyophilized to give Ac-PHSCN-NH ₂ hydrochloride as a fluffy white solid: ¹ H NMR (300 MHz, DMSO-d6) δ9.00 (s, 1H), 8.57-8.26 (m, 2H), 8.21-8.03 (m, 2H), 7.45-7.36 (m, 2H), 7.13 (s, 1H), 7.09 (s, 1H), 6.94 (s, 1H) , 4.79-4.59 (m, 1H), 4.50-4.25 (m, 4H), 3.74-3.56 (m, 3H, water peak and overlap), 3.25-3.15 (m, 2H, water peak and overlap) , 3.11-2.98 (m, 1H), 2.88-2.72 (m, 2H), 2.57-2.38 (m, 2H, DMSO peak and overlap), 2.02 (s, 3H), 1.92-1.65 (m, 4H) HPLC: 93% monomer, 7% dimer.

Example V
Materials and methods used in formulation and testing
Formulation
Ac-PHSCN-NH ₂ (50 mg / ml) was formulated in a solution containing 5OmM citric acid and the components shown in Table 1 below. This solution was lyophilized and dispensed into vials for ready use with 1 mL of water.

An accelerated stability test was performed by storing the stability test solution at 40 ° C. for 4 days and at 55 ° C. for 34 days. Accelerated stability experiments are performed by forcing degradation to occur using extreme conditions of temperature and / or humidity and quickly testing the relative stability of a particular formulation (general storage conditions Is 22 ° C, 4 ° C, or -20 ° C for most drugs). This data can then be used to select from a number of test formulations the formulation that is most likely to be stable. After reconstitution with water, each formulation was tested by reverse phase HPLC using a standard mobile phase such as water / methanol or water / acetonitrile. This method allows for the separation of Ac-PHSCN-NH ₂ from Ac-PHSCN-NH ₂ of the degradation products or disulfide linked dimer, such as fragments of Ac-PHSCN-NH _2.

Calculation of potency Potency was calculated as the area under the peak of Ac—PHSCN—NH _{2 in} the HPLC chromatogram against the standard curve.

Purity calculation purity was calculated as Ac-PHSCN-NH ₂ area under the peak as a percentage of the integrated area of the whole chromatogram.

Calculation of% dimer
The relative amount of Ac-PHSCN-NH ₂ dimer was calculated as the area under the dimer peak as a percentage of the integrated area of the entire chromatogram.

Example VI
Stability of various formulations of Ac-PHSCN-NH ₂ The stability of peptide formulations was expressed in potency (mg / mL) and purity (% monomer and dimer%). The results are shown in Tables 2 and 3 below. Table 3 shows the potency normalized to the potency of the solution at t = 0; the column indicated by N indicates the normalized value.

Example VII
Selection of preferred formulations The following summarizes the characteristics of peptide formulations.
A. The formulations in solutions 1-4 have the following characteristics:
1. Inconsistent cake shape, most collapsed in lyophilizer;
2. The cake volume decreased during the accelerated stability experiment;
3. It took longer to prepare than expected due to the collapse of the cake.
B. The formulation in solution 6 has the following characteristics:
During the accelerated stability experiment, the color changed to brown, probably due to the reaction between glucose and glycine.
C. The formulation in solution 5 has the following characteristics:
1. good quality cake, white, quick re-preparation, low moisture content;
Solution 5 is composed of 50 mg / mL peptide, 50 mg / mL glycine, 5 OmM citric acid,
pH 5.0 and lyophilized (1 mL);
2. Tough performance in lyophilizer, uniform appearance with no disintegration in any sample;
3. Appearance and reconstitution ability were maintained during accelerated stability experiments;
4. Stability comparable to (± 2%) or better than other formulations was maintained.

Example VIII
Real-time stability data are for the preferred intravenous formulation (100 mg Ac-PHSCN-NH ₂ (= ATN-161), 50 mM citric acid, 50 mg / mL glycine, pH 5.0; 2 mg solution of 50 mg / mL ATN-161 Was lyophilized from -20 ° C. or 2-8 ° C. when stored at -20 ° C. or 2-8 ° C., which was within specification (ie, greater than 90% ATN-161) for 3 years. Based on these results, a low pH formulation with sufficient bulking agent (approximately 50 mg / ml glycine) and low water content in the vial after lyophilization provides the desired quality and stability of the ATN-161 drug It has been found. The water content can be varied by optimizing the lyophilization cycle using methods well known in the art. Similar parameters will be followed to develop further formulations of ATN-161 or acid addition salts or analogs thereof.

  All references cited above, whether specifically incorporated or not, are hereby incorporated by reference in their entirety. Citation of the above documents is not an admission that any of them is pertinent prior art. All statements regarding dates or representations regarding the contents of these documents are based on information available to the applicant and do not constitute any approval as to the validity of the dates or contents of these documents.

  Having fully described the present invention, those skilled in the art will understand the spirit and scope of the present invention without undue experimentation within the broad range of equivalent parameters, concentrations and conditions. It will be understood that it can be practiced without departing.

Claims (43)

(a) Peptide Pro-His-Ser-Cys-Asn, an analogue thereof, or the peptide or a salt of the analogue,
(b) at least one additional compound that stabilizes the peptide, analog or salt against spontaneous tandem dimerization or higher oligomerization,
A composition obtained by formulating using   2. The composition of claim 1, wherein the peptide is capped at its N-terminus and C-terminus by an N-terminal cap and a C-terminal cap, respectively.   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-end cap is an acetyl group.   The composition according to any one of claims 1 to 4, wherein the further compound inhibits, prevents or reverses the formation of disulfide bonds between sulfhydryl groups of Cys residues.   The composition according to any one of claims 1 to 4, wherein the further compound is a biocompatible acid buffer having a pK of about 5.   4. The composition of claim 3, wherein in the presence of the buffer, the pH of the solution is higher than 3.0 and equal to or lower than 7.5.   The composition according to claim 6, wherein the acid buffer is citric acid, acetic acid, or 2- (N-morpholino) ethanesulfonic acid (MES).   9. The composition of claim 8, wherein the acid buffer is citric acid at a concentration of about 25 mM.   10. A composition according to any one of claims 6 to 9, wherein the buffer is supplemented with excipients and glycine as a bulking agent.   11. The composition of claim 10, wherein the concentration of glycine is about 50 mg / ml.   The composition according to any one of claims 6 to 9, wherein the buffer comprises citric acid and acetic acid.   The composition according to any one of claims 6 to 8, wherein the buffering agent also contains Tris.   14. The composition of any one of claims 1 to 13, comprising (i) the peptide or a salt of the peptide or analog, (ii) about 50 mM citric acid, and (iii) about 50 mg / ml glycine. object.   A lyophilized form of 100 mg peptide or salt of the peptide or analog, 50 mM citric acid, 50 mg / ml glycine lyophilized from 2 mL of pH 5.0 solution, present in a container or vial. 14. The composition according to 14.   The composition according to any one of claims 1 to 15, further comprising one or more reducing agents.   17. The composition of claim 16, wherein the reducing agent comprises dithiothreitol, β-mercaptoethanol, or glutathione.   18. The composition of claim 17, wherein the concentration of the one or more reducing agents does not exceed about 10 mM.   The composition according to any one of claims 16 to 18, further comprising a biocompatible non-thiol antioxidant.   20. A composition according to any one of the preceding claims comprising a cryoprotectant present in a cryoprotective amount.   21. The composition of claim 20, wherein the molar ratio of cryoprotectant to peptide is from about 50 to 600 moles of cryoprotectant per mole of peptide.   The cryoprotectant is one or more sugars, one or more amino acids, one or more methylamines, one or more lyophobic salts, and / or one or more polyols according to claim 20 or 21 The composition as described.   23. The composition of any one of claims 20-22, wherein the cryoprotectant 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 combinations thereof.   23. The composition of claim 22, wherein the cryoprotectant is a non-reducing sugar.   26. The composition of claim 25, wherein the sugar is trehalose or sucrose.   27. A composition according to any one of claims 1 to 26, which is sterile and formulated for in vivo administration. (a) a first container comprising the composition according to any one of claims 1 to 27 in solution or lyophilized form;
(b) optionally a second container containing a diluent or reconstitution liquid for the lyophilized composition, and
(c) optionally, (i) instructions for use of the solution, or (ii) re-preparation and / or use of the lyophilized composition;
A product or kit comprising:   30. The product 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.   29. A product or kit according to claim 28, wherein the first container and the optional second container are bottles, vials, syringes, or test tubes.   29. A product or kit according to claim 28, wherein the first container and the optional second container are multi-use containers.   32. A product or kit according to any one of claims 28 to 31 wherein the composition is in lyophilized form.   A method for inhibiting angiogenesis in a subject comprising administering to the subject the composition according to any one of claims 1 to 27, wherein said peptide or analog is effective for antiangiogenesis. The method above, wherein the method is administered in an amount.   A method of treating cancer in a subject by inhibiting angiogenesis, comprising administering to the subject the composition according to any one of claims 1 to 27, wherein said peptide or similar Such a method, wherein the body is administered in an amount effective for treating cancer.   A method of treating Crohn's disease in a subject by inhibiting angiogenesis, comprising administering to the subject the composition according to any one of claims 1 to 27, wherein the peptide or The method above, wherein the analog is administered in an amount effective to treat Crohn's disease.   28. Use of a composition according to any one of claims 1 to 27 in a medicament for administration to a subject to suppress unwanted angiogenesis.   40. Use according to claim 36 for treating cancer by administration to a subject suffering from cancer.   38. Use according to claim 36 for treating Crohn's disease by administration to a subject suffering from Crohn's disease.   39. Use according to any one of claims 36 to 38, wherein the peptide, analog, or salt of the peptide or analog is administered in an anti-angiogenic effective amount.   28. Use of a composition according to any one of claims 1 to 27 in the manufacture of a medicament for administration to a subject to suppress unwanted angiogenesis.   42. Use according to claim 41 in the manufacture of a medicament for administration to a subject suffering from cancer to treat cancer.   42. Use according to claim 41 in the manufacture of a medicament for administration to a subject suffering from Crohn's disease to treat Crohn's disease.   43. Use according to any one of claims 40 to 42, wherein the peptide, analog, or salt of the peptide or analog is administered in an anti-angiogenic effective amount.