Formulations of peptides inhibiting the tsp-cd47 interaction

Abstract

The present invention relates to a formulation of a peptide inhibitor of the TSP-CD47 interaction in association with at least one buffer and at least one basic amino acid. The invention further relates to the therapeutic uses of such formulation.

Description

FORMULATIONS OF PEPTIDES INHIBITING THE TSP-CD47INTERACTIONFIELD OF INVENTION

[0001] The present invention relates to a pharmaceutical formulation comprising a peptide inhibitor of the interaction between the CD47 receptor and thrombospondin proteins, as well as to the use of such formulation to treat diseases or disorders.BACKGROUND OF INVENTION

[0002] The international application W02013007933 discloses peptide inhibitors that can antagonize the bond between a CD47 receptor and a protein belonging to the thrombospondin family (TSP).

[0003] In a particularly advantageous aspect of the W02013007933 disclosure, the antagonists, such as the antagonist TAX2, fix only onto the domain of the TSP that interacts with the CD47 receptor, thereby leaving the other domains of the TSP and the entire extracellular domain of the CD47 free so that they can bind their natural ligands. Such antagonists present a great therapeutic potential, in particular in the case of cancer.

[0004] However, the W02013007933 application does not disclose any formulations of such antagonists.

[0005] Thus, there is a clear need for therapeutic formulations of TSP-CD47 binding antagonists that have the characteristics necessary to provide safe and effective treatment. In particular, the development of peptide containing formulations for parenteral use presents unique challenges; the poor chemical and physical stability of peptides in solution limits formulation options. Moreover, macromolecules such as proteins, antibodies, and small peptides should present sufficient bioavailability when delivered to the subject to be treated. Therapeutics to be used for peptide delivery must meetInternational Council on Harmonization (ICH) and United States Pharmacopeia (USP) guidelines governing formulation heterogeneity, stability, viscosity, and pH to ensure safety as well as effective delivery of the active pharmaceutical ingredient to the subjects to be treated.

[0006] Consequently, there is a need for pharmaceutical formulations of TSP-CD47 binding antagonists, such as TAX2, that have desirable concentration, stability as well as characteristics suitable for therapeutic administration while respecting the clinical and regulatory prerequisites.

[0007] Advantageously, the invention provides a formulation of peptidic TSP-CD47 antagonists wherein the antagonist is present at a therapeutically efficient load, with a minimal possible amount of excipients that do not impart any effect on the specific action of the inhibitor effect as a TSP-CD47 binding antagonist that maintains the extracellular domain of the CD47 free.

[0008] Furthermore, the nature of the formulation facilitates the solubilization of the active pharmaceutical ingredient (API), i.e., the TSP-CD47 binding antagonist such as the TAX2 peptide, while ensuring its physicochemical stability as indicated by the absence of precipitation of the API and the absence of its degradation giving rise to API degradation products or impurities. According to a more advantageous aspect of the invention, the disclosed formulation ensures not only the short-term but also the longterm stability of the API, thereby ensuring the regulatory compliance of the formulation as well as an improved product shelf-life stability.SUMMARY

[0009] The invention relates to a composition comprising an antagonist peptide of the bond between a CD47 receptor and a protein belonging to a thrombospondin (TSP) family having an amino acid sequence of SEQ ID NO: 1 in association with at least one buffer and at least one basic amino acid selected from histidine, arginine and lysine.

[0010] It was surprisingly found by the inventors that such formulation of said antagonist peptide ensures not only its solubility and short-term stability, but also its long-term stability, as well as the regulatory compliance of the formulation.

[0011] In some embodiments, the composition further comprises at least one pharmaceutically acceptable surfactant.

[0012] Typically, the at least one pharmaceutically acceptable surfactant is non-ionic.

[0013] According to some embodiments, the at least one pharmaceutically acceptable surfactant is a triblock-polymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).

[0014] In some embodiments, the non-ionic surfactant is a triblock-polymer that presents an average molecular weight ranging from 8000 Da to 9000 Da, preferably about 8400 Da.

[0015] In some embodiments, the composition presents a pH ranging from 4.5 to 7.5.

[0016] The buffer comprised in the composition can be selected from phosphate buffer, citrate buffer, acetate buffer, lactate buffer, aspartate buffer and mixtures thereof, preferably the buffer is citrate buffer.

[0017] According to some embodiments, the basic amino acid is histidine.

[0018] The antagonist peptide of the bond between a CD47 receptor and a protein belonging to a TSP family can be solubilized to such extent that according to some embodiments the composition comprises said antagonist peptide in an amount ranging from 5 to 50 mg / mL.

[0019] The at least one basic amino acid, preferably histidine, can be present in an amount ranging from 0.1% to 1.5% w / v, preferably from 0.2% to 0.5% w / v in weight relative to the total volume of the composition.

[0020] According to some specific embodiments, the antagonist peptide of the bond between a CD47 receptor and a protein belonging to a TSP family presents the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 3, preferably the amino acid sequence SEQ ID NO: 3.

[0021] The invention further relates to a pharmaceutical composition, preferably a parenteral pharmaceutical composition, comprising the composition described hereinabove.

[0022] The invention further relates to said pharmaceutical composition for use as a drug.

[0023] According to some indicative embodiments, the pharmaceutical composition is for use in the treatment of cancer. According to some embodiments, the cancer may be selected from ovarian cancer, colorectal cancer, pancreatic cancer, melanoma, glioblastoma, follicular thyroid cancer, breast cancer, preferably selected from ovarian cancer, colorectal cancer, and pancreatic cancer.DEFINITIONS

[0024] “About” preceding a value is used to indicate that the value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used herein, the terms “about” and “approximately” may be used as equivalents.

[0025] “Antagonist peptide of the bond between a CD47 receptor and a protein belonging to a thrombospondin (TSP) family” also referred to herein as “antagonistpeptide” or “peptide” refers to the peptide that comprises or consists of the amino acid sequence SEQ ID NO: 1 consisting ofSEQ ID NO: 1 : R1-R2-R3-S-Q-L-L-K-G-R4-R5-R6 wherein R1 to R6 correspond to amino acids. The peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 1 X1X2X3SQLLKGX4X5X6, wherein Xi to Xe correspond to any amino acid. The antagonist peptide, or peptide, binds to a thrombospondin (TSP) protein, and more specifically to the C-terminal domain of the TSP protein which interacts with the CD47 receptor. This specific binding leaves the other domains of the TSP protein, as well as the entire extracellular domain of the CD47 receptor, free to interact with their respective other natural ligands. This allows to limit potential off-target effects of the antagonist peptide. The antagonist peptide inhibits the binding of the CD47 receptor with the TSP protein, thus inhibiting the anti-apoptotic effect of the interaction between the CD47 receptor and the TSP protein. According to some preferred embodiments, the antagonist peptide consists of the amino acid sequence as set forth in SEQ ID NO: 2 (IEVSQLLKGDAS) or as set forth in SEQ ID NO: 3 (CEVSQLLKGDAC), more preferably the antagonist peptide consists of the amino acid sequence as set forth in SEQ ID NO: 3. “TAX2” refers to the cyclic peptide consisting of the amino acid sequence as set forth in SEQ ID NO: 3, wherein the cyclization of said peptide is achieved by means of a disulfide bridge between two amino acids of the cysteine type (C). TAX2 presents an isoelectric point at a pH of 3.93 and is negatively charged at a pH of more than 4.0.

[0026] “API” is an active pharmaceutical ingredient, i.e., the antagonist peptide such as TAX2 in the present disclosure.

[0027] “Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to an excipient or carrier that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, such as a human. It includes any and all solvents, such as, for example, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. A pharmaceutically acceptable excipient or carrier refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. For human administration, preparations should meet sterility, pyrogenicity, general safety and puritystandards as required by the regulatory offices such as the EMA (European Medicines Agency) or FDA (US Food and Drug Administration).

[0028] “Subject” refers to a warm-blooded animal, more preferably a mammal. The term “mammal” refers here to any mammal, including humans. In some embodiments, a subject may be a “patient”, who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of the targeted disease or condition. In some embodiments, the subject according to the present disclosure suffers or is prone to suffer from a cancer.

[0029] “Treatment” or “treating” refers to a therapeutic (or curative) treatment, to a prophylactic (or preventative) treatment, or to both a therapeutic (or curative) treatment and a prophylactic (or preventative) treatment, wherein the object is to prevent, reduce, slow down (lessen), or cure one or more of the symptom(s) or manifestation(s) of the targeted pathologic condition or disorder, such as a cancer or an associated disorder or condition. For instance, those in need of treatment include those already suffering from a cancer, as well as those prone to develop a cancer, or those in whom a, disorder or condition associated to cancer, is to be prevented.DETAILED DESCRIPTION

[0030] According to a first aspect, the invention relates to a composition comprising: a peptide that antagonizes the bond between a CD47 receptor (herein antagonist peptide) and a protein belonging to a thrombospondin (TSP) family or antagonist peptide, as disclosed in W02013007933, the peptides’ description being incorporated herein by reference, and in particular as defined herein, in particular a peptide having an amino acid sequence of SEQ ID NO: 1 described herein; in association with at least one buffer and at least one basic amino acid.

[0031] The invention relates to a composition comprising a peptide, at least one buffer, and at least one basic amino acid, wherein the peptide inhibits the interaction between the CD47 receptor and a protein belonging to a thrombospondin (TSP) family, and wherein the peptide consists of the amino acid sequence as set forth in SEQ ID NO: 1.

[0032] The antagonist peptide presents an amino acid sequence SEQ ID NO: 1 : Rl- R2-R3-S-Q-L-L-K-G-R4-R5-R6, wherein R1 to R6 correspond to amino acids.

[0033] In some embodiments:- Rl, R3 and R5 each independently correspond to one polar amino acid chosen from isoleucine (I) and / or leucine (L) and / or valine (V) and / or alanine (A);- R2 and R4 each independently correspond to a negatively charged polar amino acid chosen from glutamic acid (E) and / or aspartic acid (D); and / or- R6 corresponds to an uncharged polar amino acid including a hydroxyl radical chosen from serine (S) or threonine (T).

[0034] The antagonist peptide, also referred to as peptide, presents an amino acid sequence as set forth in SEQ ID NO: 1 : X1X2X3SQLLKGX4X5X6, wherein Xi to Xe correspond to any amino acid.

[0035] In some embodiments:Xi, X3 and X5 each independently correspond to one polar amino acid selected from isoleucine (I) and / or leucine (L) and / or valine (V) and / or alanine (A);X2 and X4 each independently correspond to a negatively charged polar amino acid selected from glutamic acid (E) and / or aspartic acid (D); and / orXe corresponds to an uncharged polar amino acid including a hydroxyl radical selected from serine (S) or threonine (T).

[0036] The antagonist peptide, or peptide, may consist of the amino acid sequence as set forth in SEQ ID NO: 4: X1X2X3SQLLKGX4X5X6, wherein Xi to Xe is any amino acid.

[0037] The antagonist peptide, or peptide, may consist of the amino acid sequence as set forth in SEQ ID NO: 5: X1X2X3SQLLKGX4X5X6, whereinXi is I, L, V or A,X2 is E or D,X3is I, L, V or A,X4 is E or D,X5 is I, L, V or A, andXe is S or T.

[0038] The antagonist peptide, or peptide, may consist of the amino acid sequence as set forth in SEQ ID NO: 6: X1X2X3SQLLKGX4X5X6, whereinXi is I, L, V, A or C,X2 is E or D,X3is I, L, V or A,X4 is E or D,X5 is I, L, V or A, andX6is S, T or C.

[0039] The antagonist peptide, or peptide, may consist of the amino acid sequence as set forth in SEQ ID NO: 7: X1EVSQLLKGDAX2, wherein

[0040] According to some preferred embodiments, the antagonist peptide of SEQ ID NO: 1 consists of the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 3.

[0041] According to some preferred embodiments, the antagonist peptide, or peptide, of SEQ ID NO: 1 consists of the amino acid sequence as set forth in SEQ ID NO: 2 or as set forth in SEQ ID NO: 3.

[0042] In a preferred manner, at least two amino acids are cysteine residues, more preferably R1 and R6 are cysteine residues.

[0043] In a preferred embodiment, at least two amino acids are cysteine residues, more preferably Xi and Xe are cysteine residues.

[0044] Thus, more preferably the antagonist peptide presents the amino acid sequence SEQ ID NO: 3, even more preferably, the antagonist peptide is TAX2, a cyclic peptide consisting of the amino acid sequence of SEQ ID NO: 3, wherein the cyclization of said peptide is achieved by means of a disulfide bridge between two amino acids of the cysteine type (C).

[0045] In some embodiments, the antagonist peptide, or peptide, present the amino acid sequence as set forth in SEQ ID NO: 3, even more preferably, the antagonist peptide is TAX2, a cyclic peptide consisting of the amino acid sequence as set forth in SEQ IDNO: 3, wherein the cyclization of said peptide is achieved by means of a disulfide bridge between two amino acids of the cysteine type (C).

[0046] The antagonist peptide, or peptide, may consist of the amino acid sequence as set forth in SEQ ID NO: 2, or as set forth in SEQ ID NO: 3.

[0047] The antagonist peptide, or peptide, may consist of the amino acid sequence as set forth in SEQ ID NO: 2. The antagonist peptide, or peptide, may consist of the amino acid sequence as set forth in SEQ ID NO: 3.

[0048] Basic amino acids present basic side chains at neutral pH. Basic amino acids that are present in the composition of the invention are typically selected from the group consisting of histidine, arginine and lysine. In some embodiments, the at least one basic amino acid is selected from histidine and arginine. According to some preferred embodiments the at least one basic amino acid is histidine. According to some preferred embodiments, the composition comprises from 0.1% to 1.5% w / v, preferably from 0.2% to 0.5% w / v, typically about 0.4% w / v of at least one basic amino acid selected from histidine, arginine, and lysine, preferably histidine; the % being expressed in weight relative to the total volume of the composition.

[0049] Antagonist peptides of the invention, and in particular TAX2, have shown sufficient solubility in common laboratory-use buffer systems such as Tris (tris(hydroxymethyl)aminomethane) buffering systems. Without willing to be bound by a theory, the selection of the buffer in association with the selection of the at least one basic amino acid used to formulate the present composition ensures not only the solubility but also the stability of the antagonist peptide. It is understood that a buffer is a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it, thereby aiming at maintaining the pH of the composition substantially stable.

[0050] In some embodiments, the buffer is selected from phosphate, citrate, aspartate, acetate, lactate, succinate, maleate ammonium bicarbonate, tartrate buffers, and a mixture thereof, preferably the buffer is selected from phosphate, citrate, acetate, lactate, aspartate and mixtures thereof, even more preferably the buffer is a citrate buffer. As disclosedherein, a buffer may be designated by the weak acid or the weak base. Thus, unless otherwise specified, a buffer designated by the weak acid, indicates the buffer constituted by the pair of the weak acid and its ionized (base) form. Likewise, unless otherwise specified, a buffer designated by the base, indicates the buffer constituted by the pair of the base and its ionized (acid) form. For instance, citric acid or citrate buffer designates the buffer constituted by the pair of the weak citric acid and its ionized (basic) form citrate. The ionized forms of the acids / bases disclosed herein may be in the form of a salt with any pharmaceutically acceptable counterions such as alkali (e.g., sodium or potassium), alkaline earth (c.g, calcium or magnesium), ammonium, chloride, carbonate, or phosphate.

[0051] Thus, a citric acid or citrate buffer can be interchangeably used herein since they both designate the buffer comprising the pair of citric acid with a citrate salt, i.e., a citrate anion with a pharmaceutically acceptable cation, typically the citrate buffer designating the pair of citric acid and sodium citrate.

[0052] The composition comprises a buffer in an amount sufficient to adjust the pH of the composition to the desired pH value. In some embodiments, the composition comprises a buffer, as described above, in an amount ranging from 0.02% to 0.5% w / v, preferably from 0.05% to 0.20% w / v, such as for example about 0.10% w / v in weight relative to the total volume for the composition.

[0053] Typically, the composition presents a pH, that is superior to the isoelectric point of the antagonist peptide (e.g., of about 3.9 for TAX2) thereby supplying a composition whose pH is suitable for parenteral administration and wherein TAX2 is predominantly in its ionized form and thus more soluble in aqueous media. Accordingly, in some embodiments the pH of the composition ranges from 4.5 to 7.5. According to an exemplary embodiment, the pH of the composition is about 5.0.

[0054] According to a first variant, the sought pH is achieved by the addition of the suitable amount of buffer, as it is in the purview of the skilled artisan to calculate the amount of the buffer to be added so as to obtain a composition with the targeted pH value. According to a second variant, the composition is obtained by mixing the buffer with theantagonist peptide, the at least one basic amino acid and, when applicable, the other excipients disclosed herein, and then the pH is adjusted by adding a pharmaceutically acceptable strong base, such as ammonia, sodium hydroxide or sodium carbonate, or pharmaceutically acceptably acid, such as hydrochloric acid, in a sufficient amount so as to fine-tune the pH of the composition to the targeted pH value.

[0055] According to preferred embodiments, the composition further comprises a solubility enhancer, in a particular a surfactant. In some embodiments, the surfactant is in an amount ranging from 0.1 to 2.0% w / v, preferably from 0.2 to 1.0% w / v, more preferably from 0.3 to 0.5% w / v, typically about 0.4% w / v in weight relative to the total volume of the composition.

[0056] According to preferred embodiments the at least one surfactant is a non-ionic surfactant.

[0057] Preferably the at least one pharmaceutically acceptable surfactant is a triblock- polymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Even more preferably, the at least one pharmaceutically acceptable surfactant is a triblock-polymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) presenting an average molecular weight ranging from 8000 Da to 9000 Da, preferably about 8400 Da, such as for example Poloxamer 188 (CAS N° 9003- 11-6), commercially available as Kolliphor® P 188 by Sigma-Aldrich®.

[0058] The antagonist peptide composition can typically further comprise at least one pharmaceutically acceptable vehicle. Preferably, the at least one pharmaceutically acceptable vehicle is water.

[0059] According to second aspect, the invention relates to a pharmaceutical composition comprising the composition according to any one of the above embodiments in association with at least one pharmaceutically acceptable excipient. Said at least one excipient may be the vehicle as described above or any one of the pharmaceuticallyacceptable ingredients in the purview of the skilled artisan such as for example the water vehicle mentioned herein above.

[0060] Thanks to the selection of the at least one buffer, the at least one basic amino acid and preferably the non-ionic surfactant as described above, the antagonist peptide can be solubilized in an amount of up to 50 mg per mL of the composition or the pharmaceutical composition of the invention. Thus, in some embodiments the antagonist peptide is in an amount ranging from 5 to 50 mg, preferably from 8 to 20 mg, more preferably from 10 to 15 mg per mL of the composition or the pharmaceutical composition of the invention. According to an alternative expression, the TAX2 is present in the present compositions in an amount ranging from 0.5% to 2.0% w / v, preferably from 1.0 to 1.5% w / v, typically about 1.2% w / v in weight relative to the volume of the total composition. According to an advantageous aspect of the invention, the selection of the excipients as described above, not only leads to the solubilization of the peptide antagonist as described herein, but also the peptide antagonist as well as the overall composition of the invention are physicochemically stable for at least two preferably at least three months at room temperature (25°C), advantageously at least 6 months at fridge storage conditions (2- 8°C), and more than 8, typically more than 12 months under freezer storage conditions (- 20°C). According to one aspect, by physicochemically stable is meant an aggregation (precipitation) and / or an API (%) variation of less than 2% compared to the freshly prepared composition, preferably the API (%) variation being less than 0.7% compared to the freshly prepared composition.

[0061] Advantageously, the solubility of the antagonist peptide does not evolve after freezing-thawing cycles of the composition and the antagonist peptide does not undergo any substantial chemical degradation. Still more advantageously, the antagonist peptide solubility and chemical stability ensured by the present composition is obtained by enabling the minimal possible number of excipients and is compatible with filtering and injecting devices that may be used in the preparation of the pharmaceutical composition prior to its administration.

[0062] According to some embodiments, the composition or the pharmaceutical composition of the invention comprises or consists of:- from 0.5% to 2.0% w / v, preferably from 1.0 to 1.5% w / v, of an antagonist peptide as described above; at least one basic amino acid selected from histidine, arginine and lysine, preferably histidine, and at least one buffer selected from phosphate buffer, citrate buffer, acetate buffer, lactate buffer, aspartate buffer and mixtures thereof, preferably the buffer is citrate buffer; and- preferably further comprises at least one non-ionic pharmaceutically acceptable surfactant.

[0063] According to some embodiments, the composition or the pharmaceutical composition of the invention comprises or consists of:- from 0.5% to 2.0% w / v, preferably from 1.0 to 1.5% w / v, of the antagonist peptide as described above; at least one basic amino acid selected from histidine, arginine and lysine, preferably histidine, at least one buffer selected from phosphate buffer, citrate buffer, acetate buffer, lactate buffer, aspartate buffer and mixtures thereof, preferably the buffer is citrate buffer; at least one non-ionic pharmaceutically acceptable surfactant selected from a triblock-polymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), preferably presenting an average molecular weight ranging from 8000 Da to 9000 Da, more preferably about 8400 Da; and- water.

[0064] According to some embodiments, the composition or the pharmaceutical composition of the invention comprises or consists of:- from 0.5% to 2.0% w / v, preferably from 1.0 to 1.5% w / w, typically about 1.2% w / v of the antagonist peptide;- from 0.1% to 1.5%, preferably from 0.2% to 0.5% of at least one basic amino acid selected from histidine, arginine and lysine, preferably histidine,- from 0.02% to 0.5% w / v, preferably from 0.05% to 0.20% w / v, at least one buffer selected from phosphate buffer, citrate buffer, acetate buffer, lactate buffer, aspartate buffer and mixtures thereof, preferably the buffer is citrate buffer; and from 0.1 to 2.0% w / v, preferably from 0.2 to 1.0% w / v, more preferably from 0.3% to 0.5% w / v, typically about 0.4% w / v of at least one non-ionic pharmaceutically acceptable surfactant selected from a triblock-polymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), preferably presenting an average molecular weight ranging from 8000 Da to 9000 Da, more preferably about 8400 Da, such as Pol oxamer 188, and- water 100% Q.S.P., i.e., in a sufficient amount to reach 100% of the composition volume. the % in weight (w / v) being expressed in weight relative to the total volume of the composition.

[0065] The manufacturing of the antagonist peptide formulation described herein involves a carefully designed process to ensure the quality, consistency, and pharmaceutical suitability of the composition in particular the parenteral pharmaceutical composition of the invention.

[0066] Thus, the invention further relates to a process for producing the compositions according to the invention, used to prepare the disclosed formulation.

[0067] In some embodiments, the process for producing the compositions according to the invention comprises the step of mixing the antagonist peptide with at least one buffer and at least one basic amino acid as detailed in any one of the above embodiments. Optionally, the process may further comprise the addition of at least one pharmaceutically acceptable surfactant, preferably at least one pharmaceutically acceptable non-ionic surfactant as described hereinabove. In some embodiments, the process comprises the steps of i. solubilizing the at least one basic amino acid, preferably histidine in water, then ii. adding to the solution of the at least one basic amino acid, the at least one buffer and optionally at least one pharmaceutically acceptable surfactant, theniii. adding the antagonist peptide to the solution obtained in step (ii) leading to a composition according to the invention, iv. optionally adjusting the pH of the composition to 4.5 to 7.5, and v. optionally filtering the composition of step (iii) or step (iv).

[0068] Typically, steps (i), (ii), (iii) can be carried out by any means known in the art, typically by agitation such as for example vortexing. According to some embodiments, the antagonist peptide, the at least one buffer and at least one basic amino acid, and when applicable the at least one pharmaceutically acceptable surfactant are added in an amount sufficient to obtain their respective concentrations according to the invention.

[0069] The process may comprise further steps that may be needed with respect to the quality and regulatory prerequisites, in particular for parenteral pharmaceutical compositions; such as for example filtering and / or sterilization steps.

[0070] According to a further aspect, the invention relates to the composition or the pharmaceutical composition of the invention for its use as a drug. The invention also relates to the use of the composition or the pharmaceutical composition of the invention for the manufacture of a drug.

[0071] The invention relates to the composition or the pharmaceutical composition as described herein for use as a drug or as a medicament. The invention also relates to the use of the composition or pharmaceutical composition as described herein for the manufacture of a drug or a medicament.

[0072] Furthermore, the invention relates to the composition or the pharmaceutical composition of the invention for its use in the treatment of cancer. Advantageously, the formulation allows the effective liberation of the formulated antagonist peptide, without negatively affecting its biological effects.

[0073] The invention also relates to the composition or the pharmaceutical composition as described herein for use in the treatment of thrombotic disorders.

[0074] The invention further relates to the composition or the pharmaceutical composition as described herein for use in the treatment of cancer or thrombotic disorders.

[0075] Alternatively, the invention relates to a method for treating cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount, as defined by the health expert, of the composition or the pharmaceutical composition of the invention.

[0076] In addition, the invention relates to a method for treating thrombotic disorders, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition or the pharmaceutical composition as described herein.

[0077] Furthermore, the invention relates to a method for treating cancer or thrombotic disorders, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition or the pharmaceutical composition as described herein.

[0078] The invention further relates to the use of the composition or the pharmaceutical composition as described herein, for the manufacture of a medicament for the treatment of cancer.

[0079] The invention also relates to the use of the composition or the pharmaceutical composition as described herein, for the manufacture of a medicament for the treatment of thrombotic disorders.

[0080] The invention also relates to the use of the composition or the pharmaceutical composition as described herein, for the manufacture of a medicament for the treatment of cancer or thrombotic disorders.

[0081] In some embodiments, the cancer is selected from ovarian cancer, colorectal cancer, pancreatic cancer, melanoma, glioblastoma, follicular thyroid cancer, breast cancer, preferably selected from ovarian cancer, colorectal cancer, and pancreatic cancer.

[0082] In some embodiments, the thrombotic disorder is selected from heart attack, acute ischemic stroke, transient ischemic attack, peripheral arterial disease, atherosclerosis, mesenteric ischemia, deep vein thrombosis, pulmonary embolism, and phlebitis.TABLE OF SEQUENCESEXAMPLES

[0083] The present invention is further illustrated in a non-limitative manner by the following examples.Example 1: Solubility-enhancing excipients

[0084] The aim of this example was to determine the solubility of TAX2 in different excipients generally used for drug product formulations in order to select best candidates for future formulation development for TAX2.

[0085] The solubility of TAX2 peptide was performed by screening the effect of excipients using a HTS (high throughput screening) method. The compound was exposed to the excipients under continuous shaking and samples were taken after 24 hours. The supernatant was separated from excess solid by centrifugation and the assay was determined at day-0 and day-1 respectively using an HPLC-UV method. All experiments were performed at room temperature.

[0086] The solubility data from the HTS experiment indicate that TAX2 peptide is highly soluble in albumin, buffers and basic amino acids, while it exhibits low solubility in sugars and most of the amino acids.Materials and Methods

[0087] TAX2 peptide (99.12% purity) was supplied by PolyPeptide Laboratories AB and submitted to high throughput screening with the following excipients at concentrations based on the FDA’s Inactive Ingredient Database (IID).

[0088] Agilent 1100 High Performance Liquid Chromatography (HPLC) (Agilent Technologies, Santa Clara, CA) system, equipped with DAD spectrometer detector, was used. The compounds were separated using a XBridge C 18 column (2.1 x50 mm, 5pm) at 45°C. The mobile phase was 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B), and the flow rate was 1 mL / min using a gradient mode (Table 1). Calibration standards range was 5.00 pg / mL to 500 pg / mL and injection volume was 5.00 pL. Absorbance of the compounds was monitored at the wavelength 220 nm.Table 1. Gradient of the mobile phase for the HPLC analysis

[0089] A stock solution of TAX2 peptide was prepared at a concentration of 1.00 mg / mL in 0.1% TFA (Trifluoroacetic acid) in water and was further diluted with 0.1% TFA in water.

[0090] Calibration standards used for the construction of the calibration curve were prepared from the stock solution by serial dilution.

[0091] Then, a fixed weight (80 mg) of TAX2 peptide was taken into a 50 mL vial and dissolved with 16 mL water. The solution was distributed into 32 wells (each well contains 0.5 mL) in a 96-well plate and then the solution was dried by freeze drying method (-20°C temperature for 24 hours). Excipients (from Table 2) were added (0.2 mL) into the wells and the well plate was vigorously shaken for 24 hours at 1000 rpm on a vortex Multi-TB. The maximum concentration in every well would be around 12.5 mg / mL, if TAX2 is completely soluble.

[0092] Solutions were then centrifuged at 10000 rpm for 10 minutes to separate the solid particles and the supernatant was collected for HPLC analysis.

[0093] Blank samples (solvents / excipients without compounds) were prepared in parallel. The same dilution and analysis procedures were applied to the blanks. During the analysis, calibration samples were analyzed before the study samples.Results

[0094] The results of the HTS are presented in Table 2 below.Table 2. Concentration of TAX2 in the HTS samples at day 0 and the following day (day-1).

[0095] TAX2 peptide was poorly soluble in aqueous media comprising sugars such as sorbitol, maltose, mannitol, sucrose or trehalose that are commonly used in parenteralformulations.

[0096] Interestingly, the solubility of TAX2 was boosted by the presence of basic amino acids such as Histidine and Arginine. Advantageously, the presence of a basic amino acid not only enhanced the aqueous solubility of TAX2 but also maintained the TAX2 concentration on the following day (Day-1), suggesting that no short-term precipitation or degradation of TAX2 took place. On the opposite, TAX2 peptide was poorly soluble in the presence of the acidic amino acid glutamic acid, as well as in the presence of the polar amino acids asparagine, glutamine, serine, and in the presence of the nonpolar amino acids alanine, glycine, methionine, proline, and valine.

[0097] Solubility enhancing agents such as glycerin or surfactants were also screened in Example 1. Glycerin and Polysorbates enhanced the aqueous solubility of TAX2 to a limited extent. Furthermore, the amount of TAX2 in the glycerin and polysorbate samples was considerably reduced on Day-1, suggesting that some TAX2 precipitation and / or chemical degradation took place.

[0098] Albumin presented a considerable solubilization effect. However, as set in Example 2, comparative formulations comprising albumin showed a more pronounced TAX2 aggregation at 2-8°C storage and TAX2 degradation impurities during storage at room temperature.

[0099] Interestingly, the non-ionic poloxamer-188, not only improved significantly the TAX2 aqueous solubility, but also prevented any short-term precipitation and / or chemical degradation phenomena.Example 2: Composition of formulations

[0100] The purpose of this assay was to develop an IV injection formulation of TAX2 peptide at concentration of about 10 mg / mL. Based on the HTS screening of Example 1, three liquid injection formulations were prepared. The formulations were prepared successfully, and stability of formulations was evaluated up to one week at three different temperatures (-20°C; 2-8°C; and room temperature, RT). Further, day 10 formulation samples stored at RT were analyzed for impurity profiling and formulations at all threetemperatures were analyzed for potential aggregation.

[0101] TAX2 peptide formulation Fl was the most stable for all conditions tested for at least 7 days.Materials and Methods

[0102] TAX2 formulations were diluted (at a concentration of 200 pg / mL) with 0.1% TFA in water and aliquots were taken in two replicates for dilution in order to analyze them by HPLC-UV.

[0103] TAX2 powder formulations were dissolved in water (at a concentration of around 200 to 400 pg / mL) and aliquots were taken in two replicates for dilution in order to analyze them by HPLC-UV.

[0104] Blank samples (solvents without compounds) were prepared in parallel. The same dilution and analysis procedures were applied to the blanks. During the analysis, calibration samples were analyzed before the study samples.

[0105] For impurity profiling and aggregation analysis samples were injected undiluted together with corresponding placebo samples for comparison.

[0106] The samples were analyzed using the HPLC method and gradient presented in the materials and methods of Example 1.

[0107] The composition of the prepared TAX2 (~10 mg / mL) sample formulations is presented in the hereinbelow Table 3.Table 3. Compositions of a formulation according to the invention (Fl) and of comparative formulations (F2-F3)Results

[0108] Appearance of the formulations F1-F3 were recorded daily. All formulations appeared stable at -20°C and 2-8°C temperature.

[0109] However, upon storage at 2-8°C, the aggregation analysis showed that comparative formulations F2 and F3 presented higher TAX2 aggregation (1.14% and 1.47%) compared to the formulation according to the invention Fl (0.93%).

[0110] Summary of impurity profiling of day 10 samples stored at room temperature (RT) is presented in Table 4. Briefly, the purity of the formulations varied from 94.7% to 98.8% and was in the order of F2<F3«F1 (increasing order).Table 4. Summary of impurity profiling at day 10 of formulations F1-F3 stored at room temperature.

[0111] Interestingly, the formulation Fl according to the invention presented the least TAX2 aggregation and impurities compared to the comparative formulations F2 / F3 under storage conditions of 2-8°C and room temperature respectively.Example 3: Freeze-thaw stability

[0112] The aim of this example was to assess the stability of TAX2 formulations under transport conditions. The properties of the liquid Fl formulation were determined after freeze-thaw cycle at 10.0 mg / mL doses, i.e., presence of precipitation and concentration of the solutions as well as the aggregation. Two replicates of 10.0 mg / mL TAX2 peptide in the Fl formulation was subjected to freeze-thaw cycle. The samples were placed in the ultra-freezer at -80°C for 48 hours, and then moved to freezer at -25°C for another 48 hours, eventually samples were allowed to thaw in the fridge at 2-8°C for three days.

[0113] All samples were analyzed after freeze-thaw cycle, the concentrations were thenassayed for each vial by HPLC-UV using a calibration curve in the range of 5.00 to 15.0 mg / mL at 220 nm. Aggregation was also analyzed after the cycle by HPLC-UV using SEC method at a concentration of 10.0 mg / mL.

[0114] Formulation Fl was found to be stable under the tested temperature conditions.Example 4: Filter and syringe compatibility study

[0115] Filter and syringe compatibility study has been performed on liquid Fl formulation by using three sorts of filters, presented hereinbelow, to ascertain if sterile filtration and injection is compatible with TAX2 in formulation according to the invention.

[0116] Sterile filters:PES filter (25 mm, 0.22 pm):Polyether sulfone, Millipore® Lot: R0AB28756 PP filter (17 mm, 0.20 pm): Polypropylene, Thermo Fisher® Lot: 00297609 PTFE filter (13 mm, 0.20 pm): Polytetrafluoroethylene, PALL® Lot:21791619

[0117] Syringes:BD MEDICAL® 1 mL Plastipak® insulin syringe with needle.BD MEDICAL® 0.5 mL Plastipak® insulin syringe with needle.HENKE- JECT® single-use ImL syringe w / Luer tip with TERUMO® Agani needle G27.

[0118] Filter and syringe compatibility study was performed on formulation Fl in duplicate.

[0119] For the filter compatibility, the first 500 pL were discarded and analysis was done on remaining 2000 pL filtered samples. Assay for formulation Fl was determined directly before and after filtration (PES, PP, PTFE) by using reversed phase gradient HPLC.

[0120] For the syringe compatibility, samples were taken before and after using the syringe for assay and related substances determination. Placebo formulation was also tested by using the syringes to monitor any impact of the syringe on placebo formulation.

[0121] All samples were analyzed before and after filtration, the concentrations were then assayed for each vial by HPLC-UV using a calibration curve in the range of 5.00 to15.0 mg / mL at 220 nm. The results are presented in Table 5 and Table 6 respectively.Table 5. TAX2 assay (%) past the filtration with PES, PP and PTFE filtersTable 6. TAX2 assay (%) past the filtration with the assessed syringes

[0122] All tested filters and syringes were found to be compatible with TAX2 Fl formulation.Example 5: Long-term stability

[0123] The stability of the Fl formulation (assay and TAX2 (%)) according to the invention was evaluated up to six months at room temperature (RT, 25°C) and up to 36 months at -20°C and 2-8°C. The appearance of the formulation, were also monitored and the results are presented at Table 7.Table 7. Evolution of Appearance, pH, Assay (%), aggregation and amount of TAX2 (%) of the Fl formulation according to the invention at 25°C, 2-8°C and -20°C assessed upon preparation (TO), at one week (lw), 1 month (Im), 6 months (6m), 12 months (12m), 18 months (18m), 24 months (24m), and up to 36 months (36 m). CS: clear solution, *: some crystals, NA: Not applicable.

[0124] The Fl formulation of the invention was found to be stable at -20°C, and 2-8°C temperatures after twelve months with assay between 99.9% to 103%. The amount of the API (TAX2 (%)) was maintained stable (less than 0.6% deviation) after 24 months, and even after 36 months, both at -20°C, and at 2-8°C. Similarly, the pH was found stable both at -20°C and 2-8°C for up to 36 months.Example 6: Additional composition of formulations

[0125] The purpose of this assay was to develop additional IV injection formulations of TAX2 peptide at a concentration of about 10 mg / mL. Two liquid injection formulations were prepared, and their stability was evaluated up to one month at three different temperatures (-20°C, 2-8°C and 25°C). In addition, at day 10 formulation samples stored at -20°C, 2-8°C and 25°C were analyzed for impurity profiling and for potential aggregation.

[0126] The composition of the two prepared TAX2 (~10 mg / mL) sample formulations is presented in Table 8 below.Table 8. Compositions of two formulations F4 and F5 according to the invention.Materials and Methods

[0127] For the stability study, formulation samples were analyzed upon preparation (TO), at 1 day, 2 days, 1 week and 1 month by HPLC-UV. Appearance and assay (%) were determined with every time point at three different temperatures (-20°C, 2-8°C and 25°C). Impurities and aggregation were determined at day 10 at three different temperatures (-20°C, 2-8°C and 25°C).

[0128] TAX2 formulations F4 and F5 were diluted (at a concentration of 200 pg / mL) with 0.1% TFA in water and aliquots were taken in two replicates for dilution in order to analyze them by HPLC-UV. Blank samples (solvents without compounds) were prepared in parallel. The same dilution and analysis procedures were applied to the blanks. During the analysis, calibration samples were analyzed before the study samples. For impurity profiling and aggregation analysis, samples were injected undiluted together with corresponding blank samples for comparison. F4 and F5 formulation samples were analyzed using the HPLC method and gradient described in the material and methods section of Example 1.Results

[0129] The stability of the F4 and F5 formulations according to the invention was evaluated up to 1 month at -20°C, 2-8°C and 25°C. The appearance of the formulation was also monitored and the results are presented at Table 9 below.Table 9. Evolution of Appearance, pH and Assay (%) of the F4 and F5 formulations according to the invention at -20°C, 2-8°C and 25°C assessed upon preparation (TO), at 1 day, 2 days, 7 days, and 1 month. CS: clear solution.

[0130] Both F4 and F5 formulations of the invention were found to be stable at -20°C, 2-8°C and 25°C temperatures after 30 days with assay between 97.0 and 98.6% for theF4 formulation and between 97.3 and 99.5% for the F5 formulation. In addition, pH of both F4 and F5 formulations were found to be stable at -20°C, 2-8°C and 25°C temperatures after 30 days.

[0131] The total number of impurities, percentage of TAX2 purity and percentage of impurities of the F4 and F5 formulations samples stored at -20°C, 2-8°C and 25°C were evaluated after 10 days. Summary of impurity profiling of day 10 samples stored at -20°C,2-8°C and 25°C is presented in Table 10 below.Table 10 Summary of impurity profiling at day 10 of formulations F4 and F5 stored at -20°C, 2-8°C and 25°C.

[0132] Both F4 and F5 formulations exhibited low percentage of impurities and high TAX2 purity at all tested temperatures (i.e., -20°C, 2-8°C and 25°C) at 10 days. In addition, no aggregation was observed.

[0133] Overall, the data presented in the different examples demonstrate the stability and purity of the Fl, F4 and F5 formulations as described herein.

Claims

CLAIMS1. A composition comprising an antagonist peptide of the bond between a CD47 receptor and a protein belonging to a thrombospondin (TSP) family having an amino acid sequence of SEQ ID NO: 1 in association with at least one buffer and at least one basic amino acid selected from histidine, arginine and lysine.

2. The composition according to claim 1, further comprising at least one pharmaceutically acceptable surfactant.

3. The composition according to claim 2, wherein the at least one pharmaceutically acceptable surfactant is non-ionic.

4. The composition according to any one of claims 1 to 3, wherein the at least one pharmaceutically acceptable surfactant is a triblock-polymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).

5. The composition according to claim 4, wherein the triblock-polymer presents an average molecular weight ranging from 8000 Da to 9000 Da, preferably about 8400 Da.

6. The composition according to any one of claims 1 to 5, said composition presenting a pH ranging from 4.5 to 7.5.

7. The composition according to any one of claims 1 to 6, wherein the buffer is selected from phosphate buffer, citrate buffer, acetate buffer, lactate buffer, aspartate buffer and mixtures thereof, preferably the buffer is citrate buffer.

8. The composition according to any one of claims 1 to 7, wherein the basic amino acid is histidine.

9. The composition according to any one of claims 1 to 8, wherein the antagonist peptide of the bond between a CD47 receptor and a protein belonging to a TSP family is in an amount ranging from 5 to 50 mg / mL.

10. The composition according to any one of claims 1 to 9, wherein the at least one basic amino acid is in an amount ranging from 0.1% to 1.5% w / v, preferably from 0.2% to 0.5%w / v in weight relative to the total volume of the composition.

11. The composition according to any one of claims 1 to 10, wherein the antagonist peptide of the bond between a CD47 receptor and a protein belonging to a TSP family presents the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 3, preferably the amino acid sequence SEQ ID NO: 3.

12. A pharmaceutical composition, preferably a parenteral pharmaceutical composition, comprising the composition according to any one of claims 1 to 11.

13. The pharmaceutical composition of claim 12 for use as a drug.

14. The pharmaceutical composition of claim 12 for use in the treatment of cancer.

15. The pharmaceutical composition for use according to claim 14, wherein the cancer is selected from ovarian cancer, colorectal cancer, pancreatic cancer, melanoma, glioblastoma, follicular thyroid cancer, breast cancer, preferably selected from ovarian cancer, colorectal cancer, and pancreatic cancer.

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