Method of preparing parenteral formulations and formulations prepared therefrom

Abstract

The present invention refers to a pharmaceutical formulation comprising: a) a hydrophobic peptide or protein; b) a water-soluble organic surfactant selected from the list consisting of non-ionic surfactants, anionic surfactants, amphoteric surfactants, amino acids, amphipathic glycosides, water-soluble polymer compounds and tertiary amino compounds; wherein a) and b) are dispersed in a salt solution forming a colloidal suspension having particles having an average particle size of less than 100 nm cumulant radius as measured by dynamic light scattering, wherein the salt solution comprises a Tris buffer, a Sodium Bicarbonate buffer, or a mixture thereof; wherein the pH of the composition is in the range of between 7.37 to 10, preferably between 7.5 and 10, more preferably between 8.5 and 10, and wherein the formulation is suitable for parenteral administration.

Description

[0001] Method of preparing parenteral formulations and formulations prepared therefrom.

[0002] This application claims the benefit of European Patent Application No. 24382841.5 filed on July 30, 2024.

[0003] TECHNICAL FIELD

[0004] The invention relates to pharmaceutical formulations suitable for parenteral administration, preferably containing Disitertide or Disitertide related compounds or pharmacologically acceptable salts thereof, as well as methods for producing the same and uses thereof.

[0005] BACKGROUND ART

[0006] Disitertide is a hydrophobic active pharmaceutical ingredient, for which the invention provides an improved solubility and stability conditions in aqueous formulations, compatible with parenteral administration in potential patients. For example, there may be provided optimized aqueous composition that comprises Disitertide, and a different adjusted parameters of its aqueous formulations included pH, salts and buffer concentration, solubilizing, stabilizing, antioxidants, lyophilization excipients and compatible organic solvents. The composition may be directly administered or further lyophilized and resuspended with a desired aqueous diluent for infusion fluid as parenteral administration to a subject for clinical prevention of radiotherapy-induced healthy tissue damage including fibrosis. The compositions may be useful for the treatment and / or prevention of diseases or conditions that are sensitive to Transforming Growth Factor beta (TGF-P) inhibition as molecular mechanism target, such as fibrotic diseases, malignancies, or immunomodulatory alterations.

[0007] Disitertide is an active pharmaceutical ingredient consisting in the acetic salt of a 14 mer peptide inhibitor of TGF-p, derived from human Betaglycan (TGF-p type III receptor, encompassing amino acids 730-743: sequence TSLDASIIWAMMQN). Disitertide presents a widely demonstrated inhibitory activity against TGF-p biological activity, but its highly significant hydrophobic profile makes the compound incompatible with parental delivery, which is the only viable route of administration for many clinical indications. Oral administration is also restricted due to the low stability of Disitertide against digestive proteases. The same situation applies to other hydrophobic peptides or proteins that are intended to be parenterally administered.

[0008] The development of a hydrophobic peptide / protein formulation such as a Disitertide formulation for parenteral routes of administration is a major technological challenge that must overcome its extremely low solubility in aqueous medium. Parenteral formulations imply multiple controlled elements and parameters like pH, salt / buffer, different excipients, viscosity, osmolarity, particle size (in case of colloidal suspensions), active compound concentration, etc.

[0009] US2012 / 315256 discloses a topical pharmaceutical composition comprising a TGF-pi inhibitor peptide suitable for ophthalmic application, such as eyedrops, ointments, creams, etc. Particularly, liquid pharmaceutical dosage forms comprise particles of size higher than 10,000 nm in cumulant radius and thus are not suitable for parenteral administration.

[0010] US2023 / 414760 discloses stable pharmaceutical formulations of different antibodies, which are hydrophilic compounds and thus, water soluble compounds.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention presents a rational for pharmaceutical formulations that overcomes the aqueous solubility limitations and aggregation tendency of hydrophobic peptide / proteins such as Disitertide to enable their parenteral administration and allow the medical uses of such formulations.

[0013] The present invention details a process of parenteral formulation elements, parameters selection and optimization, to allow the solubilization of a hydrophobic peptide / protein such as Disitertide as a stable colloidal solution with controlled particle size and avoiding aggregation tendency, while maintaining the hydrophobic peptide / protein stability. In particular, if the hydrophobic peptide / protein is Disitertide, both its stability and biological activity as a TGF-p inhibitor are maintained.

[0014] Thus, a first aspect of the invention refers to a pharmaceutical composition or formulation comprising: a) a hydrophobic peptide or protein; b) a water-soluble organic surfactant selected from the list consisting of non-ionic surfactants, anionic surfactants, amphoteric surfactants, amino acids, amphipathic glycosides, water-soluble polymer compounds, and tertiary amino compounds; wherein a) and b) are dispersed in a salt solution forming a colloidal suspension having particles having an average particle size of less than 100 nm cumulant radius as measured by dynamic light scattering, wherein the salt solution comprises a Tris buffer, a Sodium Bicarbonate buffer, or a mixture thereof; wherein the pH of the composition is in the range of between 7.37 to 10, preferably between 7.5 and 10, more preferably between 8.5 and 10, still more preferably between 9.0 and 10, still more preferably between 9.5 and 10, and wherein the formulation is suitable for parenteral administration. In a second aspect of the invention, the formulation or composition as defined in accordance with the first aspect of the invention or with any of the preferred embodiments of the invention, is for use in diseases prevention or therapy.

[0015] In a third aspect of the invention, the formulation or composition as defined in accordance with the first aspect of the invention or with any of the preferred embodiments of the invention, is for use, via parental administration, in diseases prevention or therapy.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] Other objects and advantages of the present invention will be apparent from the following detailed description and accompanying drawings, in which:

[0018] FIG. 1. Disitertide (1 mg / ml) water solubility as a function of pH. (A) optic microscopic images 4x augmentation at different pHs including Tyndall effect (T), and macroscopical precipitation (P) analysis results. (B) Turbidity of Disitertide solutions sample, presented in (A), measured as absorbance at 415 nm solutions as function of pH, R2= 0,85.

[0019] FIG. 2. Disitertide (1 mg / ml) solubility, (A) in different salt solutions and buffers at three different initial pHs (7, 8.5 and 10), illustrated as optic microscopic images, 4x augmentation, and (B) DLS Prometheus Panta analyzed cumulant radius (nm) particle size and polydispersity index (PDI) results. Water: H2O, PBS: Phosphate buffered saline, Be: Sodium Bicarbonate, TRIS: Tris(Hydroxymethyl)aminomethane. PP: Prometheus Panta particle size analysis as cumulant radius (nm), OR: out of range particle size (above the particles size detection range of the Prometheus Panta equipment: > 10 .m); PDI: Polydispersity Index.

[0020] FIG. 3. Ionic strength effect over Disitertide (1 mg / ml) solubility, measured as consecutive dilutions of salt solutions and buffers. (A) Optic microscopic images, 4x augmentation, including pH values and DLS Prometheus Panta analyzed cumulant radios (nm) particle size and polydispersity index (PDI) results. (B) Disitertide particle size cumulant radius (nm) respect Sodium bicarbonate (NaBC) and Tris buffers concentrations with the corresponding microscopic images. TRIS: Tris(Hydroxymethyl)aminomethane; PBS: Phosphate buffered saline; PP: Prometheus Panta particle size analysis as cumulant radius (nm); PDI: polydispersity index; OR: out of range particle size (above 10 .m).

[0021] FIG. 4. Disitertide (1 mg / ml) particle size and thermal stability data from Prometheus Panta (NanoTemper Technologies) in the presence or absence of excipient copolymer Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (at 0.1 and 0.05%), in Sodium Bicarbonate (NaBC) 50mM or Tris (Hydroxymethyl)aminomethane (TRIS) 25mM buffers, before (PI - darker line) and after lyophilization (PL - lighter line). The first column presents data of particles size cumulant radius (nm) and polydispersity index as size distribution curve width. The following columns present data of Disitertide's structure (Ratio of 350 / 330 nm fluorescence and its first derivative) and aggregation trend (particle size cumulant radius evolution in nm) as a function of a temperature ramp from 25->95->25°C in the control buffers and in the presence of 0.05 and 0.1% of copolymer Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). RP: relative percentage; PI: prepared immediately; PL: post-lyophilization; Rt (350 / 330nm): fluorescence ratio 350 / 330 nm; Rt (1st der): fluorescence ratio first derivative; CR (nm): Cumulant radius in nanometers; T (°C): Temperature in Celsius grades.

[0022] FIG. 5. Disitertide (1 mg / ml) thermal stability data from Prometheus Panta (NanoTemper Technologies) in the presence or absence of excipient copolymer Poly(ethylene glycol)- block-poly(propylene glycol)-block-poly(ethylene glycol) (at 0.25, 0.125, 0.0625, 0.03125 and 0.015625%), in Sodium Bicarbonate (NaBC) 50mM or Tris (Hydroxymethyl)aminomethane (TRIS) 25 mM buffers, in immediately prepared samples (A) or after lyophilization (B). Including (following the order by rows) data of Disitertide's structure (Ratio of 350 / 330 nm fluorescence and its first derivative), turbidity, aggregation trend (particle size cumulant radius evolution in nm) and scattering as a function of a temperature ramp from 25->95°C. PI: prepared immediately; PL: post-lyophilization; Rt (350 / 330nm): fluorescence ratio 350 / 330 nm; Rt (1st der): fluorescence ratio first derivative; Trb (mAU): turbidity in miliabsorbance units; CR (nm): Cumulant radius in nanometers; DLS: dynamic light scattering in counts units. T (°C): Temperature in Celsius grades.

[0023] FIG. 6. Total Gibbs free energy (DG) in the context of colloidal aqueous solutions as function of liquid surface tension (g) and particle volume (bulk) free energy (DGv). (A) Evolution of different free energy parameters (liquid surface free energy, solute particles volume and total free energy as addition of the previous two) as function of particles size (r radius) increase. (B) Gibbs Free energy (DG) evolutions respect Disitertide (1 mg / ml) particle size (cumulant radius in nm) in a true solution, control buffers (NaBC: Sodium Bicarbonate 50 mM, and TRIS: Tris (Hydroxymethyl)aminomethane 25mM), and different formulations containing solubilizing excipients listed in Table 1 b.

[0024] FIG. 7. Antioxidant activity of Disitertide in the presence or absence of different amino acids- based antioxidant excipients in Tris - Sodium bicarbonate buffer (TBC). (A) antioxidant activity of different formulations and controls of Disitertide (0.1 mg / ml) measure as absorbance at 500 nm and Ascorbic acid equivalent values (mg / 100ml), after 15 minutes of incubation in the Lugol / CD colorimetric assay. (B) Ascorbic acid standard curves (0 to 5 mg / 100ml) and 500 nm absorbance values and graphics. AAE: Ascorbic Acid Equivalents; 0 F-aa: Aminoacids formulation controls w / o Disitertide; CD: Highly Branched Cyclic Dextrin; TBC: Tris 50mM + Sodium Bicarbonate 50mM buffer; Na BC : Sodium Bicarbonate. L-Cys: L-Cysteine; PVP K12: poly[1-(2-oxopyrrolidin-1-yl)ethylene] MW 3000-7000 Da. FIG. 8. Disitertide's in vitro activity in a TGF-p reporter gene HEK-293 cell line assay. (A) Bars diagram representation of Disitertide dose dependent inhibition (0 to 200 mg / ml) over TGF-p activity, measured as colorimetric reaction (absorbance 655 nm) mediated by TGF- P inducible HEK-293 secreted embryonic alkaline phosphatase (SEAP), in a formulation consisting of copolymer, Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol) and L-mannitol in Tris buffer. (B) Linear graph representation of Disitertide dose dependent inhibition in a formulation consisting of copolymer Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and L-mannitol in Tris buffer. (C) Linear graph representation of Disitertide dose dependent inhibition in a formulation consisting of copolymer Poly(ethylene glycol)-block-poly(propylene glycol)- block-poly(ethylene glycol) and Isomaltulose in Tris buffer. (D) Linear graph representation of Disitertide dose dependent inhibition in a formulation consisting of copolymer, 2-{2-[3,4- bis(2-hydroxyethoxy)oxolan-2-yl] -2- (2-hydroxyethoxy) ethoxyjethyl dodecanoate, Glutamine and Trehalose in Tris buffer. 0: controls without Disitertide are indicated in the graphs with an empty circle symbol. CP2MOx : Copolymer 2-methyloxirane I oxirane; Isom: Isomaltulose; Mann: Mannitol; PoES-ML: 2-{2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate ; GLUT: Glutamine; TRH: Trehalose; TRIS: Tris buffer 25mM; PI: prepared immediately; PL: post-lyophilization.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] As used herein “acetic salt” shall be understood as an element of a pharmaceutical active compound where an acetic acid (CH3COOH) molecule yields a hydrogen ion to an amine or other organic basic group of the active pharmaceutical ingredient generating the neutralization of the basic group and the acetate anion (CH3COO“).

[0027] As used herein “water-soluble” shall be understood as condition of a solute when generates a homogenous mixture with water at a molecular or ionic level, meaning that the molecules or ions of the substance interact with water molecules to the extent that disperse uniformly throughout the water, making them indistinguishable from the solvent and with particles size at molecular level (nanometric scale).

[0028] As used herein “water-soluble organic surfactant” shall be understood as water-soluble amphiphilic (amphipathic) molecules that lower the interfacial tension between a liquid solvent and a solid solute (water and Disitertide in this case) improving solid solubility in the solvent.

[0029] As used herein “non-ionic surfactant” shall be understood as surfactants that do not contain dissociable functional groups, which means that they do not separate into ions in water solutions. As used herein “anionic surfactant” shall be understood as surfactants that comprise saturated or weakly unsaturated hydrocarbon chains to which a hydrophilic group presents a negative charge on their hydrophilic molecular region end, generally a strong acid such as a sulfate [-O-SO3] or sulfonate [-SO3], is linked.

[0030] As used herein “amphoteric surfactant” shall be understood as surfactants with ionic charge that can change between anionic properties, the isoelectric neutral stage and the cationic properties depends on the environmental pH value.

[0031] As used herein the term “solvent” shall be understood as liquid substance present in the largest amount able to dissolve or disperse solutes at molecular, ionic or nanometric particle size level.

[0032] As used herein the term “polar solvent” shall be understood as a solvent which molecules present a significant electric dipole moment, meaning the presence of molecular regions of partial positive and negative charge due to an uneven distribution of electrons. This polarity allows polar solvents to interact strongly with other polar molecules or ions, effectively dissolving them through dipole-dipole interactions, hydrogen bonding, or ion-dipole interactions. Examples: water, ethanol, acetone, dimethyl sulfoxide, etc.

[0033] As used herein the term “amphipathic compounds” shall be understood as organic chemical compounds containing both polar (water-soluble) and nonpolar (not water-soluble) portions in its structure. It may also relate to a chemical compound having both hydrophobic and hydrophilic regions.

[0034] As used herein the term “non-polar solvent” shall be understood as solvent which molecules present reduced to no permanent electric dipole moment, meaning the absence of charged regions in the molecules and the inability to engage in strong dipole-dipole interactions or hydrogen bonding, making them effective at dissolving non-polar substances through van der Waals forces (dispersion forces). Examples: Hexane, Benzene, Toluene, Chloroform, Carbon Tetrachloride, etc.

[0035] As used herein the term “hydrophobic peptide or protein” shall be understood as amino acids linear sequences below (peptides) or above (proteins) 50 amino acids in length, containing relative high number of hydrophobic non-polar residues like Alanine, Valine, Leucine, Isoleucine, Methionine, Phenylalanine, Tryptophan or Tyrosine, with GRAVY (Grand Average of Hydropathy) values above zero. In terms of GRAVY score Hydrophobicity is calculated as the sum of hydropathy values of all the amino acids, divided by the number of total residues of the peptide / protein sequence) where positive values (>0) correspond to general hydrophobic peptides or proteins and negative values (<0) correspond to general hydrophilic peptides or proteins. Where Disitertide presents a GRAVY value of 0.46. Examples of hydrophobic peptides or proteins that present very low affinity to water and poor or absence water solubility are listed herein as: Disitertide, Gramicidin A, Cyclosporine, p-Amyloid, Melittin, Magainin, Dynorphin A, Dermcidin, Cecropin A, Maximin H5, Indolicidin, Piscidin, Thanatin, LL-37, Brevinin-1 , Protegin-1 , Dermaseptin, Cecropin, Buforin, Mastoparan, Some collagen derived peptides like Gly-Pro- Hyp, Octreotide, Liraglutide, Glatiramer acetate, Enfuvirtide, Brimonidine, Exenatide, Desmopressin, Semaglutide, Apolipoproteins, Melanin-concentrating hormone, Neuropeptide Y, p-Endorphin, Corticotropin-releasing hormone, Encephalins, Galanin, Vasopressin, Bradykinin, Dalbavancin, Leuprorelin, Teriparatide, Teduglutide, Cetrotide, Oxytocin, Polymyxins, Daptomycin, Bacitracin, Tyrocidine, Insulin, Glucagon, Erythropoietin, Growth Hormone, p-Casein, Fibrinogens, Keratins, Albumins, Collagens, Interferons, some monoclonal antibodies (Bevacizumab, Cetuximab, Ipilimumab), some enzymes (a-Chymotrypsin, L-asparaginase, Lipases, membrane proteins (Na7K+- Transporting ATPase Subunit-a, Rhodopsin, Cytochrome P450 and B6f, Nav channel, ATP- binding Cassette Transporter A1 , Aquaporins, Voltage-Dependent Anion Channel 1 , Transferrin Receptor Protein 1 , NADH-ubiquinone oxidoreductase chain 1), Cholesterol esterase, Fatty Acid-Binding Protein, Platelet Glycoprotein 4, Myelin Basic Protein, Peripherin, Acetylcholinesterase, Carbonic anhydrase), Chimeric proteins (Imiglucerase, Abatacept, Elosulfase-a), all type of lectin proteins (agglutinins), etc.

[0036] As used herein the term “colloidal suspension” shall be understood as a thermodynamically stable system in which small particles, generally ranging from 1 nanometer to 1 micrometer in size, are evenly homogeneously dispersed throughout a continuous phase, typically a liquid solvent. These particles remain uniformly distributed without settling due to gravity, thanks to mechanisms such as electrostatic or steric stabilization. Colloidal suspensions exhibit properties like the positive Tyndall effect, where light is scattered by the particles, and Brownian motion, where particles move randomly due to molecular collisions. This type of suspension does not dissolve over time in the absence of external energy but can aggregate.

[0037] As used herein the term “average particle size” shall be understood as the mean cumulant radius of particles in a sample, calculated by averaging the sizes of individual particles within the sample. This measurement is typically determined using techniques such as dynamic light scattering (DLS), laser diffraction, or electron microscopy. The average particle size cumulant radius provides a representative dimension of the particles.

[0038] As used herein the term “parenteral administration” shall be understood as the delivery of substances into the tissues and circulatory system via routes other than the digestive tract, typically involving injection or infusion. This method includes intravenous, intraarterial, intramuscular, subcutaneous, and intradermal injection, as well as intrathecal, epidural, intraosseous, intracisternal, intravitreal, intraarticular, intraperitoneal, intracardiac, intrapleural, intracerebroventricular, and intravesical administration, facilitating direct delivery of medications or nutrients into the bloodstream or tissues, ensuring rapid and controlled absorption.

[0039] As used herein the term “acetic salt of SEQ ID...” shall be understood as final result of the reaction of an acetic acid molecule (CH3COOH) with an amine group of a protein or peptide sequence, where the carboxyl group (COOH) of the acetic acid donates a proton (H+) to the peptide, involving an amine group (NH2) in the peptide. This results in the formation of an acetate ion (CH3COO“) paired with the protonated form of the peptide.

[0040] As used herein the term “% wt / wt” shall be understood as percent weight / weight, a concentration measurement that expresses the mass of a substance as a percentage of the total mass of the mixture. It is calculated by dividing the mass of the solute by the total mass of the solution or mixture and then multiplying by 100. This unit is commonly employed to specify the concentration of components in formulations, ensuring precise and consistent composition. Example: 1 % wt / wt of a solute in aqueous solutions is equivalent to 1 gram of solute in a final water weight of 100 grams, considering that water density at room temperature (20-25°C) is 0.9982 to 0.9970 g / ml the final volume of the solution is approximately equal to 100 ml.

[0041] As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the” also include the plural of the noun. Thus, for instance, if an item (such as a drug) is selected from a group consisting of a list of drugs, it is understood that it includes also a mixture of at least two drugs from the list.

[0042] The term "and / or" means that any one of the options to which it relates are possible or at least two options take place at the same time.

[0043] As used herein the term “Tris buffer” shall be understood as Tris(hydroxymethyl)aminomethane buffer, with chemical formula of Tris is (HOCH2)3CNH2, a widely used chemical buffer that maintains a stable pH in biological and chemical reactions. It is effective in the pH range from 7.0 to 9.0 and used in a concentration from 5 to 300 mM.

[0044] As used herein the term “Sodium bicarbonate buffer” (NaBC) shall be understood as a solution of sodium bicarbonate (NaHCO3) in water that helps maintain a stable pH. It acts as a weak base, neutralizing acids and stabilizing pH levels, commonly used in biological and medical applications, particularly to maintain the pH in the range of 7.0 to 8.5 and used in a concentration range from 5 to 150mM.

[0045] Examples of solubilizing agents useful in the present invention are: Dodecan-1-ol, ethoxylated; D-Glucopyranose, oligomeric, C8-16(even numbered) alkyl glycosides; 2- hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine 3a,12a-dihydroxy- 5p-cholan-24-oate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate; 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethan-1-ol; (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid; Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); poly[1-(2- oxopyrrolidin-1-yl)ethylene]; [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2- yl] (1 R,4S,5R,9S,10R,13S)-5,9-dimethyl-14-methylidene-13-[(2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9] hexadecane-5- carboxylate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine Dodecyl Sulfate; 2-[3-(dodecanoylamino) propyl-dimethylazaniumyl] acetate; Alcohols, C12-14 (even numbered), ethoxylated < 2.5 EO, sulfates, sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine salts; 2-[bis(2-hydroxyethyl) amino]ethan-1-ol; 2-{2-[3,4- bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate; and 4- (2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane.

[0046] As used herein “solubilizing agents” shall be understood as substances able to enhance the solubility of poorly soluble drug compounds in a given solvent, typically water. These agents facilitate the dissolution of the drug, making it more available for therapeutic action when administered systemically. From a thermodynamic perspective, solubilizing agents work by reducing the Gibbs free energy of the system, making the dissolved state of the drug more energetically favorable. This involves a combination of enthalpic, and entropic changes facilitated by the specific interactions between the solubilizing agent and the drug molecules in a water-based solvent.

[0047] As used herein the term “Disitertide” shall be understood as the acetic salt of the peptide sequence (H2N)-TSLDASIIWAMMQN-(COOH), having the amine group (-NH2) terminal end in the threonine (T) extreme, and the carboxylic group (-COOH) terminal end in the asparagine (N) extreme.

[0048] The present invention confronts the problem of providing a formulation of hydrophobic peptide / protein formulation, such as a Disitertide's formulation, suitable for parenteral administration. Parenteral formulations imply multiple controlled elements and parameters like pH, salt / buffer, different excipients, viscosity, osmolarity, particle size (in case of colloidal suspensions), active compound concentration, etc. The present invention details a process of parenteral formulation elements, parameters selection and optimization, to allow the solubilization of a hydrophobic peptide / protein such as Disitertide as a stable colloidal solution with controlled particle size and avoiding aggregation tendency, while maintaining the hydrophobic peptide / protein (such as Disitertide) stability and biological activity (in the case of Disitertide, as a TGF-p inhibitor). As an example, in Table 1a a selection of excipients is listed.

[0049] In an embodiment,

[0050] Table 1b below summarizes results of selected excipients after screening of efficient solubilizers of Disitertide at low excipients concentrations, including the following information in the table: chemical name, CAS number, molecular weight, molecular formula, structural formula (with noted hydrophilic and hydrophobic regions), Buffers, and Prometheus Panta equipment results (best lower effective excipient %, colloidal solution particle size radius (nm), polydispersity index (PDI), and diffusion coefficient in um2 / s) both in immediately prepared and post lyophilization samples. Selected excipients with Lower excipient efficient percentage ranges between 0.5 and 0.0015%, colloidal particle size cumulant radius values remain under 100 or even under 50 nm in some cases, polydispersity index values are under 0.5, in many cases under 0.3 and in some cases below 0.2. Diffusion coefficient ranges from 2.45 to 6.18, while in control buffers (Sodium Bicarbonate 50 mM and Tris 25 mM) in the absence of excipients PDI use to range from 1.5 to 2.5 values, indicating a higher propensity of aggregation.

[0051] Identified efficient excipients compounds include amphipathic surfactants (nonionic, anionic and amphoteric) monomeric or polymeric, inert polymers, alkaline agents, amino acids, amino alcohols and natural glucosides. All these efficient solubilizer excipients were selected between groups of other chemical compounds of these categories, also tested but with no solubilizer effect over Disitertide in these buffers and conditions (see table 4). The efficient selected tensoactives, detergents or surfactants (anionic, cationic, non-ionic, amphoteric), are defined as molecules, with some exceptions, with amphiphilic nature used as excipients, with solubilizing activity over Disitertide in solution, where the hydrophobic component of the molecule might vary among a methyl group, linear aliphatic saturated chains (from 8 to 16 carbons), or aliphatic branched structures like tert-butyloxycarbonyl radical (BOC), dioctyl group or polyethylene chains; cyclic aliphatic saturated or unsaturated structures like benzyl group, diterpenic structures (ent-Kaurene), Tert octyl phenyl group, tetramethyl butyl benzyl group or any of these chemical groups combinations or polymerized derivatives thereof. And the hydrophilic component of the amphiphilic excipient molecule may also vary among a cyclic glucose, succinate, hydroxyl or carboxyl groups; sodium, potassium, magnesium, zinc, calcium or monoethanolamine salts of sulfonate or sulfate; sulfonic acid group; sorbitan ethoxylated derivative groups, polyoxyethylene moieties or any combination of these chemical groups or their polymerized derivatives thereof. Table 1a

[0052] Table 1b

[0053] V: average. PDI: Polydispersity Index. NaBC: Sodium Bicarbonate. TRIS: Tris hydroximethyl-aminomethane The hydrophobic region of the molecule is indicated in dark grey, and the hydrophilic region, in light grey.

[0054] CAS number and molecular weight correspond to the main compound: cocam idopropyl betaine.

[0055] The condition with 50 mM Sodium Bicarbonate was discarded due to a high increase in the solution's pH. The condition with 25 mM TRIS was discarded due to the inability to dissolve Disitertide. The condition with 50 mM Sodium Bicarbonate was discarded due to the inability to dissolve Disitertide.

[0056] Table 2 presents solubility data [visual aspect, formulation particles size, polydispersity index and diffusion coefficient, before and after lyophilization in Sodium Bicarbonate (NaBC) and / or Tris buffer] of these commercial solubilizing excipients or products compared with two examples of selected compounds from the invention. Note that all commercial products were evaluated at least at 1 % concentration (as suggested ranges of use by the manufacturers), below this concentration percentage, solubilizing effect decrease drastically with all products in NaBC buffer. Some of the commercial solubilizing agents are not able to solubilize Disitertide, as demonstrated in the case of 2-(2-Ethoxyethoxy) ethanol in NaBC buffer and after lyophilization, in Gattefosse excipients in all conditions and Nonionic, ionic and Zwitterionic surfactants mix (Invitrogen) that generates Disitertide solutions with visible insoluble particles. In post-lyophilization samples of 11 -hydroxy- 17-[2- ethoxy]i5 heptadecanoic acid in TRIS there is an efficient solubilization effect over 0.125%, also with Poly(vinyl caprolactam-co-vinyl acetate-co-poly(ethylene glycol)) above 0.25% in NaBC and above 0.125% in Tris there is a significant solubilizing effect over Disitertide, and in the case of Glycerol polyethylene glycol ricinoleate the solubilizing effect is restricted to immediate preparation in NaBC over 0.5% and post-lyophilization over 0.5%. Considering the high percentages of commercial excipients used, and that particle size radius above 100 nm in radius and polydispersity index above 0.2 is defined as not an optimal solubilization state, the screening approach of the present invention yield superior outcomes, as demonstrated with the chosen examples (4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane and Sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2- sulfonate) using excipients percentages 5 to 30 times lower than those required with commercial solubilizer excipients, that is extent to even 200 timer lower required excipient percentage presence in the case of Alcohols, C12-14(even numbered), ethoxylated < 2.5 EG, sulfates, sodium salts or 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol where a 0.005% of the excipient is enough to reduce the Disitertide particle radius size below 100nm.

[0057] Table 2 R: out of range (above the particles size detection range of the Prometheus Panta equipment: > 10 mm).

[0058] Table 3 presents data of a T ris buffer 25mM based formulations, in the presence of 4-(2,4,4- trimethylpentan-2-yl) phenol, formaldehyde, oxirane (Tyloxapol) 0.065% and with different lyophilization agents (sugars: trehalose, isomaltulose, lactose, glucose and sucrose; polyols: mannitol and glycerol) at two different percentages. Samples analysis includes data of colloidal solutions particle size (nm), polydispersity index, Diffusion coefficient (mm2 / s) and thermal stability, measured as temperature (°C) of first inflexion point turbidity onset curve. Tyloxapol clearly reduces particle size and polydispersity index, and increases diffusion coefficient before and after lyophilization, thermal stability in the presence of Tyloxapol increases from 51.19 to 62.30 °C for inflexion of turbidity curve upon temperature. Evaluated lyophilization agents reveal that some are able to decrease particle size radius and PDI, like D-Mannitol at 5%, and / or increase thermal stability like Isomaltulose or sucrose, after lyophilization.

[0059] All samples with pH around 8 in TRIS buffer, Tyndall +, and transparent except for certain slight cloudiness in TRIS control sample. EOF-p-O CP: 4-(2,4,4-trimethylpentan-2-yl) phenol; formaldehyde; oxirane, also referred as Ethylene oxide-formaldehyde-p-octylphenol copolymer.

[0060] PDI: Polydispersity index.

[0061]

[0062] All samples with pH around 8 in TRIS buffer, Tyndall +, and transparent except some slight cloudiness in TRIS control sample.

[0063] 0: controls; EOF-p-O CP: 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane, also referred as Ethylene oxide-formaldehyde-p-octylphenol copolymer.

[0064] Table 4 summarizes solubility tests of Disitertide (1 mg / ml) with negative solubilization outcomes in the presence of different excipient types, after vortex and sonication, that includes different range results with particles size radius above 100 nm: negative results like Disitertide solutions presenting visible particles to the naked eye, that implies presence of particles above 40-50 microns of diameter, as for instance the case of solubilization test in PBS, sodium chloride 0.9%, ethanol, tartaric acid, para-amino benzoic acid, Sodium Lauryl Glutamate or Ammonium Lactate. Negative solubility outcomes also include colloidal turbid suspensions with particle size from 1 to 40 microns in diameter, like solutions containing Glutamic acid, Taurine, Sodium Alginate, and is also considered as negative results those suspension with particles between 0.2 to 1 micron in diameter (particles from 100 to 500 nm in radius) as for example solutions containing N,N-Dimethylethanolamine, polyethylene glycol 400, Decanoic acid or L-Lysine. Positive results in Disitertide solubilization are define as transparent colloidal solutions with particles size below 100 nm in cumulant radius, as measured using Dynamic Light Scattering technology in the Prometheus Panta equipment (NanoTemper Technologies, GmbH, Munich, Germany).

[0065] Table 4. Example of unsuccessful Disitertide solubilization attempts with different excipients.

[0066] It must be considered that the development and identification of a suitable formulation for the solubilization of Disitertide, in an acceptable dosage form compatible with clinically acceptable intravenous administration, is a very intricated and highly complex process. Solubility-enhancing pH ranges have been identified (Example 1 ; reference example), but these ranges alone do not resolve the issue due to their incompatibility with direct human administration, caused by excessive alkalinity. Moreover, among the potential saline / buffer compositions, only two have been found to facilitate reasonable pre-solubilization of Disitertide; most compositions are detrimental to solubilization even at favorable pH levels (Example 2; reference example). Additionally, due to the salting-in / out effect, an optimal range of salt concentrations exists within the selected buffers, deviations from this optimal concentration, whether higher or lower, adversely affect the solubilization of Disitertide, as demonstrated in Example 3 (reference example). At this step of development, Disitertide colloidal particle size is above 100 nm radius, not stable and out of the acceptable range for clinical administration. The presence of additional excipients is required in order to thermodynamically reduce the required energy to maintain stable lower particle sizes (Example 7). From the several excipients tested, only few (Example 4 and 7) were able to reduce the particle size in colloidal suspension of Disitertide, even commercial alternatives designed to improved hydrophobic compounds, including active pharmaceutical ingredients like insoluble proteins among others, fail to properly dissolve Disitertide or required substantial higher concentrations to approach similar results, being comparatively less efficient in solubilizing properties compared to the excipients indicated in the present invention (Example 5). Effective identified excipients (example 4) are necessary but not sufficient to generate the final solution, the combination with a proper pH and specific saline composition and concentration is required for the final optimal result. Selected excipients used in combination with other buffers or pH range do not perform with the same effectiveness or even are unable to dissolve Disitertide to a minimal extent. In further analysis, the functional intravenous formulation must present an adequate thermostability (Example 6), inferring equivalence to over time stability, also chemically stable due to the presence of antioxidant elements (Example 9), and compatible with lyophilization process (Table 3) and thereof compatible with lyophilization bulk agents (Example 8). Antioxidant and lyophilization agents should not negatively affect the solubility of Disitertide or even improve it as the case of Mannitol effect after lyophilization in table 3. In the end, but of cri ti ca I re I e va n ce , the final formulation must retain the biological activity of Disitertide with no significant variations, in order to provide its preventive or therapeutic clinical activity (Example 10). Therefore, the present invention is a definition of multi-parametric elements scope complex combination, required to obtain a clinically scalable intravenous formulation of Disitertide in the state of colloidal solution with clinically acceptable particle size.

[0067] The term "lipophilic" is used herein to describe drugs which are lipid-soluble and hydrophobic, i.e. which are insoluble or sparingly soluble in water.

[0068] The expression "parenteral" as used herein refers to routes of administration other than through the gastrointestinal tract or lungs, and to formulations for use in administering drugs by such routes. Thus, "parenteral" as used herein includes, for example, intramuscular, subcutaneous intra-articular (i.e. into the joint, which in turn includes intra-synovial, i.e. into the synovial fluid) and, especially, intravenous routes and formulations. The words "parenteral" and "injectable" are used interchangeably herein.

[0069] As already indicated, numerous drugs suffer from problems associated with their lack of water solubility and / or lack of stability in water. These lipophilic and / or water-labile drugs cannot be practically formulated as aqueous parenteral solutions. Consequently, the drugs are either unavailable for injection at the present time, or they are available for injectable use only in combination with undesirable organic vehicles. Injection of such vehicles is undesirable because of the systemic and local toxicity which can result, some of the organic solvents commonly used as vehicles include dimethylacetamide (DMA), dimethylsulfoxide (DMSO), propylene glycol (PG), benzyl alcohol and ethanol. Examples of the toxicity associated with these solvents include central nervous system depression, nystagmus, lymphocytosis, liver and kidney damage, blood disorders, jaundice, weight loss, anemia, convulsions, hallucinations, mutagenic effects, cyanosis, hypotension, bronchial spasms, cardiac standstill and death.

[0070] Moreover, parenteral administration of lipophilic or water-labile drugs in organic vehicles can result in precipitation of the drug at and / or near the injection site and / or in the lungs or other organs, which in turn leads to increased toxicity. Precipitation of drugs in the lungs, for example, has led to severe respiratory distress and even death in laboratory animals. On the other hand, when lack of a suitable solvent results in the fact that the drug is only available as an oral formulation, then bioavailability becomes a concern since drugs are frequently less bioavailable from oral delivery forms than they are from parenteral, especially intravenous, forms.

[0071] Among the lipophilic and / or water-labile drugs which are contemplated for use in aqueous parenteral formulations in accordance with the present invention, the following drugs are preferably included within the scope of the present invention: Disitertide, Gramicidin A, Cyclosporine, B-Amyloid, Melittin, Magainin, Dynorphin A, Dermcidin, Cecropin A, Maximin H5, Indolicidin, Piscidin, Thanatin, LL-37, Brevinin-1 , Protegin-1 , Dermaseptin, Cecropin, Buforin, Mastoparan, some collagen derived peptides like Gly-Pro-Hyp, Octreotide, Liraglutide, Glatiramer acetate, Enfuvirtide, Brimonidine, Exenatide, Desmopressin, Semaglutide, Apolipoproteins, Melanin-concentrating hormone, Neuropeptide Y, p- Endorphin, Corticotropin-releasing hormone, Encephalins, Galanin, Vasopressin, Bradykinin, Dalbavancin, Leuprorelin, Teriparatide, Teduglutide, Cetrotide, Oxytocin, Polymyxins, Daptomycin, Bacitracin, Tyrocidine, Insulin, Glucagon, Erythropoietin, Growth Hormone, p-Casein, Fibrinogens, Keratins, Albumins, Collagens, Interferons, some monoclonal antibodies (Bevacizumab, Cetuximab, Ipilimumab), some enzymes (a- Chymotrypsin, L-asparaginase, Lipases, membrane proteins (Na7K+-Transporting ATPase Subunit-a, Rhodopsin, Cytochrome P450 and B6f, Nav channel, ATP-binding Cassette Transporter A1 , Aquaporins, Voltage-Dependent Anion Channel 1 , Transferrin Receptor Protein 1 , NADH-ubiquinone oxidoreductase chain 1), Cholesterol esterase, Fatty Acid- Binding Protein, Platelet Glycoprotein 4, Myelin Basic Protein, Peripherin, Acetylcholinesterase, Carbonic anhydrase), Chimeric proteins (Imiglucerase, Abatacept, Elosulfase-a), and lectin proteins (agglutinins).

[0072] In one particularly preferred embodiment of the present invention, the drug contemplated for use in the instant parenteral formulations is the hydrophobic peptide or protein of SEQ ID NO 1 (TSLDASIIWAMMQN) or any pharmaceutically acceptable salt thereof; or any peptide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID No 1 and capable of retaining at least 50% of the capacity to inhibit the activity of TGF-p in an in vitro cell line bioassay with respect to SEQ ID No 1.

[0073] Therefore, as mentioned above, a first aspect of the invention refers to a pharmaceutical composition or formulation comprising: a) a hydrophobic peptide or protein; b) a water-soluble organic surfactant selected from the list consisting of non-ionic surfactants, anionic surfactants and amphoteric surfactants, amino acids, water-soluble polymer compounds and tertiary amino compounds; wherein a) and b) are dispersed in a salt solution forming a colloidal suspension having particles having average particle size of less than 100 nm cumulant radius as measured by dynamic light scattering, wherein the salt solution comprises a Tris buffer and / or a Sodium Bicarbonate buffer; wherein the pH of the composition is in the range of between 7.37 to 10, preferably between 7.5 and 10, more preferably between 8.5 and 10, still more preferably between 9.0 and 10, still more preferably between 9.5 and 10, and wherein the formulation is suitable for parenteral administration.

[0074] In a preferred embodiment of the first aspect of the present invention, the hydrophobic peptide or protein has a GRAVY (Grand average of hydropathicity index) value above zero and is selected from any one of the list consisting of: Disitertide, Gramicidin A, Cyclosporine, P-Amyloid, Melittin, Magainin, DynorphinA, Dermcidin, CecropinA, Maximin H5, Indolicidin, Piscidin, Thanatin, LL-37, Brevinin-1 , Protegin-1 , Dermaseptin, Cecropin, Buforin, Mastoparan, some collagen derived peptides like Gly-Pro-Hyp, Octreotide, Liraglutide, Glatiramer acetate, Enfuvirtide, Brimonidine, Exenatide, Desmopressin, Semaglutide, Apolipoproteins, Melanin-concentrating hormone, Neuropeptide Y, p-Endorphin, Corticotropin-releasing hormone, Encephalins, Galanin, Vasopressin, Bradykinin, Dalbavancin, Leuprorelin, Teriparatide, Teduglutide, Cetrotide, Oxytocin, Polymyxins, Daptomycin, Bacitracin, Tyrocidine, Insulin, Glucagon, Erythropoietin, Growth Hormone, p- Casein, Fibrinogens, Keratins, Albumins, Collagens, Interferons, some monoclonal antibodies (Bevacizumab, Cetuximab, Ipilimumab), some enzymes (a-Chymotrypsin, L- asparaginase, Lipases, membrane proteins (Na+ / K+-Transporting ATPase Subunit-a, Rhodopsin, Cytochrome P450 and B6f, Nav channel, ATP-binding Cassette Transporter A1 , Aquaporins, Voltage-Dependent Anion Channel 1 , Transferrin Receptor Protein 1 , NADH- ubiquinone oxidoreductase chain 1), Cholesterol esterase, Fatty Acid-Binding Protein, Platelet Glycoprotein 4, Myelin Basic Protein, Peripherin, Acetylcholinesterase, Carbonic anhydrase), Chimeric proteins (Imiglucerase, Abatacept, Elosulfase-a), and lectin proteins (agglutinins).

[0075] In another embodiment of the first aspect of the invention, the hydrophobic peptide or protein is a natural or synthetic peptide, linear or cyclic, or a salt thereof, the peptide comprising from 7 to 30 amino acids in length, comprising in their sequence 50% or more hydrophobic amino acids selected from the group consisting of leucine, alanine, glycine, valine, proline, phenylalanine, isoleucine, tryptophan, and methionine. Preferably, said hydrophobic amino acids are selected from the group consisting of alanine, leucine, isoleucine, tryptophan, and methionine. In another embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, said peptide exhibit an aqueous solubility of less than 1 mg / mL within a physiological pH range (7.0-7.5), and an isoelectric point within the acidic to neutral pH range (pH 2.5-7.5).

[0076] In another preferred embodiment of the first aspect of the present invention, the hydrophobic peptide or protein is of SEQ ID NO 1 (TSLDASIIWAMMQN) or any pharmaceutically acceptable salt thereof; or any peptide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID No 1 and capable of retaining at least 50% of the capacity to inhibit the antiproliferative activity of TGF-p in an in vitro proliferation cell line (Mv-1-Lu) bioassay with respect to SEQ ID No 1. As a side note, in vitro Mv-1-Lu TGF-p blocking activity bioassay consist briefly of: Mv-1-Lu cells (American Type Culture Collection Rockville, MD) were cultured (5x103 cells / well) in a 96-well flat-bottomed plate (Costar Corporation CA, USA) at 37 °C, 5% CO2 overnight in complete medium (RPMI 1640 containing L-glutamine and supplemented with 5% fetal calf serum and antibiotics). TGF-p inhibitor Peptides (50 to 200 pg / ml) were tested by triplicate in the presence of 200 pg / ml of added TGF-pi (R&D, Minneapolis, MN). After 12h incubation, 1 pCi of (methyl-3H)thymidine (Amersham Life Science, Buckinghamshire, UK) was added per well and incubated for 12 h. Cells were harvested (Titertek Cell Harvester Inc., Sterling, USA) and radioactivity measured on a beta scintillation counter (Top count, Packard, Meriden, CT, USA). As positive and negative controls, Mv-1-Lu cells grown in absence or presence of human TGF-pi (Roche Diagnostics, Mannheim, Germany), were used respectively. Inhibition of TGF-pi was calculated using the formula: % inhibition of TGF-p = 100 (cpm Pep cpm NC) / (cpm PC cpm NC) where Pep = peptide tested; NC = (negative control): cells in the presence of TGF-pi , but without peptide; PC = (positive control): cells containing neither TGFpi nor peptide, cpm: counts per minute. Alternatively, other in vitro bioassays suitable for measuring TGF-p activity, and therefore also applicable to assessing the inhibitory effect of this cytokine, can be equivalently employed to evaluate the inhibitory capacity of Disitertide and derived peptide sequences. Such assays include, but are not limited to wound healing assay, spheroid formation or invasion assays, TGF-p induced reporter gene assays (e.g., luciferase, HEK-Blue™ [secreted embryonic alkaline phosphatase], green or red fluorescent protein), fibroblast-to-myofibroblast transition assays, Transwell migration and invasion assays, and collagen gel contraction assays.

[0077] In another preferred embodiment of the first aspect of the present invention, the hydrophobic peptide or protein is of SEQ ID NO 1 (TSLDASIIWAMMQN) or any functional variant thereof having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID No 1 , such as SEQ ID NO 2 (TSLMIWTMM) or SEQ ID NO 3 (TSLDATMIWTMMA), and capable of retaining at least 50% of the capacity to inhibit the antiproliferative activity of TGF-p in an in vitro proliferation cell line (Mv-1-Lu) bioassay with respect to SEQ ID No 1 .

[0078] In another preferred embodiment of the first aspect of the present invention, the hydrophobic peptide or protein is of SEQ ID NO 1 (TSLDASIIWAMMQN) or any functional variant thereof comprising or consisting of SEQ ID NO 4 (TSLXXSIIWXMMXX), wherein X is understood as any amino acid, preferably SEQ ID NO 1 (TSLDASIIWAMMQN).

[0079] In another preferred embodiment of the first aspect of the present invention, the hydrophobic peptide or protein is an acetic salt of SEQ ID NO 1 (TSLDASIIWAMMQN), preferably at a concentration in the composition or formulation of less than 20 mg / ml, preferably less than 10 mg / ml, more preferably between 1 mg / ml and 20 mg / ml, more preferably between 1 mg / ml and 10 mg / ml, more preferably between 2 mg / ml and 5 mg / ml, more preferably between 3 mg / ml and 4 mg / ml.

[0080] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the salt solution comprises or consists of a sodium bicarbonate solution at a concentration equal or below 150 mM, preferably equal or below 100 mM, more preferably between 25 and 75 mM, preferably at a concentration of 50 mM.

[0081] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the salt solution comprises or consists of a Tris buffer at a concentration equal or below 300 mM, preferably equal or below 100mM, more preferably between 10 and 50 mM, preferably at a concentration of 25 mM.

[0082] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the salt solution comprises or consists of a Tris buffer and a Sodium Bicarbonate solution at concentrations equal or below 100 mM of each buffer, preferably between 5 and 25 mM of each buffer.

[0083] From hereinafter, any one of the above-mentioned dosages for the tris buffer and / or Sodium bicarbonate solution shall be referred to herein as “buffer concentration of the invention”. It is herein noted that a much-preferred concentration for the Tris buffer is 25 mM, and a much- preferred concentration for the Sodium Bicarbonate is 50 mM.

[0084] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the formulation comprises a water-soluble organic surfactant selected from the group consisting of: Dodecan-1-ol, ethoxylated; D-Glucopyranose, oligomeric, C8-16(even numbered) alkyl glycosides; 2-hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine 3a, 12a- dihydroxy-5p-cholan-24-oate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate; 2-[4-(2,4,4- trimethylpentan-2-yl)phenoxy]ethan-1-ol; (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid; Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); poly[1-(2-oxopyrrolidin-1-yl)ethylene]; [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S)-5,9-dimethyl-14-methylidene-13- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo

[0085] [11.2.1.01 ,10.04,9] hexadecane-5-carboxylate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine Dodecyl Sulfate; 2-[3-(dodecanoylamino) propyl- dimethylazaniumyl] acetate; Alcohols, C12-14 (even numbered), ethoxylated < 2.5 EO, sulfates, sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine salts; 2- [bis(2-hydroxyethyl) amino]ethan-1-ol; 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate; and 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane; as well as any pharmaceutical acceptable salt thereof. For example, sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine salts. In the present document, to avoid any misinterpretation of the written scope of each chemical compound, different chemical compounds are separated by semicolons (;) and the internal chemical elements of each compound are separated by commas (,).

[0086] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of poly[1-(2-oxopyrrolidin-1-yl)ethylene]; 2-hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12- trimethyltridecyl)chroman-6-yl succinate; (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid; 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate; Sodium; 1 ,4-bis(2-ethylhexoxy)-1 ,4- dioxobutane-2-sulfonate; Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol); and pharmaceutical acceptable salt thereof.

[0087] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the salt solution comprises or consists of a Tris buffer, wherein the pH is from 8.5 to 10, preferably, 8.6.

[0088] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the Tris buffer is at a concentration from 10 to 50 mM, preferably at a concentration of 25 mM, particularly, wherein the pH is from 8.5 to 10, preferably, 8.6.

[0089] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the salt solution comprises or consist of a sodium bicarbonate buffers a concentration from 40 to 60 mM, preferably at a concentration of 50 mM, particularly, wherein the pH is from 8.5 to 10, preferably, 8.6.

[0090] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the salt solution comprises a Tris at a concentration from 5 to 25 mM and sodium bicarbonate is at a concentration from 5 to 25 mM, preferably at a concentration of Tris from 5 to 10 mM such as of 6.25 mM and sodium bicarbonate from 5 to 15 mM such as of 12.5 mM, particularly, wherein the pH is from 8.5 to 10, preferably, 8.6.

[0091] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of poly[1-(2- oxopyrrolidin-1-yl)ethylene], wherein said compounds comprises or consists of present in the formulation at a concentration from 0.001% to 0.5%, preferably from 0.005% to 0.25% (%wt / wt).

[0092] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of 2-hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate, wherein said compound is present in the formulation at a concentration from 0.005% to 0.5% (%wt / wt), preferably from 0.015% to 0.05% (%wt / wt).

[0093] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of (2S)-2-amino-5- (diaminomethylideneamino) pentanoic acid, wherein said compound is present in the formulation at concentration from 0.05% to 0.5% (%wt / wt), preferably from 0.05% to 0.25% (%wt / wt).

[0094] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of 2-{2-[3,4-bis(2- hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate, wherein said compound is present in the formulation at a concentration from 0.0005% to 0.05% (%wt / wt), preferably from 0.001 % to 0.005% (%wt / wt).

[0095] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of sodium; 1 ,4-bis(2- ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate, wherein said compound is present in the formulation at a concentration from 0.05% to 0.5% (%wt / wt), preferably from 0.1% to 0.25% (%wt / wt).

[0096] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), wherein said compound is present in the formulation at a minimum concentration from 0.005% to 0.05% (%wt / wt), preferably from 0.005% to 0.01 % (%wt / wt).

[0097] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of a mixture of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), poly[1-(2- oxopyrrolidin-1-yl)ethylene], and 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate.

[0098] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of a mixture of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), (2S)-2- amino-5-(diaminomethylideneamino) pentanoic acid, and 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate.

[0099] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant comprises or consists of a mixture of (2S)- 2-amino-5-(diaminomethylideneamino) pentanoic acid, poly[1-(2-oxopyrrolidin-1- yl)ethylene], and 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate.

[0100] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the pharmaceutical formulation further comprises an antioxidant agent selected from the group consisting of L-Arginine, L-Histidine, L-Lysine, b-Alanine, L-Methionine, L- Tryptophan, N-Acetyl L-Cysteine, L-Cysteine, Taurine, Betaine, Sarcosine, L-Carnitine. In a particular embodiment, the antioxidant is at a concentration from 0.01% to 0.05% (%wt / wt), particularly L-Histidine. In a more particular embodiment, the antioxidant is L-Histidine.

[0101] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the pharmaceutical formulation further comprises lyophilization agents selected from trehalose, isomaltulose, lactose, glucose, sucrose, D-mannitol and glycerol, particularly D-Mannitol. In a particular embodiment, the lyophilization agent is at a concentration from 2.5% to 5% (%wt / wt). In a more particular embodiment, the lyophilization agent is D-Mannitol.

[0102] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of 2- [4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol, wherein said compounds are present in the formulation at a minimum concentration of about 0.003% (%wt / wt), preferably in the range of from 0.003% to 0.5% (%wt / wt), preferably in the range of from 0.003% to 0.05% (%wt / wt), and wherein the salt solution is a T ris buffer at preferably any of the concentrations defined in the buffer concentration of the invention. In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of: sodium Dodecan-1-ol, ethoxylated; Sodium 3a,12a-dihydroxy-5p-cholan-24-oate; sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4- dioxobutane-2-sulfonate; Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol); 2-[3-(dodecanoylamino)propyl-dimethylazaniumyl]acetate; Alcohols, C12-14(even numbered), ethoxylated < 2.5 EO, sulfates, sodium salts; 2-[bis(2- hydroxyethyl)amino]ethan-1-ol; 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane, wherein said compound is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), more preferably in the range of from 0.005% to 0.25% (%wt / wt), wherein the salt solution is a Sodium Bicarbonate and / or Tris buffer at preferably any of the concentrations defined in the buffer concentration of the invention.

[0103] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of 2- hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate, wherein said compound is present in the formulation at a minimum concentration of about 0.01% (%wt / wt), preferably in the range of from 0.015% to 0.5% (%wt / wt), preferably in the range of from 0.015% to 0.05% (%wt / wt), and the salt solution is a Tris buffer at preferably any of the concentrations defined in the buffer concentration of the invention.

[0104] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of D- Glucopyranose, oligomeric, C8-16 (even numbered) alkyl glycosides and wherein said compound is present in the formulation at a minimum concentration of about 0.02% (%wt / wt), preferably in the range of from 0.02% to 0.5% (%wt / wt), preferably in the range of from 0.02% to 0.05% (%wt / wt), and the salt solution is preferably a Tris buffer at preferably any of the concentrations defined in the buffer concentration of the invention.

[0105] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of Sodium Dodecyl Sulfate; and 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde oxirane, wherein said compounds is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), preferably in the range of from 0.005% to 0.05% (%wt / wt), and the salt solution is: a. a Tris buffer, preferably at any of the concentrations defined in the buffer concentration of the invention, if 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane is selected; or b. a sodium bicarbonate buffer, preferably at any of the concentrations defined in the buffer concentration of the invention, if sodium Dodecyl Sulfate is selected.

[0106] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of Dodecan-1-ol, ethoxylated; Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol); and 2-[3-(dodecanoylamino)propyl-dimethylazaniumyl]acetate; and wherein said compound is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), wherein the salt solution is a Sodium Bicarbonate and / or Tris buffer at preferably any of the concentrations defined in the buffer concentration of the invention.

[0107] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of Sodium Dodecyl Sulfate; and 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane; and wherein said compound is present in the formulation at a minimum concentration of about 0.005%, preferably in the range of from 0.005% to 0.5% (%wt / wt), and the salt solution is a Tris buffer if sodium Dodecyl Sulfate is selected, wherein the salt solution is preferably at any of the concentrations defined in the buffer concentration of the invention.

[0108] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of D- Glucopyranose, oligomeric, C8-16(even numbered) alkyl glycosides; 2-{2-[3,4-bis(2- hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate, and 4-(2,4,4- trimethylpentan-2-yl) phenol, formaldehyde, oxirane; wherein said compound is present in the formulation at a minimum concentration of about 0.02%, preferably in the range of from 0.05% to 0.5% (%wt / wt), and wherein the salt solution is preferably a Sodium bicarbonate buffer or a Sodium bicarbonate or Tris buffer if 2-{2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]- 2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate is selected, wherein the salt solution is preferably at any of the concentrations defined in the buffer concentration of the invention.

[0109] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of 2- hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate; poly[1 - (2-oxopyrrolidin-1-yl)ethylene]; and ([(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S)-5,9-dimethyl-14-methylidene-13- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2- yl]oxytetracyclo[11.2.1.01 ,10.04,9]hexadecane-5-carboxylate; wherein said compound is present in the formulation at a minimum concentration of about 0.01 %, preferably at a minimum concentration of 0.05%, preferably in the range of from 0.05% to 0.5% (%wt / wt), and wherein the salt solution is a Sodium bicarbonate buffer if 2-hydroxyethyl 2, 5,7,8- tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate or [(2S,3R,4S,5S,6R) -3,4,5- trihydroxy-6-(hydroxymethyl) oxan-2-yl] (1 R,4S,5R,9S,10R,13S) -5,9-dimethyl -14- methylidene -13- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy -6- (hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9] hexadecane-5-carboxylate are selected and a Tris buffer if poly[1-(2-oxopyrrolidin-1-yl)ethylene] is selected, wherein the salt solution is preferably at any of the concentrations defined in the buffer concentration of the invention.

[0110] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is from the group consisting of (2S)-2-amino- 5-(diaminomethylideneamino) pentanoic acid, and [(2S,3R,4S,5S,6R) -3,4,5- trihydroxy-6- (hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S) -5,9-dimethyl-14-methylidene-13- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9]hexadecane-5-carboxylate; wherein said compounds are present in the formulation at a minimum concentration of about 0.001%, preferably in the range of from 0.005% to 0.5% (%wt / wt), and wherein the salt solution is a Tris buffer if [(2S,3R,4S,5S,6R)- 3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S)-5,9-dimethyl-14- methylidene-13-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9] hexadecane-5-carboxylate is selected, and wherein the salt solution is preferably at any of the concentrations defined in the buffer concentration of the invention.

[0111] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the water-soluble organic surfactant is selected from the group consisting of sodium 3a,12a-dihydroxy-5p-cholan-24-oate, Sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4- dioxobutane-2-sulfonate, and poly[1-(2-oxopyrrolidin-1-yl)ethylene]; wherein said compounds are present in the formulation at a minimum concentration of about 0.5% and wherein the salt solution is a T ris buffer if Deoxycholic acid sodium salt or Sodium 1 ,4-bis(2- ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate are selected and a carbonate buffer if poly[1-(2- oxopyrrolidin-1-yl)ethylene] is selected, preferably wherein the buffers are present at any of the concentrations defined in the buffer concentration of the invention.

[0112] Please note that this aspect of the invention includes any of the formulations indicated in table 1.

[0113] In another preferred embodiment of the first aspect of the present invention, optionally in combination with any one of the previous preferred embodiments of the first aspect of the invention, the formulation has a lyophilization stabilizer, and the stabilizer is selected from Glucose, Trehalose, Mannitol, Sucrose, Lactose, Inositol, Xylose, Xylitol, Isomaltulose, Sorbitol, Maltitol, Mannose, Fructose, Proline, Arginine, Histidine or Glycine in a percentage presence of 0.1 to 10%, more preferably in a percentage from 0.5 to 5%. In accordance with this particular embodiment, the composition of the first aspect of the invention is a lyophilized composition or formulation preferably with neutral or positive effect of the lyophilization bulking agent over Disitertide solubility, or any other hydrophobic protein or peptide of interest, colloidal particle size and stability, respect the formulation prior lyophilization process. Lyophilization, freeze-drying, process briefly consist of a rapid sample freezing (< -50°C) and pressure reduction (< 0.2 mBar) to sublimate the ice sample content (primary drying) and gradually increase temperature to remove residual moisture (secondary drying). This process is required to preserve the drug's stability and extends its shelf life for intravenous administration. The manufacture of the formulation for freeze- drying requires to combine all its components (water, salts, pH adjustments, all excipients, and active pharmaceutical ingredient) including the lyophilization agent in a percentage that generates the final adequate outcome, defined as a stable uniformly porous surface dry matrix, without signals of shrinkage or cracking, that maintains the active ingredient integrity, biological activity and can be easily reconstituted in water for parenteral administration. Table 3 presents some example results of an efficient solubilizing formulation in the presence of two percentages of different lyophilization agents, with different outcomes after freeze-drying and resuspension in water comparing with initial samples, also in table 2 the variations after this process could be observed. The presence of an adequate freeze-drying agent, in the appropriate quantity, is necessary to maintain the solubility and stability qualities of the starting formulation, or ideally optimize these required qualities, after dry sample resuspension prior its clinical practice administration.

[0114] In a second aspect of the invention, the formulation or composition as defined in accordance with the first aspect of the invention or with any of the preferred embodiments of the invention, is for use in diseases prevention or therapy.

[0115] In a third aspect of the invention, the formulation or composition as defined in accordance with the first aspect of the invention or with any of the preferred embodiments of the invention, is for use, via parental administration, in a method of treatment of fibrosis-based diseases selected from the group consisting of lung fibrosis induced by environmental exposure to chemicals, allergens and particles, smoking, therapeutic severe effect after chemo and / or radiotherapy and antibiotics, asthma, chronic obstructive pulmonary disease, polymyositis, bronchitis, rhinosinusitis, mucositis, autoimmune disease like rheumatoid arthritis, progressive systemic sclerosis, Sjogren's syndrome or sarcoidosis, adult respiratory distress syndrome, chronic or severe infections like tuberculosis or viruses, Idiopathic pulmonary fibrosis, and adult respiratory distress syndrome. Also including Cardiac fibrosis induced by chronic hypertension, primary pulmonary hypertension, polymyositis, familial pulmonary hypertension, pre-eclampsia, atherosclerosis, restenosis, and hypertrophic cardiomyopathy and congestive heart failure, myocardial infarction, cardiomyopathies. Additionally comprising other cardiovascular diseases such as hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome), Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome. Likewise, Renal fibrosis induced by chronic kidney disease (diabetic nephropathy and / or hypertension), glomerulonephritis, chronic pyelonephritis, obstructive uropathy, polycystic kidney disease, ischemia-reperfusion injury, nephrotoxins, interstitial nephritis (drugs or recurrent infections), autoimmune diseases, chronic allograft nephropathy or genetic conditions (autosomal dominant polycystic kidney disease, Alpor syndrome, tuberous sclerosis complex, nephronophthisis, etc.). In addition, Liver fibrosis induced by chronic viral hepatitis, chronic alcohol consumption, autoimmune hepatitis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, biliary or sclerosing cholangitis, chemo- or radiotherapy, genetic diseases (hemochromatosis, Wilson's disease, a-1 antitrypsin deficiency, chronic biliary obstruction, drugs induce injury, parasites infections (schistosomiasis, echinococcosis, dietary or environment toxins, metabolic disorders and chronic cholestasis. Moreover, ophthalmic fibrosis induced by proliferative vitreoretinopathy, diabetic retinopathy, retinal vein occlusion, uveitis, retinal detachment, retinitis pigmentosa, infections, surgical adverse complications, inherited retinal disorders, glaucoma or dry and wet age-related macular degeneration. Further, also including pancreatic fibrosis generated through chronic pancreatitis, cystic fibrosis, or diabetes. Also including clinically relevant deep connective tissues related to deep skin and muscles fibrosis induced by profound wounds (surgical interventions: keloids and hypertrophic scars), third grade burns (sun, chemicals, electrical, cold and hot), radiotherapy, autoimmune diseases (scleroderma, lupus, rheumatoid arthritis). Including other connective tissue diseases such as Marfan syndrome, Marfan-like disorders, also addressing skeletal and muscular disorders like Camurati-Engelmann disease, fibrodysplasia ossificans progressiva, Hunter-Thompson and Grebe-type chondrodysplasias, osteoporosis, sclerosteosis, Van Buchem disease, brachydactyly, symphalangism, and Duchenne muscular dystrophy; post-surgical or inflammatory stenosis affecting trachea, intestines, ureters, urethra, bile ducts, vascular vessels, lymphatic vessels and spinal canal; oncological diseases selected from the group consisting of gliomas, neuroendrocrine tumors, blastomas, melanomas, germ cell tumors, mesothelioma, carcinomas (adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, basal cell carcinomas), sarcomas, myelomas, leukemias, lymphomas, and mixed tumor types (carcinosarcomas, adenosquamous carcinomas, teratomas) originated at or metastasized to brain, spinal cord, meninges, peripheral nerves, retina, salivary glands, head and neck tissues, thyroid, oral mucosa, throat, lung, breast, esophagus, stomach, small intestine, colon, rectum, pancreas, liver, bile ducts, blood, bone marrow, vascular or lymphatic vessels, lymph nodes, pleural / peritoneal membranes, bones, cartilage, adipose tissue, skeletal muscle, smooth muscle, skin, Kidney, bladder, prostate, testicle, ovarium, uterus, endometrium, cervix, vulva,. Also including hereditary rare cancer syndromes like juvenile polyposis syndrome, hereditary nonpolyposis colorectal cancer.

[0116] In a preferred embodiment of the third aspect of the invention, the composition is Disitertide and is for use in the treatment of diseases or conditions mediated by TGF-p biological activity. This growth factor mediates a wide range of pathological effects, including diseases where TGF-p presents a clear and pivotal role inducing fibrosis, tumor progression, angiogenesis and potent immunomodulatory effects, among others physio-pathological cascades. Parenteral administration of Disitertide is intended to prevent or treat clinical conditions like all that implies a profibrotic component, immunomodulatory alterations, infectious diseases, tumoral processes including metastases, cardiovascular diseases, neurological disorders, developmental disorders and metabolic and bone conditions. On the other hand, the parenteral formulations of Disitertide in accordance with the present invention are intended for the treatment of pathologies related to fibrosis-based diseases that present an extracellular matrix alteration in connective tissues involving deposition of large amounts of proteins (collagens, fibronectins, elastins, laminins, etc.) with altered organization. These alterations are induced by long-term chronic inflammation and affect the mechanical and physiological properties of tissues and organs. Fibrotic based diseases include, but are not limited, to lung fibrosis induced by environmental exposure to chemicals, allergens and particles, smoking, therapeutic severe effect in chemoradiotherapy and antibiotics, asthma, chronic obstructive pulmonary disease, polymyositis, bronchitis, rhinosinusitis, mucositis; autoimmune disease like rheumatoid arthritis, progressive systemic sclerosis, Sjogren's syndrome or sarcoidosis, adult respiratory distress syndrome, chronic or severe infections like tuberculosis or viruses, and Idiopathic pulmonary fibrosis, adult respiratory distress syndrome. Also including cardiac fibrosis induced by chronic hypertension, primary pulmonary hypertension, polymyositis, familial pulmonary hypertension, pre-eclampsia, atherosclerosis, restenosis, hypertrophic cardiomyopathy and congestive heart failure, myocardial infarction, cardiomyopathies. Additionally comprising other cardiovascular diseases such as hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome), Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome. Renal fibrosis induced by chronic kidney disease (diabetic nephropathy and / or hypertension), glomerulonephritis, chronic pyelonephritis, obstructive uropathy, polycystic kidney disease, ischemia-reperfusion injury, nephrotoxins, interstitial nephritis (drugs or recurrent infections), autoimmune diseases, chronic allograft nephropathy or genetic conditions (autosomal dominant polycystic kidney disease, Alpor syndrome, tuberous sclerosis complex, nephronophthisis, etc.). In addition, Liver fibrosis induced by chronic viral hepatitis, chronic alcohol consumption, autoimmune hepatitis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, biliary or sclerosing cholangitis, chemo- or radiotherapy, genetic diseases (hemochromatosis, Wilson's disease, a-1 antitrypsin deficiency, chronic biliary obstruction, drugs induce injury, parasites infections (schistosomiasis, echinococcosis, dietary or environment toxins, metabolic disorders and chronic cholestasis. Moreover, ophthalmic fibrosis induced by proliferative vitreoretinopathy, diabetic retinopathy, retinal vein occlusion, uveitis, retinal detachment, retinitis pigmentosa, infections, surgical adverse complications, inherited retinal disorders, glaucoma or dry and wet age-related macular degeneration. Additionally, pancreatic fibrosis generated through chronic pancreatitis, cystic fibrosis, or diabetes. Also including clinically relevant deep connective tissues related to deep skin and muscles fibrosis induced by profound wounds (surgical interventions: keloids and hypertrophic scars), third grade burns (sun, chemicals, cold, electrical, hot), radiotherapy, autoimmune diseases (scleroderma, lupus, rheumatoid arthritis). Including other connective tissue diseases such as Marfan syndrome, Marfan-like disorders, also addressing skeletal and muscular disorders like Camurati-Engelmann disease, fibrodysplasia ossificans progressiva, Hunter-Thompson and Grebe-type chondrodysplasias, osteoporosis, sclerosteosis, Van Buchem disease, brachydactyly, symphalangism, and Duchenne muscular dystrophy. Post-surgical or inflammatory stenosis affecting trachea, intestines, ureters, urethra, bile ducts, vascular vessels, lymphatic vessels and spinal canal. Also, is clinically relevant the fibrotic processes related with peritoneal walls sclerosis induced peritoneal dialysis and the peritoneal postsurgical adhesions (WO 089443 A2 (2010) - “Pharmaceutical compositions comprising TGF-beta 1 inhibitor peptides”, WO 031135 A1 (2000): “TGF(b)1 Inhibitor Peptides”, Guo W wt al. Targeting the TGF-p signaling pathway: an updated patent review (2021-2023)”. Expert Opinion on Therapeutic Patents Volume 34, 2024 issue. Ren L.L. et al. “Transforming growth factor-p signaling: From tissue fibrosis to therapeutic opportunities”. Chem Biol Interact. 2023 Jan 5:369:110289, Biernacka A. et al. “TGF-p signaling in fibrosis”. Growth Factors. 2011 Oct;29(5):196-202. Kumari L.A. et al. “New rationales for using - inhibitors in radiotherapy.” Int J Radiat Biol. 2007 Nov-Dec;83(11-12):803-11.).

[0117] Additionally, the parenteral formulations of Disitertide are intended for the treatment of oncological diseases where TGF-p mediates several major induction effects over tumoral progression and survival tumor microenvironment mechanisms like tumorigenesis, tumor invasiveness, angiogenesis, local immunosuppression, tumor stromal induction and metastasis, among others. These oncological clinical conditions include, but are not limited to, gliomas, neuroendrocrine tumors, blastomas, melanomas, germ cell tumors, mesothelioma, carcinomas (adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, basal cell carcinomas), sarcomas, myelomas, leukemias, lymphomas, and mixed tumor types (carcinosarcomas, adenosquamous carcinomas, teratomas) originated at or metastasized to brain, spinal cord, meninges, peripheral nerves, retina, salivary glands, head and neck tissues, thyroid, oral mucosa, throat, lung, breast, esophagus, stomach, small intestine, colon, rectum, pancreas, liver, bile ducts, blood, bone marrow, vascular or lymphatic vessels, lymph nodes, pleural / peritoneal membranes, bones, cartilage, adipose tissue, skeletal muscle, smooth muscle, skin, Kidney, bladder, prostate, testicle, ovarium, uterus, endometrium, cervix, vulva, among others. Also including hereditary rare cancer syndromes like juvenile polyposis syndrome, hereditary nonpolyposis colorectal cancer, Bannayan-Riley-Ruvalcaba syndrome, and Cowden syndrome (US 0326243 A1 (2017) - “Methods and Systems for Treating or Preventing Cancer”. WO 101225 A1 (2009) - “Pharmaceutical composition for cancer treatment”, WO 048857 (2007) - “Use of TGF-BETA 1 inhibitor peptides in the preparation of an immune response modulating agent”.), Ali S. et al. “TGF-p signaling pathway: Therapeutic targeting and potential for anti-cancer immunity”. Eur J Pharmacol. 2023 May 15:947:175678., and Saunier E.F. et al. “TGF beta inhibition for cancer therapy”. Curr Cancer Drug Targets. 2006 Nov;6(7): 565-78.). In some embodiments, parenteral formulations of Disitertide are intended to provide improvement in anemia forms cause by Ineffective or altered erythropoiesis and cardiovascular diseases, including but not limited to Myelodysplastic Syndromes, a- and b- thalassemia, chronic inflammation related anemias, anemia in chronic kidney disease, aplastic anemia, Diamond-Blackfan anemia (WO 079591 A2 (2017) - “Methods for Increasing Red Blood Cell Levels and Treating Ineffective Erythropoiesis”, Zhou L. et al. “Transforming growth factor-beta in hematopoiesis: recent insights and clinical implications”. Current Opinion in Hematology, 2014. 18(4), 267-272. and Suragani, R. N. V. S. et al. “Transforming growth factor-p superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis”. Nature Medicine, 2014. 20(4), 408-414., WO 018662 A1 (2019)- “TGF-Beta Inhibition to Treat Hematologic Symptoms of Shwachman-Diamond Syndrome”, WO 079591 A2 (2017) - “Methods for Increasing Red Blood Cell Levels and Treating Ineffective Erythropoiesis”).

[0118] TGF-p plays a crucial role in immune regulation, including immunosuppression. Some pathogens exploit TGF-p that serves as an immunosuppressant in favor of the infectious agent to evade the host immune response, enhancing their survival and persistence. Several infectious diseases caused by viruses, bacteria, fungi, protozoa, and small parasites can be treated through TGF-p inhibition to increase immune specific immune responses against the infectious agent. In this manner, the Disitertide parenteral formulations are intended for the treatment of pathologies related to microorganisms inducing immunosuppression mediated by TGF-p including, but no limited to, Hepatitis B and C virus, human immunodeficiency virus (HIV), influenza virus, Epstein-Barr virus, herpes simplex virus, Cytomegalovirus, respiratory syncytial virus, Mycobacterium tuberculosis, Helicobacter pylori, Borrelia burgdorferi, Staphylococcus pneumoniae and S. aureus, Chlamydia trachomatis, Mycoplasma spp., Candia albicans, Histoplasma Capsulatum, Cryptococcus neoformans, Blastomyces dematitidis, Aspergillus fumigatus, Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Leishmania spp., Toxoplasma gondii, Entamoeba histolytica, Giardia lamblia, Babesia spp., and also small parasites infections like Schistosoma spp., Fasciola hepatica, Echinococcus granulosus, Stronfyloides 048857 (2007) - “Use of TGF-BETA 1 inhibitor peptides in the preparation of an immune response modulating agent”, and Reed S.G. “TGF-p in infections and infectious diseases”. Microbes and Infection. Volume 1 , Issue 15, December 1999, Pages 1313-1325.).

[0119] Furthermore, the parenteral formulations of Disitertide in accordance with the present invention are intended for the treatment of pathologies related to the prevention, treatment or progression delay of Inflammatory or immune disorder like autoimmune diseases, or clinical conditions, including but not limited to systemic sclerosis, psoriasis, left-sided ulcerative colitis, inflammatory bowel disease, periodontal disease, peri-implantitis, interstitial cystitis, atrophic vaginitis, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, ankylosing spondylitis, Sjogren's syndrome, autoimmune hepatitis, arthrosis, atopic dermatitis, systemic sclerosis, scleroderma, rosacea, allergic rhinitis, chronic sinusitis, myalgic encephalomyelitis, asthma, chronic obstructive pulmonary disease, atherosclerosis, osteoarthritis, autoimmune thyroid disease (Graves' disease, Hashimoto's thyroiditis), Addison's disease, pernicious anemia, autoimmune uveitis, vasculitis, dermatomyositis, myasthenia gravis, pemphigus vulgaris, coeliac disease, primary biliary cholangitis, vitiligo, dermatomyositis, alopecia areata, Crest's syndrome, chronic pancreatitis, interstitial cystitis, chronic inflammatory demyelinating polyneuropathy, eosinophilic esophagitis, gout, sarcoidosis, rheumatic polymyalgia, Behget's disease, Crohn's disease, idiopathic thrombocytopenic purpura, severe combined immunodeficiency, cryoglobulinemia. Furthermore, including clinical situations that require controlled reduction of immune responses against certain treatments based on gene vectors, cellular or biological therapy (WO 173829 A1 (2019) - “Delivering biological drugs to tissues”, WO 048857 (2007) - “Use of TGF-BETA 1 inhibitor peptides in the preparation of an immune response modulating agent” and Prud'homme G.J. et al. “The inhibitory effects of transforming growth factor-beta-1 (TGF-beta1) in autoimmune diseases”. J Autoimmun. 2000 Feb; 14(1):23-42.). In certain embodiments, the formulations are designed to treat bone diseases associated with elevated or decrease turnover, including but not limited to osteogenesis imperfecta (Type I to XI), McCune-Albright syndrome, Gaucher disease, hyperoxaluria, Paget disease of bone, juvenile Paget disease, osteoporosis, fibrous dysplasia, bone metastasis (WO 027329 A1 (2018) - “TGF-Beta Antagonist Conjugates” WO 113123 A1 (2019) - “TGF-p receptor fusion proteins and other TGF-p antagonists for reducing TGF-p signaling”.

[0120] In particular, hereafter is referred a compilation of potential activity evidence of Disitertide systemic administration, of non-soluble suspended particles, in intended clinical applications animal models related to fibrosis efficacy proofs of concept, like fibrogenic processes inhibition in peritoneum for peritoneal dialysis solutions induce damage (Loureiro J. et al. “Blocking TGF- i protects the peritoneal membrane from dialysate-induced damage”. J Am Soc Nephrol. 2011 Sep; 22(9):1682-95 and Busnadiego O. et al. “A pathogenetic role for endothelin-1 in peritoneal dialysis-associated fibrosis”. J Am Soc Nephrol. 2015 Jan; 26(1): 173-82.) and postsurgical peritoneal adhesions (Sandoval P. et al. “Mesothelial-to-mesenchymal transition in the pathogenesis of post-surgical peritoneal adhesions”. _J Pathol. 2016 May; 239(1):48-59.), in radiotherapy induced fibrosis (Cruz- Morande S. et al. “P144 a Transforming Growth Factor Beta Inhibitor Peptide, Generates Antifibrogenic Effects in a Radiotherapy Induced Fibrosis Model”. Curr Oncol. 2022 Apr 12; 29(4):2650-2661.), in corpus cavernosum fibrosis induced by diabetes, trauma or hypertension (Li WJ. et al. “P144, A TGF-pi antagonist peptide, synergizes with sildenafil and enhances erectile response via amelioration of cavernosal fibrosis in diabetic rats”. J Sex Med. 2013 Dec; 10(12):2942-51.), in liver fibrosis as consequence of chronic liver damage and inflammation in viral hepatitis, chronic alcohol consumption, etc. (Ezquerro IJ. et al. “A synthetic peptide from transforming growth factor beta type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury”. Cytokine. 2003 Apr; 22(1-2):12-

[0121] 20.), in cardiac fibrosis due to infarction, hypertension, diabetes or idiopathic background (Hermina N. et al. “A synthetic peptide from transforming growth factor-betal type III receptor prevents myocardial fibrosis in spontaneously hypertensive rats”. Cardiovasc Res. 2009 Feb 15; 81 (3):601-9.), in prosthetic implants tissue fibrotic reactions like silicone implants (Ruiz-de-Erenchun R. et al. “Use of the transforming growth factor-betal inhibitor peptide in peri prosthetic capsular fibrosis: experimental model with tetraglycerol dipalmitate” Plast Reconstr Surg. 2005 Oct; 116(5): 1370-8. and San-Martin A. et al. “Effect of the inhibitor peptide of the transforming growth factor beta (p144) in a new silicone pericapsular fibrotic model in pigs”. Aesthetic Plast Surg. 2010 Aug; 34(4):430-7.) in renal fibrosis related to diabetes, hypertension or chronic kidney diseases (Baltanas A. et al. “A synthetic peptide from transforming growth factor-Pi type III receptor inhibits NADPH oxidase and prevents oxidative stress in the kidney of spontaneously hypertensive rats”. Antioxid Redox Signal. 2013 Nov 10; 19(14): 1607-18., Li D. et al. “TGF-pi peptide-based inhibitor P144 ameliorates renal fibrosis after ischemia-reperfusion injury by modulating alternatively activated macrophages”. Cell Prolif. 2022 Oct; 55(10): e13299. and Allam A. et al. “Omeprazole induces profibrotic gene expression in rat kidney: implication of TGF- p / Smad signaling pathway”. Drug Chem Toxicol. 2023 Nov 20:1-8.). Also, here included a compilation of potential activity evidence of Disitertide parenteral administration in intended clinical application through animal models related efficacy proofs of concept in tumours development, like primary tumours inhibition in models of colon cancer (Medina-Echeverz J. et al. “Overexpression of apolipoprotein A-l fused to an anti-transforming growth factor beta peptide modulates the tumorigenicity and immunogenicity of mouse colon cancer cells”. Cancer Immunol Immunother. 2015 Jun; 64(6):717-25.), brain tumours: glioblastoma (Gallo-Oller G. et al. “P144, a Transforming Growth Factor beta inhibitor peptide, generates antitumoral effects and modifies SMAD7 and SKI levels in human glioblastoma cell lines”. Cancer Lett. 2016 Oct 10; 381 (1):67-75.), melanoma (Diaz-Valdes N. et al. “Induction of monocyte chemoattractant protein-1 and interleukin-10 by TGFbetal in melanoma enhances tumor infiltration and immunosuppression”. Cancer Res. 2011 Feb 1 ; 71 (3):812-

[0122] 21.), and inhibition of metastasis process like lung cancer to lymph node metastasis (Salvo E. et al. “Combined targeting of TGF-pi and integrin p3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer”. Mol Cancer. 2014 May 19:13:112.), lung cancer to bone metastasis (Vicent S. et al. “A novel lung cancer signature mediates metastatic bone colonization by a dual mechanism”. Cancer Res. 2008 Apr 1 ; 68(7):2275- 85.), colorectal cancer to liver metastasis (Zubeldia IG. et al. “Epithelial to mesenchymal transition and cancer stem cell phenotypes leading to liver metastasis are abrogated by the novel TGFpi -targeting peptides P17 and P144”. Exp Cell Res. 2013 Feb 1 ; 319(3):12-22. and Medina-Echeverz J. “Harnessing high density lipoproteins to block transforming growth factor beta and to inhibit the growth of liver tumor metastases”. PLoS One. 2014 May 5; 9(5):e96799), colorectal carcinoma to lung metastasis (Wu N. et al. “LINC00941 promotes CRC metastasis through preventing SMAD4 protein degradation and activating the TGF- P / SMAD2 / 3 signaling pathway”. Cell Death Differ. 2021 Jan; 28(1):219-232. and Li T. et al. “Mesenchymal Stem Cell-Derived Exosomal microRNA-3940-5p Inhibits Colorectal Cancer Metastasis by Targeting Integrin a6”. Dig Dis Sci. 2021 Jun;66(6):1916-1927.), and inhibition of tumour angiogenesis (Serrati S. et al. “TGFbetal antagonistic peptides inhibit TGFbetal- dependent angiogenesis”. Biochem Pharmacol. 2009 Mar 1 ; 77(5):813-25. and Margheri F. et al “GDF5 regulates TGFp-dependent angiogenesis in breast carcinoma MCF-7 cells: in vitro and in vivo control by anti-TGFp peptides”. PLoS One. 2012; 7(11): e50342.), and immunotherapy effects over tumour growth in lymphoma (Llopiz D. et al. “Peptide inhibitors of transforming growth factor-beta enhance the efficacy of antitumor immunotherapy”. Int J Cancer. 2009 Dec 1 ; 125(11):2614-23), and melanoma (Diaz-Valdes N. et al. “Induction of monocyte chemoattractant protein-1 and interleukin-10 by TGFbetal in melanoma enhances tumor infiltration and immunosuppression”. Cancer Res. 2011 Feb 1 ; 71(3):812- 21 .). Also, here included a compilation of potential activity evidence of Disitertide parenteral administration in intended clinical application through animal models related efficacy proofs of concept in immunomodulatory infectious disease like malaria (Ocaha-Morgner C. et al. “Role of TGF-beta and PGE2 in T cell responses during Plasmodium yoelii infection”. Eur J Immunol. 2007 Jun; 37(6): 1562-74.), and chronic liver virus infection like hepatitis B virus (Otano I. et al. “Modulation of regulatory T-cell activity in combination with interleukin-12 increases hepatic tolerogenicity in woodchucks with chronic hepatitis B”. Hepatology. 2012 Aug; 56(2):474-83.). Not excluding other references of preclinical efficacy proofs of concept of Disitertide in cartilage damage and repair (Sanz-Ramos P. et al. “The role of Alk-1 and Alk-5 in the mechanosensing of chondrocytes”. Cell Mol Biol Lett. 2014 Dec; 19(4):659-74.), inner ear cell damage protection in noise and chemical induced deafness (Sanz L. et al. “Swept-sine noise-induced damage as a hearing loss model for preclinical assays”. Front Aging Neurosci. 2015 Feb 16; 7:7. and Murillo-Cuesta S. et al. "Transforming growth factor pi inhibition protects from noise-induced hearing loss”. Front Aging Neurosci. 2015 Mar 20; 7:32.), aortic aneurism prevention in Marfan Syndrome (Arce C. et al. “Anti-TGFp (Transforming Growth Factor P) Therapy with Betaglycan-Derived P144 Peptide Gene Delivery Prevents the Formation of Aortic Aneurysm in a Mouse Model of Marfan Syndrome”. Arterioscler Thromb Vase Biol. 2021 Sep; 41(9): e440-e452.), osteointegration of titanium implants (Sevilla P. et al. “In vitro cell response on CP-Ti surfaces functionalized with TGF-pi inhibitory peptides”. J Mater Sci Mater Med. 2018 May 23; 29(6):73., and Cirera A. et al. “Biofunctionalization with a TGFp-1 Inhibitor Peptide in the Osseointegration of Synthetic Bone Grafts: An In Vivo Study in Beagle Dogs”. Materials (Basel). 2019 Sep 27; 12(19):3168.), oxidative stress damage in kidneys with hypertensive background (Baltanas A. et al. “A synthetic peptide from transforming growth factor-Pi type III receptor inhibits NADPH oxidase and prevents oxidative stress in the kidney of spontaneously hypertensive rats”. Antioxid Redox Signal. 2013 Nov 10; 19(14): 1607-18. and Miguel-Carrasco JL. et al. “Blockade of TGF-pi signaling inhibits cardiac NADPH oxidase overactivity in hypertensive rats”. Oxid Med Cell Longev. 2012; 2012: 726940), corneal fibrosis with opacity (US 0315256 A1(2012) - Use of Transforming Growth Factor - beta 1 (TGF-pi) inhibitor peptides for the treatment of corneal fibrosis and / or haze”), treatment of dry eye, also known as keratoconjunctivitis sicca, dysfunctional tear syndrome, Syndrome de Sjogren, (WO 148155 A1 (2013) - “Compositions and methods for the treatment of dry eye disease”), and vascular retinal damage like in age related macular degeneration and retinitis pigmentosa (Recalde S. et al. “Transforming growth factor-p inhibition decreases diode laser-induced choroidal neovascularization development in rats: P17 and P144 peptides”. Invest Ophthalmol Vis Sci. 2011 Sep 9; 52(10):7090-7., and Zarranz-Ventura J. et al. “Transforming growth factor-beta inhibition reduces progression of early choroidal neovascularization lesions in rats: P17 and P144 peptides”. PLoS One. 2013 May 31 ; 8(5): e65434.).

[0123] The invention is illustrated further in the following, non-limiting examples.

[0124] EXAMPLES

[0125] EXAMPLE 1 (Reference Example)

[0126] Disitertide solubility as function of pH pH is a relative logarithmic measurement scale (-Iog10 C, where C= H+ion concentration moles per liter) in aqueous solutions indicating acidity (<7) or basicity (>7). pH is specifically a measure of the relative amount of free hydrogen (H+) and hydroxyl ions (OH-) in aqueous solutions, ranging from 0 to 14 with 7 as relative neutral value.

[0127] The net charge on organic compounds, including proteins and peptides, at any given pH is determined by the pK values (pKs) of the ionizable groups (Tanford, 1962). The net charge of a protein / peptide is zero at the isoelectric point (pl), positive at pHs below the pl, and negative at pHs above the pl. In this context, Disitertide is a 14-mer peptide (TSLDASIIWAMMQN) with an isoelectric point at pH 3.1-3.8 (upon sources, net charge=0), +1 charge at pH below 2, -1 charge at pH=7 and -2 charge at pH above approx. 9.5. Accordingly, at pH above 9.5 the net charge of Disitertide increases from -1 to -2, as a result the peptide becomes more polar and increases its water solubility. At pH range from 5-7.5 that include physiological pH (7.37-7.42), Disitertide present low polarity and extremely low water solubility. The worst pH for solubilization ranges around 3.75 where the net charge of Disitertide is close to zero. Above pH 9.5 the Aspartic residue (Asp: D) generates an extra negative charge from -1 to a final net charge of -2, increasing polarization and therefore increasing Disitertide general solubility. Values of pH were analyzed in every sample, for some assays pH was adjusted in order to establish the preferred pH for Disitertide solubility enhancement and pH interactions.

[0128] Disitertide solubility at 1 mg / ml was tested in Milli-Q® ultrapure water with different final pH values. pH was adjusted, after addition of Disitertide, with concentrated NaOH or HCI and measured with a PAL-pH DPH pHmeter (ATAGO CO., LTD.) adding 600 ml of samples over the glass electrode surface holder. Disitertide solubility was monitored by optical microscope oxion inverso OX.2053-PLPH (Euromex Ltd.) and images were recorded with digital Euromex camara CMEX DC.5000f. with the Image Focus software (Euromex, Microscopen bv, Netherlands) in a laptop computer. Solutions turbidity was measure through absorbance (optical density quantification) at 415 nm wavelength in transparent 96 well flat bottom plates in a Tekan Life Sciences equipment (Tecan Trading AG, Switzerland) model INFINITE M NANO+. Tyndall colloidal particles observation was performed with a red laser pointer (635 nm) and direct visual detection.

[0129] As illustrated in FIG. 1 Disitertide 1 mg / ml in ultrapure water at neutral pH (7) results in lack of solubility, but this solubility is a function of basic pH (presented pH values are final pH measurements, adjusted after Disitertide addition). FIG. 1A shows microscopic images of Disitertide particles at x4 augmentation at different pH with qualitative analysis of positive or negative Tyndall effect (T+ / T-) and macroscopical precipitation (P+ 1 P-). pH above 9.5 generates a translucid water solution with slight positive for colloidal particles perform with Tyndall test, and no visible precipitation. Above pH 11 the solution becomes Tyndall negative (free of colloidal particles). In FIG. 1 B presents turbidity measurements at 415nm of the same samples, showed in section A, indicating a reasonably good solubility above pH 9.5. It is noted that lower pH induces stronger insoluble aggregation density of Disitertide and pH above 9.5 generate clear solutions images (these results are restricted to ultrapure water used as solvent). Solubility of Disitertide could vary significantly in different saline solutions or buffers compositions and different concentrations of salts (see example 2; reference example).

[0130] EXAMPLE 2 (Reference Example)

[0131] Effect of salt solution / Buffers over Disitertide solubility

[0132] A salt solution is a dissolution in water of a chemical molecule made of a metal cation and a non-metal anion that dissociates in water following a chemical equilibrium of associationdissociation. A balance salt solution is defined as a salt solution with physiologic pH (7.37- 7.42) and isotonic concentration (300-312 mOsm / L), although many standard intravenous infusions in clinical use could differ to a certain extent from these osmolarity and pH values of reference.

[0133] When a salt solution is composed by an acid or a base aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa, it is called a buffer solution and is defined by its pH slightly changes when a small amount of strong acid or base is added.

[0134] Disitertide solubility enhancement with different salts and buffers solutions at different pH were monitored through macroscopical observation, microscopic images, Tyndall test, and turbidity (absorbance) to asset solubility, as described in (reference) example 1. Additional quantification of particle size analysis (cumulant radius) and polydispersity index were performed with Prometheus Panta equipment (NanoTemper Technologies, GmbH, Munich, Germany) using dynamic light scattering (DLS) measurements carried out with the Size Analysis function of the Panta Control software. Samples were transferred to capillaries (volume of =10 ml / capillary) and loaded onto the instrument sample tray. Measurements were carried out using the high sensitivity mode in the Size Analysis function of the Panta Control software.

[0135] As presented in FIG. 2, solubilization potential of different solvents / buffers were tested, at different initial pHs. It must be taken under consideration that Disitertide presents a pH acidification effect in some solutions without buffering capacity, especially in pure water. Different salt solutions and buffers generate different solubilization potential over Disitertide, concluding that the interaction between pH and salt composition of a solution or buffer has an independent effect on the solubility of Disitertide, at same pHs some salt solutions and buffers provide favorable or detrimental chemical environment for Disitertide solubilization. Examples of favorable buffers include Sodium Bicarbonate (Be), Tris and Arginine buffer. As examples of most detrimental solutions for Disitertide solubility can be found phosphate buffer saline (PBS), Ringer Lactate and saline solution among others (ver Fig. 2). Combinations of Sodium Bicarbonate and Tris buffers (TBC) maintain reduction of colloidal particles size of Disitertide at combinations in the range from 5 to 25mM of each buffer.

[0136] EXAMPLE 3 (Reference Example)

[0137] Effect of ionic strength over Disitertide solubility

[0138] A salt solution in water is defined as a solution of salts able (buffers) or not (solutions) to avoid strong pH variations. As previously established, Disitertide solubility due to its peptide nature is sensitive to the salt composition and pH of an aqueous solution, as well as the presence of other elements. Therefore, Disitertide behaves as a simple electrolyte in solution, making it susceptible to the ionic concentration of the medium, which can alter the peptide's net charge and solubility in water. Furthermore, salt ions concentration impact Disitertide solubility: at low ionic strengths, increased salt concentration typically enhances Disitertide solubility, a phenomenon known as "salting in". Conversely, at higher ionic strengths, Disitertide solubility may decrease, in an event termed "salting out". The salt concentration range between “salting in” and “salting out” varies depending on the composition of different solutions, pH or buffers.

[0139] Disitertide solubility enhancement, with different concentrations of salts and buffers solutions at same pH range, were monitored through macroscopical observation and microscopic images. Additional quantification of particle size analysis (cumulant radius) and polydispersity index (PDI) were performed with Prometheus Panta equipment (NanoTemper Technologies, GmbH, Munich, Germany) using dynamic light scattering (DLS) measurements carried out with the Size Analysis function of the Panta Control software. Samples were transferred to capillaries (volume of =10 ml / capillary) and loaded onto the instrument sample tray. Measurements were carried out using the high sensitivity mode in the Size Analysis function of the Panta Control software. FIG. 3A presents the effect of different concentrations of salt / buffer solutions at the same initial pH range from 8 to 7 along dilutions from the initial solutions. Disitertide presents a clear incompatibility with saline solution at any dilution from physiological concentration 0.9% (155 mM) to 12.5 mM, Phosphate Buffer Saline (PBS) is also an inefficient solubilizer of Disitertide although the presence of insoluble particles is lower at concentration from 1x to 0.25x. As shown in FIG. 3B, selected best Disitertide solubilizer buffers, Sodium Bicarbonate (NaBC) and Tris buffers, present an optimal solubilizing concentration around 50 mM for BC and 25 mM for Tris, presenting lower particles size and lower PDI at these concentrations, in comparison with lower or higher concentrations from these values, due to “salting in and out” effect over Disitertide solubility. Different combinations of Sodium Bicarbonate and Tris buffers, from 5 to 25mM permutations, maintain the reduction effect over Disitertide radius particle size respect these buffers independently.

[0140] EXAMPLE 4

[0141] Effect of excipients over Disitertide solubility measured as particle size in colloidal suspensions

[0142] Pharmaceutical excipients are defined as inactive substances that serve as the vehicle or medium for a drug or other active compound administration. Excipients are used together with active compounds (APIs) to generate pharmaceutical dosage forms. The excipients are considered as inert substances, i.e., they do not have any active role in therapeutics, but they can be used to support the process to produce an effective product (Dureja and Kumar, 2013), this include a wide range of excipients activities as binders, cosolvents, fillers, disintegrants, lubricants, surfactants, emulsifying agents, antimicrobials, preservatives, lubricants, suspending substances, colorants, solvents, ointment bases, flow modifiers, tonicity adjusters, solubilizer agents, antioxidants, antiaggregant agents, antifoaming substances, etc. More specifically, in parenteral formulations the main excipient types are preservatives, pH adjusters, buffers, tonicity adjuster (osmolarity), stabilizers, viscosity modifiers, antioxidants and solubilizers. In addition, these excipients activities can be categorized into four main functions: Provide bulk (scalability) to the formulation, facilitate drug bioavailability, solubility, or other key pharmacokinetic consideration, aid API handling during manufacturing and provide stability and prevent from denaturation, aggregation, oxidation, etc. Also, excipients should be ideally feasible, pharmacologically inert, not able to interact with the API, cost effective and stable for handling. Being solubility in aqueous solutions the main challenging issue in the development of Disitertide parenteral formulations, the initial and primary approach to the analysis of excipients screening was to determine their effect on Disitertide solubility measured as particles size in a range of nm in colloidal suspension, being more soluble as the particle size radius is progressively reduced in the presence of the excipient.

[0143] In order to analyze the effect of different excipients over Disitertide solubility, different excipients percentages were tested with 1 mg / ml of Disitertide in different solvents. Initially water, saline solution (NaCI 0.9%, phosphate buffer saline (PBS), Sodium Bicarbonate buffer 1 ,26% (150 mM) and TRIS Buffer (300 mM), and after buffer optimization only Sodium Bicarbonate buffer 50 mM and TRIS buffer 25 mM were tested with different excipients (around 160 compounds) and their combinations. The resulting formulations were analyzed by Optical microscope (visible and subvisible particles at micrometric range), pHmeter, Optical densitometry (absorbance 415nm) measure turbidity, laser Tyndal effect to detect qualitative colloidal suspensions and Prometheus Panta analysis to measure particle size (nanometric cumulant radius range: 1nm - 10.000nm), and polydispersity index, as well as Diffusion coefficient and thermal stability in each formulation solution.

[0144] Table 1 summarizes results of selected excipients after screening of efficient solubilizers of Disitertide at low excipients concentrations, including the following information in the table: chemical name, CAS number, molecular weight, molecular formula, structural formula (with noted hydrophilic and hydrophobic regions), Buffers, and Prometheus Panta equipment results (best lower effective excipient %, colloidal solution particle size radius (nm), polydispersity index (PDI), and diffusion coefficient in um2 / s) both in immediately prepared and post lyophilization samples. Selected excipients with Lower excipient efficient percentage ranges between 0.5 and 0.0015%, colloidal particle size cumulant radius values remain under 100 or even under 50 nm in some cases, polydispersity index values are under 0.5, in many cases under 0.3 and in some cases below 0.2. Diffusion coefficient ranges from 2.45 to 6.18, while in control buffers (Sodium Bicarbonate 50mM and Tris 25 mM) in the absence of excipients PDI use to range from 1 .5 to 2.5 values, indicating a higher propensity of aggregation. Identified efficient excipients compounds include amphipathic surfactants (nonionic, anionic and amphoteric) monomeric or polymeric, inert polymers, alkaline agents, amino acids, amino alcohols and natural glucosides. All these efficient solubilizer excipients were selected between groups of other chemical compounds of these categories, also tested but with no solubilizer effect over Disitertide in these buffers and conditions. The efficient selected tensoactives, detergents or surfactants (anionic, cationic, non-ionic, amphoteric), are defined as molecules with amphiphilic nature used as excipients, with solubilizing activity over Disitertide in solution, where the hydrophobic component of the molecule is among a methyl group, linear aliphatic saturated chains (from 8 to 16 carbons), or aliphatic branched structures like tert-butyloxycarbonyl radical (BOC), dioctyl group or polyethylene chains; cyclic aliphatic saturated or unsaturated structures like benzyl group, diterpenic structures (ent-Kaurene), Tert octyl phenyl group, tetramethyl butyl benzyl group or any of these chemical groups combinations or polymerized derivatives thereof. And the hydrophilic component of the amphiphilic excipient molecule is among a cyclic glucose, succinate, hydroxyl or carboxyl groups; sodium, potassium, magnesium, zinc, calcium or monoethanolamine salts of sulfonate or sulfate; sulfonic acid group; sorbitan ethoxylated derivative groups, polyoxyethylene moieties or any combination of these chemical groups or their polymerized derivatives thereof.

[0145] EXAMPLE 5 (Examples and Comparative Examples)

[0146] Comparison with commercial excipients / kits specifically intended for hydrophobic drugs solubilization

[0147] Active pharmaceutical ingredients (APIs) solubility, in aqueous solutions, is one of the most challenging problems in drug products development in the pharmaceutical industry, around 40% of approved drugs and 90% of under development APIs present poor water solubility (Liu X. et al. “Improving solubility of poorly water-soluble drugs by protein-based strategy: A review”. International Journal of Pharmaceutics. Volume 634, 5 March 2023, 122704) . For this reason, commercial excipients designed to optimize drug solubility are continuous development. The following commercial solubilizing excipients (Comparative Examples) or products: 2-(2-Ethoxyethoxy)ethanol (Gattefosse, topical formulations), Propane-1 , 2-diol monocaprylate and 1 ,2,3-Propanetriol, mono(3-(octanoyloxy)propyl) ether (Gattefosse, oral formulations), Nonionic, ionic and Zwitterionic surfactants mix (Invitrogen, proteins solubilizer kit), Glycerol polyethylene glycol ricinoleate (BASF, parenteral formulation) 11- hydroxy-17-[2-ethoxy]i5 heptadecanoic acid (BASF, parenteral and oral formulation), polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (BASF, oral formulation), polyamino acid-based polymer (Lubrizol Corporation, parenteral formulation); have been analyzed in comparison with some selected excipient examples of the invention (4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane and Sodium 1 ,4-bis(2- ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate) respect Disitertide solubility measured as the effect over the particle size radius and polydispersity index in colloidal suspensions of preferred buffers and pH, analyzed in the Prometheus Panta equipment (NanoTemper Technologies, GmbH, Munich, Germany). Table 2 presents solubility data [visual aspect, formulation particles size, polydispersity index and diffusion coefficient, before and after lyophilization in Sodium Bicarbonate (NaBC) and / or Tris buffer] of these commercial solubilizing excipients or products compared with two examples of selected compounds from the invention. Note that all commercial products were tested at least at 1% concentration (as suggested ranges of use by the manufacturers), below this concentration percentage, solubilizing effect decrease drastically with all products in NaBC buffer. Some of the commercial solubilizing agents are not able to solubilize Disitertide, as demonstrated in the case of 2-(2-Ethoxyethoxy) ethanol in NaBC buffer and after lyophilization, in Gattefosse excipients in all conditions and Nonionic, ionic and Zwitterionic surfactants mix (Invitrogen) that generates Disitertide solutions with visible insoluble particles. In postlyophilization samples of 11 -hydroxy-17-[2-ethoxy]is heptadecanoic acid in TRIS there is a efficient solubilization effect over 0.125%, also with Poly(vinyl caprolactam-co-vinyl acetate- co-poly(ethylene glycol)) above 0.25% in NaBC and above 0.125% in Tris there is a significant solubilizing effect over Disitertide, and in the case of Glycerol polyethylene glycol ricinoleate the solubilizing effect is restricted to immediate preparation in NaBC over 0.5% and post-lyophilization over 0.5%. Considering the high percentages of commercial excipients used, and that particle size radius above 100 nm in radius and polydispersity index above 0.2 is defined as not an optimal solubilization state, the screening approach of the present invention yield superior outcomes, as demonstrated with the chosen examples (4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane and Sodium 1 ,4-bis(2- ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate) using excipients percentages 5 to 30 times lower than those required with commercial solubilizer excipients.

[0148] EXAMPLE 6

[0149] Thermal stability of Disitertide solubility in different colloidal suspension formulations

[0150] Thermal stability stress testing is a standard approximation to evaluate a pharmaceutical product degradation analysis, that provides preliminary useful data to determine the physicochemical stability of the active pharmaceutical ingredient, potential aggregation tendency and issues with the final product under storage, regulatory compliance across product quality attributes, etc. Selected formulations, with Disitertide particle size radius below 100 nm and polydispersity index below 0.3, were analyzed in the Prometheus Panta equipment (NanoTemper Technologies, GmbH, Munich, Germany) undergoing a temperature stability gradient from 25°C to 95°C and back to 25°C (FIG. 4) while measuring the following parameters: particle size (cumulant radius), polydispersity index, fluorescence ratio (350 / 330 nm), fluorescence ratios first derivative, turbidity, and light scattering.

[0151] Particle size indicates initial grade of colloidal solubilization and aggregation trend along temperature gradient increase, polydispersity index represents the distribution of particles size (considered standard acceptable homogeneity <0.2), fluorescence ratio (350 / 330 nm) and its first derivative measure thermal structural modifications that affects Disitertide tryptophan residue aromatic ring exposure as function of the temperature gradient, turbidity is the macroscopic cloudiness of a solution due to suspended particles, and scattering is the microscopic light reflection where type and intensity depends on particles concentration and size. All these parameters could present significant variations through the temperature increase gradient.

[0152] FIG. 4 represents particles size and polydispersity index (PDI: linear histogram width) in the first graphics column, and temperature ramp (slope: 25 to 95 to 25°C) induced changes in structural state (fluorescence ratio 350 / 330 and first derivative), and aggregation trend as particles size (cumulant radius in nm) in the rest of the represented graphics. Conditions consist of Disitertide at 1 mg / ml in control Sodium Bicarbonate (NaBC) and Tris buffer prepared immediately (PI) and after resuspension of lyophilization samples (PL), in the presence or absence of Copolymer Poly(ethylene glycol)-block-poly(propylene glycol)- block-poly(ethylene glycol) (CP 2-MOxrn / Oxrn) at 0.05 and 0.1 %, as example or effective solubilizer excipient. Results show that Poly(ethylene glycol)-block-poly(propylene glycol)- block-poly(ethylene glycol) clearly reduces Disitertide particle size radius and PID compared with respective control buffers. Regarding thermal stability, Tris buffer is superior to NaBC regarding structural stability, but NaBC is superior as aggregation stabilizer respect TRIS. The lyophilization process improves the stability of Disitertide in NaBC, while its effect in Tris shows a clear trend of reduced aggregation, but only slight and inconclusive changes in structural stability. Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) in NaBC increases Disitertide’s stability to some extent, with the melting temperature (Tm) rising from 58.63°C to 62.91 °C. The rest of the parameters remain stable, except for particle size, which is unaffected up to 80°C. In Tris buffer, Poly(ethylene glycol)-block- poly(propylene glycol)-block-poly(ethylene glycol) leads to some improvement in structural stability but also causes a clear increase in aggregation (particle size) as temperature rises, more intensely in both PI and PL samples. The formulation in Tris with 0.1 % Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) presents the best overall stability profile.

[0153] FIG.5 shows temperature ramp (slope: 25 to 95°C) induced changes in structural stability state (fluorescence ratio 350 / 330 and first derivative), turbidity, aggregation trend as particles size (cumulant radius in nm), and scattering. Conditions consist of Disitertide at 1 mg / ml in control Sodium bicarbonate (NaBC) and Tris buffer prepared immediately (PI) and after resuspension of lyophilization samples (PL) in the presence or absence of Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) dilutions (0.25, 0.125, 0.0625, 0.03125 and 0.015625 %), as example or effective solubilizer excipient. In PI samples, NaBC buffer improves stability in all tested concentrations, best stability profile is obtained at lower Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol)concentrations. Although no relevant differences were detected with respect to particle size evolution along thermal ramp, Poly(ethylene glycol)-block- poly(propylene glycol)-block-poly(ethylene glycol) at 0.025 and 0.03125% maintain lower variations in particle size radius. Tris buffer presents similar behavior than NaBC, in combination with Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), but with even lower variations in structural stability and aggregation rate (particle size) in at 0.025 and 0.03125% of Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol). In PL samples NaBC improve thermal stability at low Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)concentrations, being 0.015625% the optimal one in particle size stability, but with a slight increase in turbidity. After lyophilization, Tris buffer samples behave very close to PI samples. Lower Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) concentrations are more efficient maintaining stability respect higher ones, although all tested concentrations stabilize samples better than control buffer. At high concentrations of Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), fluorescent ratio 350 / 330nm is higher although stable through the thermal ramp. In summary, lower Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) concentrations perform better, stabilizing parameter during the thermal increase, than higher ones in NaBC, both in IP and PL. In Tris buffer low concentrations stabilize particle size along the thermal ramp. These results are an example of performed thermal stability analysis with all selected excipients presented in example 4, table 1 .

[0154] EXAMPLE 7

[0155] Excipients effect over Disitertide aggregation rate in solution measured as Gibbs free energy thermodynamic potential.

[0156] The aggregation tendency of a solute in aqueous solution is a parameter inversely correlated with solubility. Aggregation potential of Disitertide, in different excipients formulations, was measured as the Gibbs aggregation free energy defined by (AG), and source from colloidal Disitertide particles size value, that was experimentally obtained at a constant temperature (25°C) using dynamic light scattering (DLS) technology in a Prometheus Panta equipment (NanoTemper Technologies, GmbH, Munich, Germany). Gibbs AG (kcal / mol) measures the difference in free energy balance between enthalpy / entropy required to modify a thermodynamic system. With respect to the aggregation trend of Disitertide, AG measures the change in free energy when molecules of Disitertide bind and generate aggregation complexes, as an indicator of spontaneous binding reaction. Negative values for Gibbs AG indicate a spontaneous solubilization reaction that releases energy with aggregation trend, and positive values indicate nonspontaneous reaction that requires external energy to allow solubilization and high aggregation trend.

[0157] AG = AH - T-AS

[0158] Where

[0159] AG = Gibbs free energy, AH = change in enthalpy, AS = change in entropy and T= temperature in K°.

[0160] There is a relationship between the Gibbs free energy and particle size in colloidal suspensions of hydrophobic compounds in aqueous solutions. This relationship can be described using concepts from thermodynamics and surface chemistry. In colloidal systems, the Gibbs free energy change associated with particle size can be influenced by surface energy contributions. For a colloidal particle, the total Gibbs free energy (AG) can be expressed as the sum of the bulk Gibbs free energy and the surface Gibbs free energy. The formula can be derived as follows:

[0161] AG — AGbulk + AGsurface

[0162] Where Bulk Gibbs Free Energy (AGbuik) is related to the volume of the particle and the bulk properties of the material.

[0163] AGbuik = VAgbuik

[0164] Where V is the volume of the particle and Agbuik is the Gibbs free energy change per unit volume of the bulk material. Assuming the particles in solution as spherical elements particle of radius r:

[0165] V = (4 rrr3) / 3

[0166] Surface Gibbs Free Energy (AGsurface) is related to the surface area of the particle and the surface tension or surface energy of the material.

[0167] AGsurface—gA

[0168] Where g is the surface tension or surface energy per unit area and A is the surface area of the particle.

[0169] For an ideal spherical particle of radius r: A = 4TTr2

[0170] Combining these expressions:

[0171] AG = 4 / 3TTr3gbuik + 4TTr2g

[0172] Where AG: Total Gibbs free energy (J), r: Radius of the particle (m), Agbuik: Bulk Gibbs free energy change per unit volume (J / m3), y: Surface tension or surface energy per unit area (J / m2),

[0173] Agbuik: 109Joules per cubic meter (typical for solid materials and compatible with hydrophobic proteins / peptides values) r (radius of the particle): 10"9meters (nanometer, typical for nanoparticles range) y: 0.0728 J / m2(surface tension of water at 25°C)

[0174] This equation shows that the Gibbs free energy of a colloidal particle in suspension is a function of its radius r. The first term represents the bulk free energy, which scales with the volume of the particle (r3), and the second term represents the surface free energy, which scales with the surface area of the particle (r2).

[0175] As the particle size decreases, the surface area to volume ratio increases, making the surface energy contribution more significant. For very small particles, of soluble solutes, the surface energy term can dominate, significantly affecting the total Gibbs free energy and thus the stability of the colloidal system. This relationship is important in understanding phenomena such as Ostwald ripening, where smaller particles dissolve and redeposit onto larger particles, driven by differences in Gibbs free energy. Understanding this balance between bulk and surface contributions is crucial for predicting the behavior and stability of colloidal suspensions. For highly hydrophobic compounds, as the case of Disitertide, the Gibbs free energy of dissolution (AGsol) is generally positive due to the low entropy of mixing and / or unfavorable interactions between the solute and solvent. This indicates that the dissolution process is not thermodynamically favorable. The hydrophobic nature of these compounds means that they tend to aggregate to minimize contact with the aqueous environment, resulting in minimal solubility.

[0176] In summary, for highly hydrophobic compounds, the dissolution process is characterized by a positive AG, indicating that the process is not spontaneous, and the compounds remain poorly soluble in water. FIG. 6A (Thanh et al., Chem. Rev. 2014, 114, 15, 7610-7630.) is a schematic representation of Gibbs free energy change in colloidal solutions nucleation and aggregation growth progress as a function of the particles size. AG represent total Gibb's energy, AGVand AGcrit are free energy and critical free energy (AG*) of the particle in aggregation. Free energy diagram for nucleation explaining the existence of an aggregation critical nucleus. AGs denotes the surface free energy, AGv denotes the bulk free energy curve, the dashed line with AG is the curve for the total free energy of the particle and rc is the critical size described by equation that differs in each different excipient combination with Disitertide in a potential function (y= 0.001 *x3) of relationship between Disitertide particle size in colloidal solution and DG (DGs) Gibbs free energy.

[0177] Disitertide tends to aggregate due to its high hydrophobicity and high propensity of interaction of its own molecules that is a reaction thermodynamically favorable. As shown in FIG. 6B, Gibbs energy is a potential function of Disitertide particle size in colloidal solutions where in a theoretical Disitertide complete solubilization, in equilibrium, DG is adjusted to zero. In the optimized Sodium Bicarbonate and Tris buffers the Gibbs energy is still very high, indicating a high tendency of Disitertide to aggregate, and the presence of selected solubilizing excipients (Example 4, table 1) reduces this energy by stabilizing the Disitertide particles in colloidal solution. Certain selected excipients, at low concentrations, can effectively reduce the natural tendency of Disitertide to aggregate. This data supports a thermodynamic explanation of the solubilizing effect of certain excipients in the context of Disitertide colloidal solutions. In this scope of temperature independent free Gibbs energy equation, relating surface tension and colloidal particles size, and assuming that DG is adjusted to trend to 0 when Disitertide is in a true solution, and DG=1000 (relative units) when Disitertide colloidal particle size in solution is equal to 100 nm in radius, Gibbs free energy (DG) in this conditions must correspond to DG values bellow 1000, preferably bellow 500, ideally below 250 for an adequate stable Disitertide colloidal solubilization.

[0178] EXAMPLE 8

[0179] Lyophilization bulking agents’ effect over Disitertide solubility referred as a colloidal particles size in solutions.

[0180] Also known as freeze-drying, lyophilization consists in the removal of water from frozen state to gaseous state in the absence of going through the liquid state. This physical process is used in parenteral drug formulation to generate water free product that prevents drugs instability and degradation, prolonging its shelf life and retarding the expiration date.

[0181] For that purpose, solutes (drug + excipients) are dissolved in an appropriate solvent and converted in ice form at a very low temperature between -50 and -90 °C. Then the surface or the obtained frozen piece is subjected to high vacuum pressure (below 0.2 mBar) and connected to a cold chamber (-55 to -60°C) while the sample / s gradually reach room temperature (22-25 °C). All this process is executed under aseptic conditions in a lyophilizer equipment (Lyomicron -55°C, Coolvacuum technologies, Reixac, Barcelona). After lyophilization, samples could be kept at RT or refrigeration (4°C) till resuspension in the same original volume with sterile water for injection prior parenteral administration to the patients. In our development different formulations were tested as prepared immediately (PI) and post lyophilization (PL) to check solubility, particle size, lyophilization outcome, and in vitro activity.

[0182] There are many variables affecting the lyophilization outcome, like lyophilized matrix collapse, retraction, detachment, shrinkage, melt back, puffing, inconsistence, etc. To avoid these issues, temperature, pressure, and timing control is essential. Furthermore, there are many excipients which function includes the stabilization of lyophilized matrix quality outcome after lyophilization. When a final formulation is obtained, lyophilization could be feasibly optimize with different specific excipients like carbohydrates like sugars (glucose, lactose, mannose, fructose, etc.) and polyols (mannitol, inositol, propylene glycol, glycerol, etc.) and many others (amino acids, polymers, buffering agents, cosolvent, etc.), even though many of these excipients have been taking into account in the previous screening of excipients oriented to increase solubility and stability of Disitertide.

[0183] Table 3 presents data of a Tris buffer 25 mM based formulations, in the presence of 4- (2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane 0.065% and with different lyophilization agents (sugars: trehalose, isomaltulose, lactose, glucose and sucrose; polyols: mannitol and glycerol) at two different percentages. Samples analysis includes data mv-1-of colloidal solutions particle size (nm), polydispersity index, Diffusion coefficient (mm2 / s) and thermal stability, measured as temperature (°C) of first inflexion point turbidity onset curve. Tyloxapol clearly reduces particle size and polydispersity index, and increases diffusion coefficient before and after lyophilization, thermal stability in the presence of Tyloxapol increases from 51.19 to 62.30 °C for inflexion of turbidity curve upon temperature. Tested lyophilization agents reveal that some are able to decrease particle size radius and PDI, like D-Mannitol at 5%, and / or increase thermal stability like Isomaltulose or sucrose, after lyophilization.

[0184] EXAMPLE 9

[0185] Antioxidants excipients as solubility stabilizers of Disitertide formulations

[0186] An antioxidant excipient is a substance added to pharmaceutical formulations to prevent oxidation and thus protect the active pharmaceutical ingredients (APIs) and other components from degradation. Oxidation is a chemical reaction that can lead to the loss of potency and efficacy of the drug, as well as the development of harmful by-products. The use of antioxidant excipients can help in maintaining the stability and self-life of pharmaceuticals. Antioxidant excipients can also contribute to prevent aggregation issues in liquid formulations, maintaining the solubility state of the pharmaceutical compound. Protein and peptide aggregation in pharmaceutical formulations can be triggered or exacerbated by oxidation, which causes changes in the molecular structure of proteins and peptides, leading to their aggregation.

[0187] Different antioxidants excipients were tested using an antioxidant assay based on Lugol reaction, where starch reacts with molecular iodine generating a blue colored solution that can be reduced in the presence of antioxidants compounds like ascorbic acid. Disitertide exerts certain antioxidants effect due to the oxidation trend of tryptophan and methionine present in Disitertide sequence. The assay consists in the incubation of Lugol solution where iodine reacts with potassium iodide generating triiodide ion (I2 + KI — > KI3). This ion reacts with cluster dextrin® (cyclic branched derivative of amylopectin from starch hydrolyzation) generating a blue colored solution. In the presence of antioxidants compounds, like Disitertide and antioxidants excipients, the intensity of this colored solution decreases in a proportionally inverse relation with the concentration of Disitertide and antioxidants.

[0188] Antioxidant assay consist of the incubation of Lugol (AGR - Labkem 00A-250 Free Iodine 1 % ± 0.02%) at 0.0125% and cluster dextrin® (highly branched cyclic dextrin, HSN) at 1 .25%, in Tris / Sodium bicarbonate (TBC) 50mM, in the presence or absence of formulated Disitertide (10ml) with different amino acids with antioxidant potential (L-Arginine, L- Histidine, L-Lysine, b-Alanine, L-Methionine, L-Tryptophan, N-Acetyl L-Cysteine, L- Cysteine, Taurine, Betaine, Sarcosine, L-Carnitine) as potential antioxidant agents and the pertinent controls (buffer NaBC, and formulations in this buffer . After 15 minutes of incubation in a black 96 well plate with translucid bottom, under stirring and protected from light, samples absorbance was read at 500 nm wavelength in a Tekan Life Sciences equipment (Tecan Trading AG, Switzerland) model INFINITE M NANO+. Antioxidant capacity was measured both as direct inhibitory activity percentage of Lugol colorimetric reaction and ascorbic acid equivalents (AAE) as antioxidant reference.

[0189] FIG.7 provides a comprehensive analysis of the antioxidant effect of Disitertide, alone or in combination of several amino acids with antioxidant potential, in section 7A absorbance at 500 nm after 15 minutes of incubation are presented, the first two columns include absorbance values of formulations in the presence or absence of Disitertide (0.1 mg / ml), Columns of AAE include values of antioxidant activity equivalence referred to ascorbic acid activity in mg / 100ml and horizontal histograms of inhibitory percentage activity of Lugol colorimetric reaction in a 0 to 100 scale, as a measure of antioxidant activity. FIG.7B presents the standard curve values and graphics of ascorbic acid dilutions from 5 to 0.5 mg / 100ml in two independent samples plates. Linearity of the ascorbic acid curves are above 99.6 % and the reproducibility between them is 98.48% reflecting the degree of sensitivity and optimization of the biochemical assay. Antioxidant assay analysis indicates that Disitertide presents a per se antioxidant activity around 1 .15 AAE (=20% Lugol reaction inhibition: LRI), some excipients present a strong antioxidant activity, from grater to minor: L-Histidine 0.01% (5.89 AAE « 95% LRI), N-Acetyl L-Cysteine (5.28 AAE « 86% LRI), L- Tryptophan 0.01% (4.5 AAE « 72% LRI) and L-Methionine 0.01% (1.69 AAE « 27% LRI), and. In the presence of Disitertide (1 mg / ml) antioxidant capacity are maintained with some reduction in the most active antioxidants, L-Histidine being the most efficient, and L- Tryptophan became more antioxidant than N-Acetyl or L-Cysteine in combination with Disitertide. Disitertide combination with L-Methionine presents more antioxidant capacity than both compounds separately. In combination with Disitertide, L-Lysine and L-Serine generate an antioxidant capacity (2.07-2.27 AAE » 38% LRI) above Disitertide alone and far from the slight pro-oxidant activity or these two amino acids when tested in the absence of Disitertide.

[0190] EXAMPLE 10 in vitro activity of formulated nanometric colloidal suspensions of Disitertide as inhibitor of TGF-p biological activity

[0191] During the development of Disitertide parenteral formulations, assessing the in vitro activity is crucial for several reasons. This stage involves evaluating the stability, solubility, and compatibility of the active pharmaceutical ingredient (API) with excipients and potential delivery systems. Testing in vitro activity provides essential data on the API's efficacy and potency in a controlled environment, ensuring that it retains its therapeutic effect when administered. It helps identify any potential interactions or degradation products that could compromise the formulation's solubility, stability, safety and efficacy. Additionally, these studies guide the optimization of formulation parameters, such as pH and osmolarity, which are critical for ensuring the drug's bioavailability and minimizing adverse reactions. Thorough in vitro evaluation is fundamental to developing a safe, effective, and stable parenteral product.

[0192] Biological activity of Disitertide formulations was evaluated using HEK-Blue™ TGF-p cells reporter gene and QUANTI-Blue™ Solution (InvivoGen Ltd., Toulouse, France) an engineered cell line from the human embryonic kidney HEK 293 cell line to detect bioactive human and murine transforming growth factor-beta (TGF-P) by monitoring the activation of the TGF- / Smad pathway and for screening TGF-p inhibitors. This cell line contains a stable reporter gene (secreted embryonic alkaline phosphatase: SEAP), inducible in the presence of TGF-p. The binding of TGF-p to its receptors on the cell surface of HEK-Blue™ TGF-p cells trigger a signaling cascade leading to the formation of a Smad3 / Smad4 complex. The heterocomplex enters the nucleus and binds SBE sites inducing the production of SEAP that can be colorimetrically read from cells supernatant, after reaction the presence SEAP, the color of QUANTI-Blue™ changes from pink to purple / blue. The purple / blue color intensity reflects the activity / levels of SEAP that can be determined quantitatively as absorbance using a spectrophotometer at 655 nm. Assays were performed following the provider instructions, briefly: 50x106HEK-Blue™ TGF-p cells per well were seeded in 100ul of DM EM complete medium in a translucid 96 well plate that undergoes incubation at 37°C, 5% CO2 and 95% or relative humidity during the whole assay. After 1-2 hours, treatments were added to the plate in additional 100 ml including proper controls, and human recombinant TGF-p (Miltenyi Biotec, Germany) at 0.2 ng / ml in the absence and presence of different concentrations of Disitertide (200, 150, 100 and 50 mg / ml) in cell culture medium, control buffer (Sodium Bicarbonate or TRIS) and corresponding formulations. After 24 h of incubation, 10 ml of each well supernatant was collected and transferred to a 96 black plate with clear bottom. After addition of 90ul of QUANTI-Blue™ Solution plates were incubated at 37°C under shaking for 15-30 minutes, then absorbance values (655 nm) were read with Tekan Life Sciences equipment (Tecan Trading AG, Switzerland) model INFINITE M NANO+. The inhibition percentage of Disitertide formulations over TGF-p activity were calculated following this formula:

[0193] % TGFp inhibition = 100 x (TGFp - Dstd I TGFp - Neg0)

[0194] Where TGF-p = Absorbance (655nm) signal cells supernatant treated with 0.2 ng / ml of human recombinant TGF-p, Dstd = Absorbance (655nm) signal in cell supernatants treated with 0.2 ng / ml of human recombinant TGF-p in the presence of certain concentration of Disitertide, and Neg 0 = Absorbance (655nm) signal in cells supernatants in the absence of recombinant TGF-p.

[0195] FIG.8 presents examples of Disitertide in vitro effect in different formulations with TRIS 25 mM as basal buffer. In figure 8A, absolute absorbance values (655nm) of cells supernatant after reaction with the substrate (QUANTI-Blue™ solution) in the absence (control) or presence of TGF-p (0.2 ng / ml) and different concentrations of Disitertide. Tested formulation consist of prepared immediately (PI) Poly(ethylene glycol)-block-poly(propylene glycol)- block-poly(ethylene glycol) 0.05% with D-Mannitol 1.25% in Tris 25mM with different concentrations of Disitertide. TGF-p (0.2 ng / ml) induce the signal of SEAP activity more than 4.5 times, and Disitertide is able to inhibit this induction in a dose dependent manner by inhibition of TGF-p blocking its interaction with cell membrane receptors and thereof its biological activity. FIG.8 also presents the inhibitory percentage activity of Disitertide (see formula above) at different concentrations over TGF-p (0.2 ng / ml) formulated in: (B) prepared immediately (PI) and post lyophilization (PL) Copolymer Poly(ethylene glycol)- block-poly(propylene glycol)-block-poly(ethylene glycol) (CP2MOx) 0.05% with D-Mannitol 1.25% in Tris 25mM (CP2MOx -Mann-TRIS); (C) prepared immediately (PI) and post lyophilization (PL) Copolymer Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol) (CP2MOx) 0.05% with Isomaltulose 0.5 % in Tris 25mM (CP2MOx - Isom-TRIS) and (D) prepared immediately (PI) and post lyophilization (PL) polyoxyethylenesorbitan monolaurate (Poesy-ML) 0.03% with Glutamine 0.006% and Trehalose 1.25 % in Tris 25mM (PoES-ML-GLUT-TRH-TRIS). All these formulations maintain Disitertide in vitro biological activity, lyophilization generates some minor reduction in activity, and in the case of CP2MOx-lsom-TRIS (C) the activity is higher compared with TRIS buffer formulation both as PI and PL. All formulation and TRIS buffer controls (0: w / o TGF-P) produce expected non-significant marginal effects over TGF-p inhibition, indicating the specific activity of Disitertide and no significant cytotoxic effects of the formulation components (excipients, salts and pH) over cells during the bioassay.

[0196] EXAMPLE 11

[0197] A pharmaceutical formulation comprising 1 mg / mL of Disitertide (Polypeptide Group, France) was prepared in 25 mM Tris (hydroxymethyl) aminomethane (Fisher scientific, Cat. No. BP152-1) buffer solution. The formulation further contained 0.02% (w / v) L-Histidine (Thermo scientific Cat. No. 166150250) as an antioxidant agent, 2.5% (w / v) D-Mannitol (Carlo Ebra, Cat. No. 460355) as a cryoprotectant and bulking agent, 0.025% (w / v) poly[1 - (2-oxopyrrolidin-1-yl)ethylene] (Thermo scientific, Cat. No. J60382.14) as a stabilizing and solubilizing polymer, 0.005% (w / v) poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol) (Panreac, Cat. No. A4974 / 0250) as a non-ionic surfactant, and 0.05% (w / v) of (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid (Acofarma, Cat. No. 225949).

[0198] In particular, the formulation excipient solution was prepared by dissolving the precise amounts of each component in 10 mL of previously prepared Tris buffer 25 mM pH 8.6 adjusted with required amount of glacial acetic acid (17.4M) (Carlo Ebra, Cat. No. 401421) in purified water (Milli-Q Advantage A10, Merck Millipore, Germany) under vortex stirring at room temperature (20-25°C). Specifically, 250 mg of D-Mannitol (2.5% w / v), 2 mg of L- Histidine (0.02% w / v), 2.5 mg of poly[1-(2-oxopyrrolidin-1-yl)ethylene] (0.025% w / v), 0.5 mg of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (0.005% w / v), and 5 mg of (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid (0.05% w / v) were sequentially added to the aqueous medium and allowed to fully dissolve with vortex stirring. After complete dissolution, 1 mL of the resulting excipient formulation was transferred to a sterile container, and 1 mg of lyophilized Disitertide (P144) (Polypeptide Group, France) was added to this 1 mL aliquot to obtain a final peptide concentration of 1 mg / mL. The mixture was gently stirred in vortex at room temperature and then subjected to brief probe sonication (Biobase UCD-250, China), consisting of 1 cycle of 10 seconds sonication at 30% amplitude, to ensure homogeneous dispersion of Disitertide and surfactant components. No further pH adjustment was necessary, and the final pH remained at value 8.6. The final peptide formulation was homogeneous with a final particle size below 100 nm in cumulant radius. The resulting formulation was subsequently filled into sterile plastic vial and freeze at -80 °C and transferred to a lyophilizer (Coolvacuum technologies, lyomicron -55 °C, Spain). The lyophilization cycle process included an internal freezing constant temperature of -58 °C. The sample was allowed to reach RT gradually, covered with ice and thermos-blocks freeze at -80°C at a pressure of 0.022 mBar for at least 24 hours, leaving the sample exposed the last two hours in the lyophilization vessel at room temperature (22 °C). Vial presents a homogeneous lyophilized cake that maintains the original shape and size of the initial formulation volume in the vial, the vial was immediately taken and sealed. The lyophilized product was stored at 2-6 °C until use. Vial reconstitution was carried out by adding one milliliter of sterile distilled Milli-Q water to the vial, in order to recover the initial conditions and concentrations of excipients and Disitertide. The final resuspended peptide formulation was clear and homogeneous with a final particle size below 100 nm in cumulant radius.

[0199] EXAMPLE 12

[0200] Similarly, pharmaceutical formulations comprising a Tris buffer 25 mM or sodium bicarbonate 50mM, wherein the pH is from 7 to 8,6, with at minimum of 0.01 % L-Histidine as antioxidant agent and 2.5% of D-Mannitol as lyophilization agent, in the presence of water-soluble organic surfactants from the list below, separately or in combinations up to three elements.

[0201] The water-soluble organic surfactants were the following ones:

[0202] • poly[1-(2-oxopyrrolidin-1-yl)ethylene], at a minimum of 0.001%.

[0203] • 2-hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate at a minimum of 0.005%.

[0204] • (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid at a minimum of 0.05%.

[0205] • 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate at a minimum of 0.0005%.

[0206] • sodium; 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate at a minimum of 0.05%.

[0207] • poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) at a minimum of 0.005%.

[0208] Specifically, formulations prepared comprised a buffer comprising 25 mM Tris or 50 mM sodium bicarbonate at a pH of 8.6 or lower, 2.5% of D-mannitol, 0,01 to 0,02% of L-histidine, and a combination of three surfactant excipients selected from:

[0209] (i) 0.005% of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), 0,001 to 0,025% of poly[1-(2-oxopyrrolidin-1-yl)ethylene], and 0,005 to 0,05% of 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate;

[0210] (ii) 0.005% of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), 0.005% of (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid, and 0,005 to 0,05% of 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate; or

[0211] (iii) 0.05% of (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid, 0,001 to 0,025% of poly[1-(2-oxopyrrolidin-1-yl)ethylene], and 0,005 to 0,05% of 2-{2-[3,4- bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate.

[0212] The formulations were prepared by dissolving the specified percentages of each excipient in the selected buffer, followed by addition of an appropriate amount of P144 to achieve a final concentration of 1 mg / mL. The pH was then adjusted to a desired value within the range of physiological pH to 8.6. The resulting solutions were subsequently homogenized by sonication to properly disperse Disitertide within the formulation.

[0213] For reasons of completeness, various embodiments of the invention are set out in the following numbered clauses:

[0214] Clause 1. A pharmaceutical formulation comprising: a) a hydrophobic peptide or protein; b) water-soluble organic surfactants selected from the list consisting of non-ionic surfactants, anionic surfactants and amphoteric surfactants, amino acids, amphipathic glycosides, water-soluble polymer compounds and / or tertiary amino compounds; wherein a) and b) are dispersed in a salt solution forming a colloidal suspension having an average particle size of less than 100 nm cumulant radius as measured by dynamic light scattering, wherein the salt solution comprising a Tris buffer and / or a Sodium Bicarbonate buffer; wherein the pH of the composition is in the range of between 7.37 to 10, preferably between 7.5 and 10, more preferably between 8.5 and 10, and wherein the formulation is suitable for parenteral administration.

[0215] Clause 2. The formulation of clause 1 , wherein the hydrophobic peptide or protein has a GRAVY (Grand average of hydropathicity index) value above zero and is selected from any one of the list consisting of Disitertide, Gramicidin A, Cyclosporine, p-Amyloid, Melittin, Magainin, Dynorphin A, Dermcidin, Cecropin A, Maximin H5, Indolicidin, Piscidin, Thanatin, LL-37, Brevinin-1 , Protegin-1 , Dermaseptin, Cecropin, Butorin, Mastoparan, some collagen derived peptides like Gly-Pro-Hyp, Disitertide, Octreotide, Liraglutide, Glatiramer acetate, Enfuvirtide, Brimonidine, Exenatide, Desmopressin, Semaglutide, Apolipoproteins, Melanin-concentrating hormone, Neuropeptide Y, b-Endorphin, Corticotropin-releasing hormone, Encephalins, Galanin, Vasopressin, Bradykinin, Dalbavancin, Leuprorelin, Teriparatide, Teduglutide, Cetrotide, Oxytocin, Polymyxins, Daptomycin, Bacitracin, Tyrocidine, Insulin, Glucagon, Erythropoietin, Growth Hormone, b-Casein, Fibrinogens, Keratins, Albumins, Collagens, Interferons, some monoclonal antibodies (Bevacizumab, Cetuximab, Ipilimumab), some enzymes (a-Chymotrypsin, L-asparaginase, Lipases, membrane proteins (Na+ / K+-Transporting ATPase Subunit-a, Rhodopsin, Cytochrome P450 and B6f, Nav channel, ATP-binding Cassette Transporter A1 , Aquaporins, Voltage- Dependent Anion Channel 1 , Transferrin Receptor Protein 1 , NADH-ubiquinone oxidoreductase chain 1), Cholesterol esterase, Fatty Acid-Binding Protein, Platelet Glycoprotein 4, Myelin Basic Protein, Peripherin, Acetylcholinesterase, Carbonic anhydrase), Chimeric proteins (Imiglucerase, Abatacept, Elosulfase-a), and lectin proteins (agglutinins).

[0216] Clause 3. The formulation of clause 1 , wherein the hydrophobic peptide or protein is of SEQ ID NO 1 (TSLDASIIWAMMQN) or any pharmaceutically acceptable salt thereof; or any peptide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID No 1 and capable of retaining at least 50% of the capacity to inhibit the antiproliferative activity of TGF-p in an in vitro proliferation cell line (Mv-1-Lu) bioassay with respect to SEQ ID No 1.

[0217] Clause 4. The formulation of clause 3, wherein the hydrophobic peptide or protein is of SEQ ID NO 1 (TSLDASIIWAMMQN) or any functional variant thereof such as SEQ ID NO 2 (TSLMIWTMM) or SEQ ID NO 3 (TSLDATMIWTMMA) capable of retaining at least 50% of the capacity to inhibit the antiproliferative activity of TGF-p in an in vitro proliferation cell line (Mv-1-Lu) bioassay with respect to SEQ ID No 1 .

[0218] Clause 5. The formulation of clause 3, wherein the hydrophobic peptide or protein is of SEQ ID NO 1 (TSLDASIIWAMMQN) or any functional variant thereof comprising or consisting of SEQ ID NO 4 (TSLXXSIIWXMMXX), wherein X is understood as any amino acid, preferably SEQ ID NO 1 (TSLDASIIWAMMQN).

[0219] Clause 6. The formulation of clause 1 , wherein the hydrophobic peptide or protein is an acetic salt of SEQ ID NO 1 (TSLDASIIWAMMQN), preferably at a concentration in the composition or formulation of less than 20 mg / ml, preferably less than 10 mg / ml, more preferably between 1 mg / ml and 20 mg / ml, more preferably between 1 mg / ml and 10 mg / ml, more preferably between 2 mg / ml and 5 mg / ml, more preferably between 3 mg / ml and 4 mg / ml. Clause 7. The formulation according to any one of the preceding clauses, wherein the salt solution comprises or consists of a Sodium Bicarbonate solution at a concentration equal or below 150 mM, preferably equal or below 100 mM, more preferably between 25 and 75 mM, preferably at a concentration of 50 mM.

[0220] Clause 8. The formulation according to any one of clauses 1 to 6, wherein the salt solution comprises or consists of a Tris buffer at a concentration equal or below 300 mM, preferably equal or below 100mM, more preferably between 10 and 50 mM, preferably at a concentration of 25 mM.

[0221] Clause 9. The formulation according to any one of clauses 1 to 6, wherein the salt solution comprises or consists of a Tris and a Sodium Bicarbonate buffer solution at concentrations equal or below 100 mM of each buffer, preferably between 5 and 25 mM of each buffer.

[0222] Clause 10. The formulation of any of the preceding clauses, wherein the formulation comprises a water-soluble organic surfactant, amino acid, water-soluble polymer compound and / or tertiary amino water-soluble organic surfactant is selected from the group consisting of:

[0223] Dodecan-1-ol, ethoxylated; D-Glucopyranose, oligomeric, C8-16(even numbered) alkyl glycosides; 2-hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12- trimethyltridecyl)chroman-6-yl succinate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine 3a,12a-dihydroxy-5p-cholan-24-oate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine 1 ,4-bis(2-ethylhexoxy)- 1 ,4-dioxobutane-2-sulfonate; 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol; (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid; Poly(ethylene glycol)- block-poly(propylene glycol)-block-poly(ethylene glycol); poly[1-(2-oxopyrrolidin-1- yl)ethylene]; [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]

[0224] (1 R,4S,5R,9S, 10R, 13S)-5,9-dimethyl-14-methylidene-13-[(2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9] hexadecane- 5-carboxylate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine Dodecyl Sulfate; 2-[3-(dodecanoylamino) propyl-dimethylazaniumyl] acetate; Alcohols, C12-14 (even numbered), ethoxylated < 2.5 EO, sulfates, sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine salts; 2-[bis(2- hydroxyethyl) amino]ethan-1-ol; 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate; and 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane; and pharmaceutical acceptable salt thereof.

[0225] Clause 11. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethan-1-ol, wherein said compounds are present in the formulation at a minimum concentration of about 0.003% (%wt / wt), preferably in the range of from 0.003% to 0.5% (%wt / wt), preferably in the range of from 0.003% to 0.05% (%wt / wt), and wherein the salt solution is a Tris buffer at preferably any of the concentrations defined in clause 8.

[0226] Clause 12. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of sodium Dodecan-1-ol, ethoxylated; Sodium 3a,12a-dihydroxy-5p-cholan-24-oate; sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4- dioxobutane-2-sulfonate; Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol); 2-[3-(dodecanoylamino)propyl-dimethylazaniumyl]acetate; Alcohols, C12-14(even numbered), ethoxylated < 2.5 EO, sulfates, sodium salts; 2-[bis(2- hydroxyethyl)amino]ethan-1-ol; 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane, wherein said compound is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), more preferably in the range of from 0.005% to 0.25% (%wt / wt), wherein the salt solution is a Sodium Bicarbonate and / or Tris buffer at preferably any of the concentrations defined in any of clauses 7 or 8.

[0227] Clause 13. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of 2-hydroxyethyl 2,5,7,8-tetramethyl-2- (4,8,12-trimethyltridecyl)chroman-6-yl succinate, wherein said compound is present in the formulation at a minimum concentration of about 0.01 % (%wt / wt), preferably in the range of from 0.01% to 0.5% (%wt / wt), preferably in the range of from 0.015% to 0.05% (%wt / wt), and the salt solution is a T ris buffer at preferably any of the concentrations defined in clause 8.

[0228] Clause 14. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of D-Glucopyranose, oligomeric, C8- 16(even numbered) alkyl glycosides and wherein said compound is present in the formulation at a minimum concentration of about 0.02% (%wt / wt), preferably in the range of from 0.02% to 0.5% (%wt / wt), preferably in the range of from 0.02% to 0.05% (%wt / wt), and the salt solution is preferably a Tris buffer at preferably any of the concentrations defined in clause 8.

[0229] Clause 15. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethan-1-ol; wherein said compound is present in the formulation at a minimum concentration of about 0.003% (%wt / wt), preferably in the range of from 0.003% to 0.5% (%wt / wt), preferably in the range of from 0.003% to 0.05% (%wt / wt), and the salt solution is a sodium bicarbonate buffer at preferably any of the concentrations defined in clause 7. Clause 16. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of Sodium Dodecyl Sulfate; and 4-(2,4,4- trimethylpentan-2-yl) phenol, formaldehyde oxirane, wherein said compounds is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), preferably in the range of from 0.005% to 0.05% (%wt / wt), and the salt solution is:

[0230] 1. a Tris buffer, preferably at any of the concentrations defined in clause 8, if 4- (2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane is selected; or

[0231] 2. a sodium bicarbonate buffer, preferably at any of the concentrations defined in clause 7, if sodium Dodecyl Sulfate is selected.

[0232] Clause 17. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of Dodecan-1-ol, ethoxylated; Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); and 2-[3- (dodecanoylamino)propyl-dimethylazaniumyl]acetate; and wherein said compound is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), wherein the salt solution is a Sodium Bicarbonate and / or Tris buffer at preferably any of the concentrations defined in any one of clauses 7 or 8.

[0233] Clause 18. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of Sodium Dodecyl Sulfate; and 4-(2,4,4- trimethylpentan-2-yl) phenol, formaldehyde, oxirane; and wherein said compound is present in the formulation at a minimum concentration of about 0.005%, preferably in the range of from 0.005% to 0.5% (%wt / wt), and the salt solution is a Tris buffer if sodium Dodecyl Sulfate is selected, wherein the salt solution is preferably at any of the concentrations defined in clause 8.

[0234] Clause 19. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of D-Glucopyranose, oligomeric, C8- 16(even numbered) alkyl glycosides; 2-{2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate, and 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane; wherein said compound is present in the formulation at a minimum concentration of about 0.02%, preferably in the range of from 0.05% to 0.5% (%wt / wt), and wherein the salt solution is preferably a Sodium bicarbonate buffer or a Sodium bicarbonate or Tris buffer if 2-{2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate is selected, wherein the salt solution is preferably at any of the concentrations defined in any of clauses 7 or 8. Clause 20. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of 2-hydroxyethyl 2,5,7,8-tetramethyl-2- (4,8,12-trimethyltridecyl)chroman-6-yl succinate; poly[1-(2-oxopyrrolidin-1-yl)ethylene]; and ([(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S)- 5,9-dimethyl-14-methylidene-13-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl]oxytetracyclo[11.2.1.01 ,10.04,9]hexadecane-5-carboxylate; wherein said compound is present in the formulation at a minimum concentration of about 0.01%, preferably at a minimum concentration of 0.05%, preferably in the range of from 0.05% to 0.5% (%wt / wt), and wherein the salt solution is a Sodium bicarbonate buffer if 2- hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate or [(2S,3R,4S,5S,6R) -3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl] (1 R,4S,5R,9S,10R,13S) -5,9-dimethyl -14-methylidene -13- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy -6-

[0235] (hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9] hexadecane-5-carboxylate are selected and a Tris buffer if poly[1-(2-oxopyrrolidin-1-yl)ethylene] is selected, wherein the salt solution is preferably at any of the concentrations defined in any one of clauses 7 or 8.

[0236] Clause 21. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of (2S)-2-amino-5-

[0237] (diaminomethylideneamino) pentanoic acid, and [(2S,3R,4S,5S,6R) -3,4,5- trihydroxy-6- (hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S) -5,9-dimethyl-14-methylidene-13- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo

[0238] [11.2.1.01 ,10.04,9]hexadecane-5-carboxylate; wherein said compounds are present in the formulation at a minimum concentration of about 0.001%, preferably in the range of from 0.005% to 0.5% (%wt / wt), and wherein the salt solution is a Tris buffer if [(2S,3R,4S,5S,6R)- 3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S)-5,9-dimethyl-14- methylidene-13-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9] hexadecane-5-carboxylate is selected, and wherein the salt solution is preferably at any of the concentrations defined in any one of clauses 7 or 8.

[0239] Clause 22. The formulation according to clause 10, wherein the water-soluble organic surfactant is selected from the group consisting of sodium 3a,12a-dihydroxy-5p-cholan-24- oate, Sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate, and poly[1-(2- oxopyrrolidin-1-yl)ethylene]; wherein said compounds are present in the formulation at a minimum concentration of about 0.5% and wherein the salt solution is a Tris buffer if Deoxycholic acid sodium salt or Sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2- sulfonate are selected and a Sodium bicarbonate buffer if poly[1-(2-oxopyrrolidin-1- yl)ethylene] is selected, preferably wherein the salt solution are present at any of the concentrations defined in any one of clauses 7 or 8. Clause 23. The formulation according to any one of clauses 1 to 22, wherein the formulation has a lyophilization stabilizer, and the stabilizer is selected from Glucose, Trehalose, Mannitol, Sucrose, Lactose, Inositol, Xylose, Xylitol, Isomaltulose, Sorbitol, Maltitol, Mannose, Fructose, Proline, Arginine, Histidine or Glycine.

[0240] Clause 24. The formulation according to any of the precedent clauses, wherein the formulation is for use in diseases prevention or therapy.

[0241] Clause 25. The formulation according to any of clauses 1 to 24, wherein the formulation is for use via parental administration in a method of treatment of fibrosis-based diseases selected from the group consisting of lung fibrosis induced by environmental exposure to chemicals, allergens and particles, smoking, therapeutic severe effect after chemo and / or radiotherapy and antibiotics, asthma, chronic obstructive pulmonary disease, polymyositis, bronchitis, rhinosinusitis, mucositis, autoimmune disease like rheumatoid arthritis, progressive systemic sclerosis, Sjogren's syndrome or sarcoidosis, adult respiratory distress syndrome, chronic or severe infections like tuberculosis or viruses, Idiopathic pulmonary fibrosis, and adult respiratory distress syndrome. Also including Cardiac fibrosis induced by chronic hypertension, primary pulmonary hypertension, polymyositis, familial pulmonary hypertension, pre-eclampsia, atherosclerosis, restenosis, and hypertrophic cardiomyopathy and congestive heart failure, myocardial infarction, cardiomyopathies. Additionally comprising other cardiovascular diseases such as hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome), Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome. Likewise, Renal fibrosis induced by chronic kidney disease (diabetic nephropathy and / or hypertension), glomerulonephritis, chronic pyelonephritis, obstructive uropathy, polycystic kidney disease, ischemiareperfusion injury, nephrotoxins, interstitial nephritis (drugs or recurrent infections), autoimmune diseases, chronic allograft nephropathy or genetic conditions (autosomal dominant polycystic kidney disease, Alpor syndrome, tuberous sclerosis complex, nephronophthisis, etc.). In addition, Liver fibrosis induced by chronic viral hepatitis, chronic alcohol consumption, autoimmune hepatitis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, biliary or sclerosing cholangitis, chemo- or radiotherapy, genetic diseases (hemochromatosis, Wilson's disease, a-1 antitrypsin deficiency, chronic biliary obstruction, drugs induce injury, parasites infections (schistosomiasis, echinococcosis, dietary or environment toxins, metabolic disorders and chronic cholestasis. Moreover, ophthalmic fibrosis induced by proliferative vitreoretinopathy, diabetic retinopathy, retinal vein occlusion, uveitis, retinal detachment, retinitis pigmentosa, infections, surgical adverse complications, inherited retinal disorders, glaucoma or dry and wet age-related macular degeneration. Further including pancreatic fibrosis generated through chronic pancreatitis, cystic fibrosis, or diabetes. Also including clinically relevant deep connective tissues related to deep skin and muscles fibrosis induced by profound wounds (surgical interventions: keloids and hypertrophic scars), third grade burns (sun, chemicals, electrical, cold and hot), radiotherapy, autoimmune diseases (scleroderma, lupus, rheumatoid arthritis). Including other connective tissue diseases such as Marfan syndrome, Marfan-like disorders, also addressing skeletal and muscular disorders like Camurati-Engelmann disease, fibrodysplasia ossificans progressiva, Hunter-Thompson and Grebe-type chondrodysplasias, osteoporosis, sclerosteosis, Van Buchem disease, brachydactyly, symphalangism, and Duchenne muscular dystrophy; post-surgical or inflammatory stenosis affecting trachea, intestines, ureters, urethra, bile ducts, vascular vessels, lymphatic vessels and spinal canal; oncological diseases selected from the group consisting of gliomas, neuroendrocrine tumors, blastomas, melanomas, germ cell tumors, mesothelioma, carcinomas (adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, basal cell carcinomas), sarcomas, myelomas, leukemias, lymphomas, and mixed tumor types (carcinosarcomas, adenosquamous carcinomas, teratomas) originated at or metastasized to brain, spinal cord, meninges, peripheral nerves, retina, salivary glands, head and neck tissues, thyroid, oral mucosa, throat, lung, breast, esophagus, stomach, small intestine, colon, rectum, pancreas, liver, bile ducts, blood, bone marrow, vascular or lymphatic vessels, lymph nodes, pleural / peritoneal membranes, bones, cartilage, adipose tissue, skeletal muscle, smooth muscle, skin, Kidney, bladder, prostate, testicle, ovarium, uterus, endometrium, cervix, vulva,. Also including hereditary rare cancer syndromes like juvenile polyposis syndrome, hereditary nonpolyposis colorectal cancer.

Claims

CLAIMS1. A pharmaceutical formulation comprising: a) a hydrophobic peptide or protein; b) a water-soluble organic surfactant selected from the list consisting of non-ionic surfactants, anionic surfactants, amphoteric surfactants, amino acids, amphipathic glycosides, water-soluble polymer compounds and tertiary amino compounds; wherein a) and b) are dispersed in a salt solution forming a colloidal suspension having particles having an average particle size of less than 100 nm cumulant radius as measured by dynamic light scattering, wherein the salt solution comprises a Tris buffer, a Sodium Bicarbonate buffer, or a mixture thereof; wherein the pH of the composition is in the range of between 7.37 to 10, preferably between 7.5 and 10, more preferably between 8.5 and 10, and wherein the formulation is suitable for parenteral administration.

2. The formulation of claim 1 , wherein the hydrophobic peptide or protein is an acetic salt of SEQ ID NO 1 (TSLDASIIWAMMQN), preferably at a concentration in the composition or formulation of less than 20 mg / ml, preferably less than 10 mg / ml, more preferably between 1 mg / ml and 20 mg / ml, more preferably between 1 mg / ml and 10 mg / ml, more preferably between 2 mg / ml and 5 mg / ml, more preferably between 3 mg / ml and 4 mg / ml.

3. The formulation according to any one of the preceding claims, wherein the salt solution comprises or consists of a Sodium Bicarbonate solution at a concentration equal or below 150 mM, preferably equal or below 100 mM, more preferably between 25 and 75 mM, preferably at a concentration of 50 mM.

4. The formulation according to any one of claims 1 to 2, wherein the salt solution comprises or consists of a Tris buffer at a concentration equal or below 300 mM, preferably equal or below 100 mM, more preferably between 10 and 50 mM, preferably at a concentration of 25 mM.

5. The formulation of any of the preceding claims, wherein the water-soluble organic surfactant is selected from the group consisting of: Dodecan-1-ol, ethoxylated; D- Glucopyranose, oligomeric, C8-16(even numbered) alkyl glycosides; 2-hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine 3a,12a-dihydroxy-5p-cholan-24-oate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate; 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan- 1-ol; (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid; Poly(ethylene glycol)- block-poly(propylene glycol)-block-poly(ethylene glycol); poly[1-(2-oxopyrrolidin-1- yl)ethylene]; [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl](1 R,4S,5R,9S, 10R, 13S)-5,9-dimethyl-14-methylidene-13-[(2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9] hexadecane-5- carboxylate; Sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine Dodecyl Sulfate; 2-[3-(dodecanoylamino) propyl-dimethylazaniumyl] acetate; Alcohols, C12-14 (even numbered), ethoxylated < 2.5 EO, sulfates, sodium, Potassium, Magnesium, Zinc, Calcium or Monoethanolamine salts; 2-[bis(2-hydroxyethyl) amino]ethan-1-ol; 2-{2-[3,4- bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate; and 4- (2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane; and pharmaceutical acceptable salt thereof.

6. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of poly[1-(2-oxopyrrolidin-1-yl)ethylene]; 2-hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate; (2S)-2-amino-5- (diaminomethylideneamino) pentanoic acid; 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2- (2-hydroxyethoxy)ethoxy}ethyl dodecanoate; Sodium; 1 ,4-bis(2-ethylhexoxy)-1 ,4- dioxobutane-2-sulfonate; Poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol); pharmaceutical acceptable salt thereof; and a combination of up to three thereof.

7. The formulation according to claim 6, wherein the salt solution comprises a Tris buffer at a concentration from 10 to 50 mM, preferably at a concentration of 25 mM.

8. The formulation according to claim 6, wherein the salt solution comprises a sodium bicarbonate buffer at a concentration from 40 to 60 mM, preferably at a concentration of 50 mM.

9. The formulation according to claim 6, wherein the salt solution comprises Tris at a concentration from 5 to 25 mM and sodium bicarbonate at a concentration from 5 to 25 mM, preferably at a concentration of 6.25 and 12.5 mM respectively.

10. The formulation according to any one of claims 6 to 9, wherein the pH is from 8.5 to 10, preferably 8.6.

11. The formulation according to any one of claims 6 to 10, wherein the water-soluble organic surfactant comprises poly[1-(2-oxopyrrolidin-1-yl)ethylene], wherein said compounds is present in the formulation at a concentration from 0.001 % to 0.5%, preferably from 0.005% to 0.25% (%wt / wt); orthe water-soluble organic surfactant comprises 2-hydroxyethyl 2,5,7,8-tetramethyl-2- (4,8,12-trimethyltridecyl)chroman-6-yl succinate, wherein said compound is present in the formulation at a concentration from 0.005% to 0.5% (%wt / wt), preferably from 0.015% to 0.05% (%wt / wt); or the water-soluble organic surfactant comprises (2S)-2-amino-5- (diaminomethylideneamino) pentanoic acid, wherein said compound is present in the formulation at concentration from 0.05% to 0.5% (%wt / wt), preferably from 0.05% to 0.25% (%wt / wt); or the water-soluble organic surfactant comprises 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]- 2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate, wherein said compound is present in the formulation at a concentration from 0.0005% to 0.05% (%wt / wt), preferably from 0.001% to 0.005% (%wt / wt); or the water-soluble organic surfactant comprises sodium; 1 ,4-bis(2-ethylhexoxy)-1 , 4- dioxobutane-2-sulfonate, wherein said compound is present in the formulation at a concentration from 0.05% to 0.5% (%wt / wt), preferably from 0.1 % to 0.25% (%wt / wt); or the water-soluble organic surfactant comprises poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), wherein said compound is present in the formulation at a minimum concentration from 0.005% to 0.05% (%wt / wt), preferably from 0.005% to 0.01% (%wt / wt).

12. The formulation according to any one of claims 6 to 11 , wherein the water-soluble organic surfactant comprises poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol), poly[1-(2-oxopyrrolidin-1-yl)ethylene], and 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate.

13. The formulation according to any one of claims 6 to 11 , wherein the water-soluble organic surfactant comprises poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol), (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid, and 2- {2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate.

14. The formulation according to any one of claims 6 to 11 , wherein the water-soluble organic surfactant comprises (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid, poly[1-(2-oxopyrrolidin-1-yl)ethylene], and 2-{2-[3,4-bis(2-hydroxy ethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate.

15. The formulation according to any one of claims 6 to 11 , further comprising an antioxidant agent selected from the group consisting of L-Arginine, L-H istidine, L-Lysine, b-Alanine, L-Methionine, L-Tryptophan, N-Acetyl L-Cysteine, L-Cysteine, Taurine, Betaine, Sarcosine, L- Carnitine, particularly, L-Histidine.

16. The formulation of claim 15, wherein the antioxidant agent is at a concentration from 0.01 % to 0.05% (%wt / wt).

17. The formulation according to any one of claims 6 to 16, further comprising a lyophilization agents selected from trehalose, isomaltulose, lactose, glucose, sucrose, D- mannitol and glycerol, particularly D-Mannitol.

18. The formulation of claim 17, wherein the lyophilization agents is at a concentration from 2.5% to 5% (%wt / wt).

19. The formulation according to claim 5, wherein the water-soluble organic surfactant is 2- [4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol, wherein said compound is present in the formulation at a minimum concentration of about 0.003% (%wt / wt), preferably in the range of from 0.003% to 0.5% (%wt / wt), preferably in the range of from 0.003% to 0.05% (%wt / wt), and wherein the salt solution is a Tris buffer at preferably any of the concentrations defined in claim 4.

20. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of sodium Dodecan-1-ol, ethoxylated; Sodium 3a, 12a- dihydroxy-5p-cholan-24-oate; sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate; Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); 2-[3- (dodecanoylamino)propyl-dimethylazaniumyl]acetate; Alcohols, C12-14(even numbered), ethoxylated < 2.5 EO, sulfates, sodium salts; 2-[bis(2-hydroxyethyl)amino]ethan-1-ol; 4- (2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane, wherein said compound is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), more preferably in the range of from 0.005% to 0.25% (%wt / wt), wherein the salt solution is a Sodium Bicarbonate and / or Tris buffer at preferably any of the concentrations defined in any of claims 3 or 4.21 . The formulation according to claim 5, wherein the water-soluble organic surfactant is 2- hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate, wherein said compound is present in the formulation at a minimum concentration of about 0.01 % (%wt / wt), preferably in the range of from 0.01% to 0.5% (%wt / wt), preferably in the range of from 0.015% to 0.05% (%wt / wt), and the salt solution is a Tris buffer at preferably any of the concentrations defined in claim 4.

22. The formulation according to claim 5, wherein the water-soluble organic surfactant is D- Glucopyranose, oligomeric, C8-16(even numbered) alkyl glycosides and wherein said compound is present in the formulation at a minimum concentration of about 0.02%(%wt / wt), preferably in the range of from 0.02% to 0.5% (%wt / wt), preferably in the range of from 0.02% to 0.05% (%wt / wt), and the salt solution is preferably a Tris buffer at preferably any of the concentrations defined in claim 4.

23. The formulation according to claim 5, wherein the water-soluble organic surfactant is 2- [4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol; wherein said compound is present in the formulation at a minimum concentration of about 0.003% (%wt / wt), preferably in the range of from 0.003% to 0.5% (%wt / wt), preferably in the range of from 0.003% to 0.05% (%wt / wt), and the salt solution is a sodium bicarbonate buffer at preferably any of the concentrations defined in claim 3.

24. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of Sodium Dodecyl Sulfate; and 4-(2,4,4- trimethylpentan-2-yl) phenol, formaldehyde oxirane, wherein said compounds is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), preferably in the range of from 0.005% to 0.05% (%wt / wt), and the salt solution is:- a Tris buffer, preferably at any of the concentrations defined in claim 4, if 4-(2,4,4- trimethylpentan-2-yl) phenol, formaldehyde, oxirane is selected; or- a sodium bicarbonate buffer, preferably at any of the concentrations defined in claim 3, if sodium Dodecyl Sulfate is selected.

25. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of Dodecan-1-ol, ethoxylated; Poly(ethylene glycol)- block-poly(propylene glycol)-block-poly(ethylene glycol); and 2-[3- (dodecanoylamino)propyl-dimethylazaniumyl]acetate; and wherein said compound is present in the formulation at a minimum concentration of about 0.005% (%wt / wt), preferably in the range of from 0.005% to 0.5% (%wt / wt), wherein the salt solution is a Sodium Bicarbonate and / or Tris buffer at preferably any of the concentrations defined in any one of claims 3 or 4.

26. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of Sodium Dodecyl Sulfate; and 4-(2,4,4- trimethylpentan-2-yl) phenol, formaldehyde, oxirane; and wherein said compound is present in the formulation at a minimum concentration of about 0.005%, preferably in the range of from 0.005% to 0.5% (%wt / wt), and the salt solution is a Tris buffer if sodium Dodecyl Sulfate is selected, wherein the salt solution is preferably at any of the concentrations defined in claim 4.

27. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of D-Glucopyranose, oligomeric, C8-16(even numbered) alkyl glycosides; 2-{2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2- hydroxyethoxy)ethoxy}ethyl dodecanoate, and 4-(2,4,4-trimethylpentan-2-yl) phenol, formaldehyde, oxirane; wherein said compound is present in the formulation at a minimum concentration of about 0.02%, preferably in the range of from 0.05% to 0.5% (%wt / wt), and wherein the salt solution is preferably a Sodium bicarbonate buffer or a Sodium bicarbonate or Tris buffer if 2-{2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy}ethyl dodecanoate is selected, wherein the salt solution is preferably at any of the concentrations defined in any of claims 3 or 4.

28. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of 2-hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12- trimethyltridecyl)chroman-6-yl succinate; poly[1-(2-oxopyrrolidin-1-yl)ethylene]; and ([(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S)- 5,9-dimethyl-14-methylidene-13-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl]oxytetracyclo[11.2.1.01 ,10.04,9]hexadecane-5-carboxylate; wherein said compound is present in the formulation at a minimum concentration of about 0.01%, preferably at a minimum concentration of 0.05%, preferably in the range of from 0.05% to 0.5% (%wt / wt), and wherein the salt solution is a Sodium bicarbonate buffer if 2- hydroxyethyl 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl succinate or [(2S,3R,4S,5S,6R) -3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl] (1 R,4S,5R,9S,10R,13S) -5,9-dimethyl -14-methylidene -13- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy -6-(hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9] hexadecane-5-carboxylate are selected and a Tris buffer if poly[1-(2-oxopyrrolidin-1-yl)ethylene] is selected, wherein the salt solution is preferably at any of the concentrations defined in any one of claims 3 or 4.

29. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of (2S)-2-amino-5-(diaminomethylideneamino) pentanoic acid, and [(2S,3R,4S,5S,6R) -3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S) -5,9-dimethyl-14-methylidene-13-[(2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo [11.2.1.01 ,10.04,9]hexadecane-5- carboxylate; wherein said compounds are present in the formulation at a minimum concentration of about 0.001 %, preferably in the range of from 0.005% to 0.5% (%wt / wt), and wherein the salt solution is a Tris buffer if [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl] (1 R,4S,5R,9S,10R,13S)-5,9-dimethyl-14-methylidene-13- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] oxytetracyclo[11.2.1.01 ,10.04,9] hexadecane-5-carboxylate is selected, and wherein the salt solution is preferably at any of the concentrations defined in any one of claims 3 or 4.

30. The formulation according to claim 5, wherein the water-soluble organic surfactant is selected from the group consisting of sodium 3a,12a-dihydroxy-5p-cholan-24-oate, Sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate, and poly[1-(2-oxopyrrolidin-1- yl)ethylene]; wherein said compounds are present in the formulation at a minimum concentration of about 0.5% and wherein the salt solution is a T ris buffer if Deoxycholic acid sodium salt or Sodium 1 ,4-bis(2-ethylhexoxy)-1 ,4-dioxobutane-2-sulfonate are selected and a Sodium bicarbonate buffer if poly[1-(2-oxopyrrolidin-1-yl)ethylene] is selected, preferably wherein the salt solution are present at any of the concentrations defined in any one of claims 3 or 4.

31. The formulation according to any one of claims 1 to 30, wherein the formulation has a lyophilization stabilizer, and the stabilizer is selected from Glucose, Trehalose, Mannitol, Sucrose, Lactose, Inositol, Xylose, Xylitol, Isomaltulose, Sorbitol, Maltitol, Mannose, Fructose, Proline, Arginine, Histidine or Glycine.

32. The formulation according to any of the precedent claims, wherein the formulation is for use in diseases prevention or therapy.

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