MXPA00006154A - Keratinocyte growth factor-2 formulations - Google Patents

Abstract

The invention is directed to liquid and lyophilized forms of Keratinocyte Growth Factor-2 (KGF-2) and derivatives thereof. This invention further relates to the formulation of KGF-2 for therapeutic use, for example, to promote or accelerate wound healing.

Description

FORMULATIONS OF THE FACTOR-2 GROWTH OF KERATINOCYTES BACKGROUND OF THE INVENTION Field of the Invention The present invention is directed to liquid and lyophilized formulations of keratinocyte growth factor-2 (KGF-2) and derivatives thereof. This invention also relates to formulations of KGF-2, especially topical and injectable formulations, which can be used for therapeutic use in indications that require development and regeneration of soft tissues.

Background of the Invention The fibroblast growth factor family has emerged as a large family of growth factors involved in the development and regeneration of soft tissues. This currently includes several members who share a varying degree of homology at the protein level, and who, with one exception, appear to have a mitogenic spectrum REF.121159 broad similar, for example, these promote the proliferation of a variety of cells of sdermal and neuroectodermal origin and / or promote angiogenesis. KGF was originally identified as a member of the FGF family by sequential homology or factor purification and cloning. The keratinocyte growth factor (KGF) was isolated as a mitogen from a cultured murine keratinocyte line (Rubin, JS et al., Proc.Na.I.Acid.Sci.U.A. 86: 802-806 (1989)) . Contrary to the other members of the FGF family, it has little activity on cells derived from mesenchyme but stimulates the growth of epithelial cells. Keratinocyte growth factor is produced by fibroblasts derived from the skin and from the fetal lung (Rubin et al. (1989)). It was found that keratinocyte growth factor mRNA is expressed in the kidney, colon and ileum of adults, but not in the brain or lung (Finch, PW et al., Sci ence 245: 752-755 ( 1989)). KGF shows the conserved regions within the FGF protein family. KGF binds to the FGF-2 receptor with high affinity.

The healing of damaged wounds is a significant source of morbidity and can result in complications such as dehiscence, anastomotic rupture and unhealed wounds. In the normal individual, the healing of wounds is carried out without complications. In contrast, impaired healing is associated with various conditions such as diabetes, infection, immunosuppression, obesity and poor nutrition (Cruse, PJ and Foord, R., Arch. Surg. 107: 206 (1973); Schrock, TR et al. , Ann, Surg., 177: 513 (1973), Poole, GU, Jr., Surgery 97: 631 (1985), Irvin, GL et al., Amm. Surg. 51: 418 (1985)). The repair of wounds is the result of biological processes and complex interactions. Three phases have been described in the normal wound healing: the acute inflammatory phase, the extracellular matrix and the collagen synthesis, and the remodeling (Peacock, EE, Jr., Wound Repair, 2nd Edition, WB Saunders, Philadelphia (1984) ). The process involves the interaction of keratinocytes, fibroblasts and inflammatory cells at the site of the wound. It is desirable to formulate polypeptides that are capable of promoting and enhancing soft tissue growth and regeneration thereof in pharmaceutical compositions that (1) are stable over prolonged periods of storage time, (2) increase the pharmacological activity or effectiveness of the polypeptide and / or (3) allow easy application or administration of the polypeptide in therapeutic regimens.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to liquid and lyophilized formulations of KGF-2 and to deletion or point mutants or substitution thereof (herein referred to as KGF-2 polypeptides). This invention further relates to the use of such formulations of KGF-2 polypeptides to promote or accelerate soft tissue development or regeneration thereof, for example in the healing of wounds, or in the treatment of mucocitis or inflammatory bowel disease. Preferred formulations of the present invention employ the novel mutant forms of KGF-2, and in one embodiment employ a deletion mutant referred to herein as KGF-2-? 33. The co-ingredients employed in the formulations (1) provide storage stability to the KGF-2 polypeptide, (2) further enhance the soft tissue healing activity of the therapeutic composition, and / or (3) provide the KGF-2 polypeptide in an active form, while allowing easy application and administration for particular therapeutic purposes. A first aspect of the present invention relates to a formulation comprising a KGF-2 polypeptide and a buffering agent having a buffering capacity of between about pH 5.0 and about 8.0. Useful buffers include phosphate, acetate, aconite, succinate, malate, carbonate and citrate buffers, citrate being preferred. A second aspect of the invention relates to a formulation comprising a KGF-2 polypeptide, a bulk agent for lyophilization and a buffering agent having a buffering capacity of between about pH 5.0 and about pH 8.0. Useful buffers include phosphate, aconite, succinate, malate, carbonate and citrate, with citrate being preferred. A third aspect of the invention relates to a formulation comprising a KGF-2 polypeptide and a thiol-containing compound, preferably onothioglycerol, capable of stabilizing the KGF-2 polypeptide. This formulation preferably includes a buffering agent having a buffering capacity of between about pH 5.0 and about pH 8.0. This formulation may also include one or more antioxidants and one or more metal chelating agents. A fourth aspect of the present invention relates to a formulation comprising a KGF-2 polypeptide, a buffer, and a high molecular weight compound that causes the formulation to gel at a certain predefined temperature. A preferred high molecular weight compound is a block copolymer of polyoxyethylene-polyoxypropylene, Pluronic or Poloxamer. A thiol-containing compound, such as monothioglycerol, can be included in the formulation to provide aggregate stability to the polypeptide. A fifth aspect of the present invention relates to a formulation comprising a KGF-2 polypeptide, a buffering agent and a thickening agent. Thickening agents are used to increase the viscosity of the formulation. Preferred thickeners are carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC), Natrosol and Carbomers.

In addition, the formulations of the present invention can also include metal chelating agents, antioxidants or thiol-containing compounds, such as ascorbic acid ester, monothioglycerol, cysteine, tocopherols, butylated hydroxyanisole, sodium sulfate, sodium bisulfite and sodium metasulfite. , and preservatives such as phenol, chlorobutane, benzyl alcohol, methylparabens and propylparabens. The formulations of the present invention can also have a nitrogen atmosphere protection over the top space of the bottle. In addition, the formulations of the present invention may include purging the buffer of the formulation with helium, argon or nitrogen.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1C illustrate the cDNA and the corresponding deduced amino acid sequence of KGF-2. The 35 or 36 initial amino acid residues represent the putative guiding sequence (underlined). Standard one-letter abbreviations for amino acids are used. Inaccuracies in sequencing are a common problem when trying to determine the polynucleotide sequences. Sequencing was performed using an Automatic DNA Sequencer 373 (Applied Biosystems, Inc.). The sequencing accuracy is predicted to be greater than 97% (SEQ ID NOs .: 1 and 2). Figures 2 (A) -2 (C) describe the stimulation of the proliferation of primary, normal epidermal keratinocytes by the KGF-2 polypeptides of the invention. Figure 2 (A) shows the stimulation of the proliferation of primary, normal epidermal keratinocytes by KGF-2. Figure 2 (B) shows the stimulation of the proliferation of normal primary epidermal keratinocytes by KGF-2-? 33. Figure 2 (C) shows the stimulation of the proliferation of primary, normal epidermal keratinocytes by KGF-2-? 28. Figure 3 shows the results of the bioactivity for the liquid formulation of KGF-2-? 33, in the stability at 10 months. Figure 4 shows the results of the bioactivity for the lyophilized preparation of KGF-2-? 33, in the stability at 9 months. Figure 5 shows the effect of monothioglycerol on the bioactivity of KGF-2.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES KGF-2 stimulates the proliferation of epidermal keratinocytes but not mesenchymal cells such as fibroblasts. Thus, a "polypeptide having similar activity to the KGF-2 protein" includes polypeptides showing the KGF-2 activity, in the keratinocyte proliferation assay described below and binds to the isoforms of the FGF 1-iiib receptor and 2-iiib. The present invention is directed to the pharmaceutical and veterinary formulations of KGF-2 polypeptides. KGF-2 polypeptides are defined herein by reference to the polypeptide of Figure 1 (SEQ ID NO: 2) or that encoded by the deposited cDNA, and include fragments, derivatives and analogs of the polypeptide of Figure 1 (SEQ ID NO .: 2) or that encoded by deposited cDNA that retains essentially the same biological function as the parent polypeptide. The polypeptides employed in the present invention can be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides. It has been found that KGF-2 polypeptides show poor activity and stability at a pH of 4.5 or less, or at a pH above about 8.0. The present inventors have discovered that the KGF-2 polypeptides are oxidized and precipitated. These polypeptides present a difficult challenge when trying to formulate them for therapeutic purposes. In order to maintain physical-chemical properties and maintain biological activity, KGF-2 polypeptides can be formulated with antioxidants, such as oxygen scavenger compounds, and / or a protein stabilizer, such as a thiol-containing compound, and / or a metal chelating agent such as EDTA. Stabilization, as used herein, refers to the maintenance of the physicochemical properties and substantial biological activity of the KGF-2 polypeptides over a given period of time. The formulations according to the present invention include the gel, the thick solution, the solution and the lyophilized forms. The formulations are also referred to herein as "pharmaceutical compositions" or "compositions." Injectable Formulations Liquid Formulations A first aspect of the present invention is directed to liquid formulations of KGF-2 polypeptides comprising: a KGF-2 polypeptide and a buffer having a buffer capacity of between about pH 5.0 and about pH 8.0, more preferably pH 5.5 a pH 6.5, more preferably pH 6.2. Useful buffers include buffers derived from phosphate, acetic acid, aconitic acid, citric acid, glutaric acid, malic acid, succinic acid and carbonic acid. Typically employed is an alkaline or alkaline earth metal salt of one of the aforementioned acids. More preferably, the buffer will be acetate or citrate, more preferably citrate. For example, the formulation may comprise a composition formed by mixing a buffer amount of citric acid or a pharmaceutically acceptable salt thereof with KGF-2-? 33 in water. The formulation may alternatively comprise a composition formed by mixing a buffer amount of acetic acid or a pharmaceutically acceptable salt thereof with KGF-2? 33 in water. Preferred concentrations of the buffer are from about 5 mM to about 50 mM. More preferably, the acetate buffer will have a concentration of about 20 mM and the citrate buffer will be about 10 mM to about 20 mM. The formulation may also include sodium chloride as a tonicity agent at a concentration of about 0.01 mM to about 150 mM, more preferably about 125 mM, and a metal chelating agent, such as EDTA, at a concentration of about 0.1 mM. to about 10 mM, more preferably to about 1 mM. In addition, a liquid formulation of the present invention may also include one or more of (a) a stabilizing amount of an antioxidant, such as ascorbate and / or (b) a protein stabilizing amount of a thiol compound, for example monothioglycerol (MTG ). Without wishing to be bound by any theory, it is believed that thiol-type compounds such as MTG serve to protect the free sulfhydryl groups present in the KGF polypeptides. The storage conditions for the liquid formulation are typically from about 2 ° C to about 8 ° C.

Alternatively, the storage conditions are at or below 20 ° C. More preferably, storage conditions are about -20 ° C. Maintaining a liquid formulation of KGF-2 in a frozen state limits the amount of oxidation to the polypeptide, which in turn results in a stable formulation of the polypeptide. Preferably, a liquid formulation comprises: 1) a therapeutically effective amount of a KGF-2 polypeptide; 2) an effective amount of a buffer having a buffer capacity of between about pH 5.0 and about pH 8.0; and 3) a pharmaceutically acceptable diluent; and :) optionally one or more of the following: a) sodium chloride as a tonicity agent, b) a chelating agent, c) a stabilizing amount of an antioxidant, and d) a stabilizing amount of a protein stabilizer.

The KGF-2 polypeptide is preferably maintained in solution. The compositions of the present invention are manufactured by mixing the above-listed ingredients with each other, preferably in concentrations and proportions as expressed herein. Antioxidants that can be used in the liquid formulation include ascorbic acid, tocopherols and butylated hydroxyanisole. In addition, stabilizers that can be used in the liquid formulation also include thiols such as cysteine, methionine and thioglycerols. Chelating agents that can be used include ethylenediaminetetraacetic acid (EDTA), or diethylenetriaminepentaacetic acid (DPTA), with EDTA being preferred. Formulations of the present invention that include antioxidants or thiols can increase the stability of KGF-2 polypeptides. This makes it possible to have a pharmaceutical product with a longer shelf life. More preferred liquid formulations comprise: 1) a KGF-2 polypeptide in a concentration range of about 0.02 to about 40 mg / ml (w / v), more preferably about 0.05 to about 30 mg / ml (w / v), even more preferably about 0.1 to about 20 mg / ml (w / v), still more preferably about 10 mg / ml, and most preferably about 0.2 to 4 mg / ml; 2) a buffer having a buffer capacity of between about pH 5.0 and about pH 8.0 at a concentration range of about 5 mM to about 50 mM, preferably about 5 mM to about 30 mM; and 3) a pharmaceutically acceptable diluent, preferably water, to bring the composition to a designated volume. Buffers useful for the formulations of the present invention include buffers derived from acetic, aconitic, citric, glutaric, malic, succinic, phosphoric and carbonic acids. Typically employed is an alkaline or alkaline earth metal salt of one of the aforementioned acids. Acetate and citrate buffers, such as acetic acid or a pharmaceutically acceptable salt thereof, or citric acid or a pharmaceutically acceptable salt thereof, are preferred. Preferred pH ranges for the formulation of the solution is from about pH 5.0 to about pH 8.0, preferably pH 5.5 to pH 6.5, and more preferably about pH 6.2. Sodium acetate or sodium citrate are the preferred buffering agents, with sodium citrate being the most preferred. To the above solution are also preferably added: 4) a chelating agent, such as EDTA, at a concentration in the range of about 0.1 mM to about 10 mM, more preferably to about 1 M; 5) sodium chloride at a concentration range of 0.01 mM to about 150 mM and more preferably to about 125 M. Optionally, a liquid formulation may also include a stabilizing amount of proteins of a compound selected from the group consisting of: a) about 0.5% to about 2% w / v of glycerol, b) about 0.1% to about 1% w / v of methionine, or c) about 0.1% to about 2% w / v of monothioglycerol. Preferred embodiments of this aspect of the present invention include a composition formed by mixing: 1) a KGF-2 polypeptide in a concentration of from about 0.02 to about 40 mg / ml (w / v), more preferably from about 0.1 up to about 20 mg / ml, and more preferably about 0.2 to 4 mg / ml. 2) 10 mM sodium citrate or sodium acetate mM; 3) 125 mM sodium chloride; 4) 1 mM EDTA; and 5) water as a diluent. More preferably, the solution formulation comprises a composition formed by mixing: 1) about 0.2 to about 4 mg / ml of a KGF-2 polypeptide; 2) 20 mM sodium acetate; 3) NaCl 125 MM; 4) EDTA 1 M; and 5) water as a diluent, wherein the solution is at a pH of about 6.2 and is stored at about -20 ° C. The present inventors have discovered that KGF-2 polypeptides are easily oxidized, aggregated and precipitated from the solution. Although the KGF-2 solution does not destroy biological activity, limiting the degree of oxidation of the product leads to a more stable product. The inventors observed that if the liquid formulation is at too low a pH the KGF-2 polypeptide will lose the biological activity. In addition, as the pH of the solution approaches pl for KGF-2, the protein will precipitate out of the solution. Thus, the inventors have determined that the liquid formulations should be maintained in the range of about pH 6.0 to about pH 7.0, and that a pH of about 6.2 is most optimal for stabilization of the KGF-2 polypeptide. In addition, the inventors surprisingly determined that a citrate buffer specifically stabilizes the KGF-2 polypeptides. However, the use of a citrate buffer having a pH of about 6.0-6.2, provides a liquid formulation that reduces aggregation of the KGF-2 polypeptide and increases stability, the formulation of the liquid polypeptide can still be subject to oxidation and precipitation of the KGF-2 polypeptides. In this way, the inventors developed a lyophilized formulation as described below.

Freeze-dried formulations A second aspect of the present invention is directed to lyophilized formulations of KGF-2 polypeptides comprising: a KGF-2 polypeptide and a buffer having a buffer capacity of between about pH 5.0 and about 8.0, more preferably pH 5.5 at pH 6.5, more preferably pH 6.2. Useful buffers include buffers derived from phosphoric, aconitic, citric, glutaric, malic, succinic and carbon acids. Typically employed is an alkaline or alkaline earth metal salt of one of the aforementioned acids. More preferably, the buffer will be phosphate or citrate, more preferably citrate. For example, the formulation may comprise a composition formed by mixing a buffering amount of citric acid or a pharmaceutically acceptable salt thereof with KGF-2? 33 in water. The preferred buffer concentration is from about 5 M to about 50 mM and more preferably about 10 mM. More preferably, the citrate buffer will be added at a concentration of about 10 mM. Also preferably included in the formulation is sodium chloride as a tonicity agent at a concentration of about 0.01 mM to about 150 mM, more preferably about 20 mM and a metal chelating agent, such as EDTA, at a concentration of about 0.01 mM to about 10 mM, more preferably about 1 mM. In addition, volume / cryoprotectant agents such as sucrose, glycine, mannitol, trehalose or other pharmaceutically acceptable bulking agents are included in the formulation. The amount of volume agent used will be such that the solution is isotonic and is in the range of about 2% to about 10% w / v. Preferred concentrations are as follows: 5% mannitol, 7% sucrose, 8% trehalose, or 2% glycine + 0.5% sucrose. More preferably, the sucrose or the sucrose / glycine mixture is used. In addition, a lyophilized formulation of the present invention may also include one or more of (a) a stabilizing amount of an antioxidant., such as ascorbate or (b) a stabilizing amount of thiol compound, for example monothioglycerol. The storage conditions for the lyophilized formulation are typically from about 2 ° C to about 25 ° C. More preferably the storage conditions are at or below about 2 ° C to about 8 ° C. The KGF-2 polypeptides are lyophilized at a concentration of about 0.02 mg / ml to about 10 mg / ml protein in the initial solution. The initial lyophilization solution preferably comprises (in addition to the KGF-2 polypeptides): 1) an effective amount of citric acid or a pharmaceutically acceptable salt thereof, preferably sodium citrate, at a concentration range of about 5 mM to about 20 mM; 2) sodium chloride at a concentration range of about 0.1 mM to about 125 mM; 3) EDTA at a concentration range of about 0.1 mM to about 10 mM; 4) one or more of sucrose, mannitol, glycine or trehalose at a concentration range of about 2% w / v to about 15% w / v; Y ) water. The preferred pH range for the lyophilization buffer is from about 5.5 to about 8.0, preferably about pH 6.2. More preferably, the lyophilization buffer comprises 10 mM sodium citrate, 20 mM sodium chloride, 1 mM disodium EDTA at pH 6.2 and 7% sucrose. Freeze-dried KGF-2 polypeptide formulations are reconstituted in sterile water to maintain isotonic conditions of approximately 290 mOs. The KGF-2 polypeptides can be reconstituted in sterile water, optionally containing a stabilizing amount of antioxidants comprising: a) about 0.01% up to about 2% w / v monothioglycerol, b) about 0.01% up to about 2% w / v ascorbic acid, c) about 0.01% up to about 2% w / v of methionine or d) combinations thereof.

The present invention includes lyophilization cycles that produce a stable KGF-2 polypeptide formulation. The lyophilization cycle is designed to maintain the KGF-2 polypeptide product below its collapse temperature during the primary drying phase. In addition, the moisture content is manipulated to be preferably less than 5%, and more preferably less than 2%. Such a protocol must be determined for any particular protein on an individual basis. An exemplary lyophilization cycle for the lyophilization formulation containing sucrose for KGF-2, according to the present invention, was determined as follows: The lyophilization formulation of the present invention provides a product with unexpectedly increased stability. Of course, the lyophilized KGF-2 formulations of the present invention are biologically stable for at least 9 months at temperatures up to 45 ° C (Figure 4). Reverse phase HPLC demonstrated that the lyophilized KGF-2 formulations of the present invention retained their physico-chemical properties for up to 8 months at temperatures of or below 45 ° C and 75% relative humidity. It has stability for this length of time at such high temperatures is very unusual for proteins.

Thickened and Gel Formulations A third aspect of the invention is directed to thick or gel formulations for KGF-2 polypeptides. 1) Thickening Agents: Thickening agents can be added to the liquid formulations described above to increase the viscosity of the resulting formulation.

A formulation that has an increased viscosity may be beneficial for topical applications where controlled release may be important, adhering to the shape of a wound or preventing spillage. Such thick formulations are employed for topical applications such as wound healing, to treat skin disorders or any other use that could be treated via the topical application of a pharmaceutical formulation of KGF-2. The thickening agent should raise the viscosity to about 50 to about 10,000 cps, more preferably about 50 to about 1,000 cps and more preferably about 200 to about 300 cps. Viscosity is measured using a rotating needle viscometer. The most preferred concentration of the thickening agent is from 0 to 5% (w / w). • The thick solution will remain liquid at all times. Examples of suitable thickening agents include, but are not limited to etherified cellulose and carbomer, water soluble (high molecular weight polymers of crosslinked acrylic acid with either allylucose or allylic ethers of pentaerythritol). Examples of etherified cellulose are well known in the art (listed in the USP) and include alkylcelluloses, hydroxyalkylcelluloses and alkylhydroxyalkylcelluloses, for example, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and the like. In a further embodiment, the topical or incisional gel may comprise about 0 to about 20% by weight of a cellulose derivative having a molecular weight of about 50,000 to about 700,000. In a preferred embodiment the cellulose derivative is present at about 2% to about 8% by weight and has a molecular weight in the range of about 80,000 to about 240,000. Preferred cellulose derivatives are hydroxypropylmethylcellulose, methylcellulose, carboxymethylcellulose, and hydroxyethylcellulose. When thickening agents are added to the injectable formulations, detailed above, salts and buffering agents may be added or removed from the formulation for optimum stability. For example, the concentration of citrate can be increased. Preferred concentrations for citrate are, for example, about 10 mM to about 500 mM citrate, more preferably about 10 mM to about 50 mM citrate and most preferably about 10 mM to about 20 mM citrate. In addition, the amount of sucrose can be decreased in the lyophilization formulation to a range of from about 0% to about 5% sucrose. The thickening agents can be added directly to a liquid formulation according to the present invention and then lyophilized. Alternatively, a lyophilized formulation according to the present invention can be reconstituted by the addition of a suitable diluent, more preferably water having a thickening agent dissolved therein. Such thick formulations could be administered by spray. An example of a thick, preferred KGF-2 polypeptide solution according to the present invention comprises a product formed by mixing: 1) a topically effective amount of a KGF polypeptide, preferably KGF-?; 2) about 10 mM to 500 mM sodium citrate buffer; 3) about 0.01 to about 150 mM sodium chloride; 4) about 0.75 to about 1.27 mM, preferably about 1 mM EDTA; 5) about 0.1% to about 7% sucrose; 6) about 0.75% to about 1.5% (w / w) of carboxymethylcellulose, or about 0.5 to about 1.5% of hydroxypropylmethylcellulose or about 0.25 to about 0.75% of hydroxyethylcellulose or about 0 to about 1% of carbomer or any combination thereof . The pH of such a formulation is more preferably pH 6.2. 2) Gelification Agents: Yet another aspect of the present invention is directed to gel formulations for KGF-2 polypeptides. The gelling agents can be added to injectable formulations of the present invention to provide a formulation that remains liquid at room temperature and solidifies when applied to the surface of the skin (at about 37 ° C). Such formulations may be useful for topical applications where controlled release, adherence to the shape of a wound or avoidance of spillage may be important. Such gel formulations are employed for topical uses such as wound healing, for treating skin disorders or any other use that could be treated via the topical application of the pharmaceutical composition of KGF-2. Gel formulations for polypeptides KGF-2 according to the present invention comprise: 1) a topically effective amount of a KGF polypeptide; 2) a buffer, 3) a pharmaceutically acceptable diluent, preferably water; and 4) a high molecular weight gel-forming compound. The viscosity of the gel formulations of the present invention may be in a range from about 1 to about 10,000 cps at room temperature, more preferably from about 20 to about 100 cps at room temperature. Viscosity is measured using a rotating needle viscometer.

The high molecular weight, gel-forming compounds used in the present invention are typically water-soluble polymers capable of forming a viscous aqueous solution, or water-swellable, or water-insoluble polymers (e.g., collagen) that can also form a water-soluble polymer. viscous solution and that gel on contact with the skin. High-molecular weight, gel-forming compounds useful can be selected from vinyl polymers, polyoxyethylene-polyoxypropylene copolymers, polysaccharides, proteins, poly (ethylene oxide), acrylamide polymers and derivatives and / or salts thereof. Other compounds that can be used to make the pharmaceutical gel formulations used in wound healing can be found in U.S. Patent No. 5,427,778, which is incorporated herein by reference in its entirety. Useful vinyl polymers (or substituted polyethylenes) include polyacrylic acid, polymethacrylic acid, polyvinylpyrrolidone and polyvinyl alcohol. Useful polysaccharides include cellulose derivatives, glycosaminoglycans, agar, pectin, alginic acid, dextran, starch (-amylase or amylopectin), and chitosan. Useful glycosaminoglycans include hyaluronic acid, chondroitin, chondroitin 4-sulfate, heparan sulfate and heparin. The glycosaminoglycans can be used to improve wound healing in combination with any other gel-forming polymer such as, for example, collagen, gelatin, fibronectin. The acrylamide polymers can be polyacrylamide or polymethacrylamide polymers. Preferred high molecular weight gel forming compounds are polyoxyethylene-polyoxypropylene block copolymers, especially those block copolymers which are designated on the market as PLURONICS (BASF) or POLAXAMERS (BSF). In a preferred embodiment, the gel of the present invention can comprise about 10 to about 60% by weight of a polyoxyethylene-polyoxypropylene block copolymer having an average molecular weight of about 500 to 50., 000 In a more preferred embodiment, the gel of the present invention may comprise about 14 to about 18% by weight of block copolymers having a molecular weight in the range of 1,000 to 15,000. The preferred block copolymers of the present invention are Pluronic F108 and Pluronic F127.

Polyoxyethylene-polyoxypropylene block copolymers (Pluronic or Poloxamer) have great potential for use in topical drug delivery systems because they exhibit reverse thermal gelation behavior, have good drug release characteristics as well as low toxicity. The gels are formed as the solution heats up. In this way, the gel is an aqueous solution of low viscosity at room temperature, but when it comes into contact with the body of a mammal and is heated by the body temperature, the viscosity increases as the solution gels. Pluronic gels can be used for the controlled administration of KGF-2 polypeptides for example, for wounds and other sites such where topical administration is desirable. The KGF-2 polypeptides can be combined with Pluronic in the liquid state and applied to the wound. The gelation occurs and effectively reduces the rate or rate at which the polypeptides are released into the wound, and thereby allows prolonged contact between the polypeptides and the wound site. The benefits of using such gel formulations include maintaining the wound and having a pharmaceutical composition that conforms to the shape of the wound, and to another site such where the compound can be applied. Preferred gel formulations for the KGF-2 polypeptides according to the present invention comprise citrate buffer and a Pluronic. The formulation may comprise an amount of citric acid or a pharmaceutically acceptable salt thereof. The gel formulation according to the present invention can also include a chelating agent, a stabilizing amount of antioxidants or thiols. The gel formulation will include a high molecular weight compound, such as a Pluronic, or etherified water soluble cellulose, and the like in an amount that will form a gel. In the gel formulation according to the present invention, the KGF-2 polypeptides are preferably in a concentration of about 0.01 mg / ml to about 10 mg / ml. Preferably, the gel formulations are formed by mixing: 1) a KGF-2 polypeptide, preferably KGF-2-? 33, in a calculated final concentration of 0.01 mg / ml to about 10 mg / ml; 2) an effective amount of a buffering agent; . 3) about 10% to about 60%, or more preferably about 14% to about 18% by weight of a polyoxyethylene-polyoxypropylene block copolymer having an average molecular weight of about 500 to 50,000; and 4) a pharmaceutically acceptable diluent, preferably water. Another preferred gel formulation comprises: 1) a pharmaceutically active amount of the KGF-2 polypeptide; 2) about 10 mM to about 500 mM sodium citrate; 3) about 0.01 mM to about 150 mM sodium chloride; 4) about 1 mM EDTA; 5) about 0.01% to about 7% sucrose; 6) about 14% to about 18% Pluronic F127; and 7) water, wherein the formulation is at a pH of about pH 6.2. More preferably, the gel formulation comprises: 1) a KGF-2 polypeptide, preferably KGF-2-? 33, at a concentration in the range of about 0.01 mg / ml to about 10 mg / ml (w / v), more preferably about 0.1 mg / ml to about 3 mg / ml, and most preferably about 0.2 mg / ml; 2) sodium citrate at a concentration range of about 5 M to about 20 mM; 3) about 10% up to approximately % (w / v), preferably about 15 to about 25, and more preferably about 16% Pluronic 127 or Poloxamer 407; 4) about 6.7% to about 7.3% sucrose, preferably about 7% sucrose; and 5) water until the capacity. The gel formulation optionally further includes one or more of the following: 6) EDTA at a concentration range of about 0.1 mM to about 10 mM. 7) a concentration range of about 0.01 mM to about 125 mM.

Preferred ranges for the gel formulation is from about pH 5.0 to about pH 8.0, preferably pH 6.2 and the resulting gel formulation must be isotonic. 3) Additional Stabilizing Agents: All of the above formulations of the present invention can benefit from antioxidants, metal chelating agents, thiol-containing compounds and other general stabilizing agents. Examples of such stabilizing agents include, but are not limited to: a) about 0.5% to about 2% w / v glycerol, b) about 0.1% up to about 1% w / v methionine, c) about 0.1% up to about 2% w / v monothioglycerol, d) about 1 mM to about 10 mM EDTA, e) about 0.01% up to about 2% w / v ascorbic acid, f) 0.003% up to about 0.02% w / v polysorbate 80, g) 0.001% to about 0.05% w / v polysorbate 20, h) arginine, i) heparin, j) dextran sulfate, k) cyclodextrins, or 1) combinations thereof Administration of KGF-2 Polypeptides The KGF-2 polypeptide formulations of the present invention can employ pharmaceutically suitable diluents that are known to be useful in pharmaceutical compositions. Such diluents include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must be adapted to the mode of administration. Preferably, the pharmaceutical compositions will be formulated according to the present invention, as indicated above. Water is a preferred diluent. The polypeptide having KGF-2 activity can be administered in pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily use of the pharmaceutical compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The therapeutically effective dose level specific to any particular patient will depend on a variety of factors including the type and degree of the response that will be achieved; the specific composition, including if any other agent is employed, if any; age, body weight, general health, sex and the patient's diet; the time of administration, the route of administration, and the rate of expression of the composition; the duration of the treatment; the drugs (such as a chemotherapeutic agent) used in combination or coincident with the specific composition; and similar factors well known in the medical arts. Suitable formulations, known in the art, can be found in Remington's Pharmaceutical Sci ences (latest edition), Mack Publishing Company, Easton, PA. The "effective amount" of KGF-2 for purposes of the present (including an effective amount of KGF-2) is thus determined by such considerations. The pharmaceutical compositions of the present invention can be administered in a convenient manner such as by the oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarticular, subcutaneous, intranasal, inhalation, intraocular or intradermal routes. Parenteral and topical administration are the preferred routes of administration. The term "parenteral" as used herein refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection, and infusion. The pharmaceutical compositions are administered in an amount that is effective for the treatment and / or prophylaxis of the specific indication. In most cases, the dose of KGF.2 is from about 1 μg / kg to about 30 mg / kg of body weight daily, taking into account the routes of administration, the symptoms, etc. However, the dose can be as low as 0.001 μg / kg. For example, in the specific case of topical administration doses, it is preferably administered from about 0.01 μg to 9 mg per cm2. In the case of intranasal and infra-ocular administration, the doses are preferably administered from about 0.001 μg / ml to about 10 mg / ml, and more preferably from about 0.05 mg / ml to about 4 mg / ml. As a general proposition, the total pharmaceutically effective amount of the KGF-2 polypeptide parenterally administered will be in the range of about 1 μg / kg / day to 10 mg / kg / day of the patient's body weight, although, as noted above, this will be subject to the discretion of the therapist. If administered continuously, the KGF-2 polypeptide is typically administered at a dose range of about 1 μg / kg / hour to about 50 μg / kg / hour, either for 1 to 4 injections per day or for continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution or bottle solution can also be used. A course of treatment of the KGF-2 polypeptide to affect the fibrinolytic system appears to be optimal if it is continued for longer than a certain number of days, 7 days in the case of the mice. The duration of treatment necessary to observe changes and the interval after treatment for the responses to occur seems to vary depending on the desired effect.

For parenteral administration, in one embodiment, the KGF-2 polypeptide is formulated in general by mixing it to the desired degree of purity, in a unit dose injectable form (suspension solution, or emulsion), with a pharmaceutically acceptable carrier, for example , one that is non-toxic to recipients at the doses and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation does not preferably include oxidizing agents and other compounds that are known to be harmful to the polypeptides. In general, the formulations are prepared by contacting the KGF-2 polypeptide uniformly and intimately with liquid carriers, or finely divided liquid carriers, or both. Then, if necessary, then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the patient's blood. Examples of such carriers include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. Suitable formulations, known in the art, can be found in Remington's Pharmaceuti cal Sci ences (latest edition), Mack Publishing Company, Easton, PA. The KGF-2 polypeptides can also be administered to the eye to treat damage to the lacrimal glands, disorders and pathologies in animals and humans such as a liquid, drops, or thick liquid, a gel. The KGF-2 polypeptides can also be intranasally administered to the nasal mucosa to treat disorders, damages and pathologies of the nasal mucosa and nasal sinus epithelia, in animals and humans as liquid drops or in a sprayed form. In general, the formulations are prepared by contacting the KGF-2 polypeptide uniformly and intimately with liquid carriers or finely divided solid carriers, or both. Then, if necessary, the product is conformed to the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the patient's blood. Examples of such carriers include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. Suitable formulations, known in the art, can be found in Remington's Pharmaceuti cal Sci ences (latest edition), Mack Publishing Company, Easton, PA. The carrier may also contain minor amounts of suitable additives such as substances that improve isotonicity and chemical stability. Such materials are non-toxic to patients at the doses and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight polypeptides (less than about ten residues), eg, polyarginine or tripeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose and its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers or PEG.

KGF-2 is typically formulated in such vehicles at a concentration of about 0.01 μg / ml to 50 mg / ml, preferably 0.01 μg / ml to 10 mg / ml, at a pH of about 5 to about 8, preferably about 6 to about about 7, more preferably about pH 6.2. It will be understood that the use of some of the excipients, carriers or prior stabilizers will result in the formation of KGF-2 salts. KGF-2 to be used for therapeutic administration, can be sterile. Sterility is easily achieved by filtration through sterile filtration membranes (e.g., 0.2 micrometer membranes). The therapeutic compositions of KGF-2 can be placed in a container having a sterile access port, for example, an intravenous solution bag or bottle having a plug pierceable by a hypodermic injection needle. KGF-2 will ordinarily be stored in single-dose or multi-dose containers, for example, sealed vials or flasks, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 3 ml bottles are filled with 1 ml of sterile aqueous KGF-2 solution by 1% (w / v) filtration and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting lyophilized KGF-2 using water for injection which may optionally include one or more antioxidants. The dosage can also be accommodated in a patient-specific manner to provide a predetermined concentration of a KGF-2 activity in the blood, as determined by an RIA technique, for example. In this way, the dosage of the patient can be adjusted to achieve regular progress through blood levels, as measured by RIA, in the order of 50 to 1000 ng / ml, preferably 150 to 500 ng / ml. KGF-2 is also properly administered by sustained release systems. Suitable examples of sustained release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films or microcapsules. Sustained-release matrices include polylactides (U.S. Patent No. 3,773,919, European Patent EP-58,881), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U. Sidman et al., Bi opolymers 22: 547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Bi omed, Ma ter. Res. 15: 167-277 (1981), and R. Langer. Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (Rl Langer et al., Td.) Or poly-D- (-) -3-hydroxybutyric acid (European Patent EP 133,988). Sustained-release KGF-2 compositions also include KGF-2 entrapped in liposomes. Liposomes containing KGF-2 are prepared by methods known per se: DE-3, 218, 121; Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Na ti. Acad. Sci. USA 77: 4030-4034 (1980); EP-52,322; EP-36,676; EP-88,046; EP-143,949; EP-142,641; Japanese Patent Application 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP-102,324. Ordinarily, the liposomes are of the small unilamellar type (approximately 200-800 Angstroms) in which the lipid content is greater than about 30 mol percent of cholesterol, with the selected portion being adjusted for optimal therapy with KGF-2. The invention also provides a pharmaceutical package or equipment comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such containers may be a notice in the form prescribed by a governmental agency that regulates the manufacture, use or sale of pharmaceutical products or biological products, whose notice reflects approval by the agency, manufacturing, use or sale for the human administration. In addition, the polypeptides, agonists and antagonists of the present invention can be used in conjunction with other therapeutic compounds. When the present inventors examined the biological activity and stability of the KGF-2 polypeptide prepared according to the formulations of the present invention, it was surprisingly discovered that the use of monothioglycerol can stabilize the KGF-2 polypeptides and can behave as an enhancing agent for KGF-2 polypeptides in wound healing. The optimal concentration range for the potentiating effect of monothioglycerol was from 0.1% to 2% w / v.

Polypeptides KGF-2 KGF-2 stimulates the proliferation of epithelial cells and epidermal keratinocytes, but not mesenchymal cells such as fibroblasts. Thus, a "polypeptide having similar activity to the KGF-2 protein" includes polypeptides showing the activity of KGF-2, in the keratinocyte proliferation assay described below and in the US application No. 08 / 910,875 and can bind to the 1-iib and 2-iiib isoforms of the FGF receptor. Although the degree of activity need not be identical to that of the KGF-2 protein, preferably, "a polypeptide having activity similar to the KGF-2 protein" shows substantially similar activity compared to the KGF-2 protein (e.g. the candidate polypeptide shows greater activity or no more than ten times less, and, preferably, no more than about two times less activity relative to the reference KGF-2 protein). The KGF-2 polypeptides used in the formulations of the present invention may or may not have the N-terminal methionine, preferably the polypeptide will lack the N-terminal methionine. The cDNA clone of KGF-2 was deposited as ATCC Deposit No. 75977 on December 16, 1994 in the North American Species Crop Collection (American Type Culture Collectisn), Depositaría de la Patent, 10801 University Blvd. Manassas, VA 20110-2209. In addition, a cDNA encoding KGF-2-? 33 inserted into an expression vector, pHE4-5, was deposited with the ATCC on January 9, 1998 as ATCC No. 209575. The terms "fragment", "derivative" "and" analogous "when referring to the polypeptide of Figure 1 (SEQ ID NO: 2) or that encoded by the deposited cDNA, means a polypeptide that retains essentially the same biological function or activity as such a polypeptide. Thus, an analog includes a proprotein that can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO: 2) or that encoded by the deposited cDNA can be (i) one in which one or more of the amino acid residues are substituted with a residue of conserved or non-conserved amino acid (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group , or (iii) one in which the mature polypeptide is fused to another compound, such as another compound to increase the half-life of the polypeptide (eg, polyethylene glycol) or (iv) one in which additional amino acids are fused to the psypeptide mature, such as a guiding or secretory sequence or a sequence that is employed for the purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are considered to be within the reach of those skilled in the art from the teachings described herein. The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires, to indicate a chain of at least amino acids coupled by peptidyl bonds. The word "polypeptide" is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide sequences or sequences herein are written from left to right and in the direction from the amino terminus to the carboxyl terminus. It will be recognized in the art that some amino acid sequences of the KGF-2 polypeptide can be varied without significant effects of the structure or function of the protein. If such sequences in the sequence are contemplated, it must be remembered that there will be critical areas on the protein that determine the activity. In general, it is possible to replace the waste that forms the tertiary structure, with the condition that waste that performs a similar function is used. In other cases, the type of residue may be completely unimportant if the alteration occurs in a non-critical region of the protein. The polypeptides of the present invention are preferably in an isolated form. By "isolated polypeptide" is meant a polypeptide removed from its native environment. Thus, a polypeptide produced and / or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended are polypeptides that have been purified, partially or substantially, from a recombinant host cell or a native source. The pharmaceutical formulations of the present invention include the KGF-2 polypeptide of SEQ ID NO. : 2 (in particular the mature polypeptide) and the deletion mutants thereof, as well as polypeptides having at least 90%, 95%, 96%, 97%, 98%, 99% similarity (more preferably at least 90%) %, 95%, 96%, 97%, 98%, 99% identity) to the polypeptide of SEQ ID NO: 2 and deletion mutants thereof, and also include portions of such polypeptides with such a portion of the polypeptide ( such as the deletion mutants described below) generally containing at least 30 amino acids and more preferably at least 50 amino acids. As is known in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence and its substituted amino acid sequence, which preserves a polypeptide to the sequence of a second polypeptide. By "% similarity" for two polypeptides, a similarity score produced by comparing the amino acid sequences by the two polypeptides is sought using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 by Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 53711) and the default settings to determine similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Appli ed Ma thema ti cs 2: 482-489, 1981) to find the best segment of similarity between two sequences. For a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a KGF-2 polypeptide, it is understood that the amino acid sequence of the polypeptide is identical to the reference sequence , except that the polypeptide sequence can include up to five amino acid alterations per 100 amino acids of the reference amino acid of the KGF-2 polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence can be deleted or substituted with another amino acid, or A number of amino acids up to 5% of the total amino acid residues in the reference sequence can be inserted into the reference sequence. These alterations of the reference sequence can occur at the amino- or carboxyl-terminal positions of the reference amino acid sequence or at any other site between those terminal positions, interspersed either individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, if any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% is identical to, for example, the amino acid sequence shown in Figure 1 [SEQ ID NO. : 2] or the amino acid sequence encoded by the deposited cDNA clone, this can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 by Unix, Genetics Computer Group, University REsearch Park, 575 Science Drive, Madison, Wl 53711). When Bestfit or any other sequence alignment program is used to determine if a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are adjusted, of course, such that the percentage Identity is calculated on the full length of the reference amino acid sequence and that empty spaces in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

Suppression Mutants of KGF-2 Native KGF-2 is relatively unstable in the aqueous state and undergoes chemical and physical degradation resulting in the loss of biological activity during processing and storage. Native KGF-2 is also prone to aggregation in aqueous solution, at elevated temperatures, and becomes inactivated under acidic conditions. Particularly preferred KGF-2 polypeptides are the deletion mutants shown below (numbering begins with the first amino acid in the (Met) protein): Thr (residue 36) -Ser (residue 208) Ser (46) -Ser (208 ) Cys (37) --Ser (208) Pro (47) --Ser (208) Gln (38) --Ser (208) Glu (48) --Ser (208) Ala (39) --Ser (208) Wing (49) --Ser (208) Leu (40) --Ser (208) Thr (50) -Ser (208) Gly (41) --Ser (208) Asn (51) - Ser (208) Gln (42) - Ser (208) Ser (52) -Ser (208) Asp (43) --Ser (208) Ser (53) -Ser (208) Met (44) --Ser (208) Ser (54) ) --Ser (208) Val (45) --Ser (208) Ser (55) -Ser (208) Ser (56) --Ser (208) Phe (57) --Ser (208) Ser (59) --Ser (208) Ser (62) --Ser (208) Wing (63) --Ser (208) Gly (64) --Ser (208) Arg (65) --Ser (208) Val (67) --Ser (208) Ser (69) --Ser (208) Val (77) --Ser (208) Arg (80) --Ser (208) Met (i; Thr (36), or Cys (37 - -His (207) Met (i; Thr (36), or Cys (37 --Val (206) Met (i; Thr (36), or Cys (37 --Val (205) Met (1 Thr (36) , or Cys (37 -Met (204) Met (i; Thr (36), or Cys (37 -Pro (203) Met (1 Thr (36), or Cys (37 -Leu (202) Met (i; Thr (36), or Cys (37 -Phe (201) Met (1, Thr (36), or Cys (37 -His (200) Met (i; Thr (36), or Cys (37 --Ala (199 ) Met (i: Thr (36), or Cys (37 - Ser (198) Met (i; Thr (36), or Cys (37 -Thr (197) Met (i; Thr (36), or Cys (37 - sn (196) Met (i; Thr (36), or Cys (37 -Lys (195) Met (i; Thr (36), or Cys (37 -Arg (194) Met (i; Thr (36), or Cys (37 -Arg (193) Met (i; Thr (36), or Cys (37-hr (192) Met (l), Thr (36), or Cys (37) -Lys (191) Met (l) ), Thr (36), or Cys (37) -Arg (188) Met (l), Thr (36), or Cys (37) -Arg (187) Met (l), Thr (36), or Cys ( 37) -Lys (183) Preferred embodiments include the N-terminal deletions Ala (63) -Ser (208) (KGF-2? 28) and Ser (69) -Ser (208) (KGF-2? 33). Other preferred N-terminal and C-terminal deletion mutants include: Ala (39) -Ser (208); Pro (47) -Ser (208); Val (77) - Ser (208); Glu (93) - Ser (208); Glu (104) - Ser (208); Val (123) -Ser (208); and Gly (138) - Ser (208). Other preferred C-terminal deletion mutants include: Met (l), Thr (36), or Cys (37) -Lys (153). Also included by the present invention are the deletion mutants having deleted amino acids from the N-terminus and the C-terminus. Such mutants include all combinations of the N-terminal deletion mutants and the C-terminal deletion mutants described above, for example, Ala (39) -His (200), Met (44) - rg (193), Ala (63) - Lys (153), Ser (69) --Lys (153), etc. Those combinations can be made using recombinant techniques known to those skilled in the art.

Thus, preferred KGF polypeptides for use in the pharmaceutical formulations of the present invention comprise N-terminal deletion mutants, including those comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) except for a deletion of at least the first 38 N-terminal amino acid residues (eg, a deletion of at least Met (1) -Gln (38)) but not more than the first 147 N-terminal amino acids of Figure 1 ( SEQ ID NO .: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the first 38 N-terminal amino acid residues (eg, a deletion of at least Met (l) -Gln (38)) but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the first 46 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the first 62 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the first 68 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the first 76 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the first 92 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the first 103 N-terminal amino acid residues but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the first 122 N-terminal amino acid residues but not more than the first 137 N-ter amino acid residues of Figure 1 (SEQ ID NO: 2) . In addition to a formulation comprising a mutant of KGF-2 with the range of N-terminal deletion mutants described above, the present invention is also directed to a formulation having all combinations of the ranges described above, for example, deletions of at least the first 62 N-terminal amino acid residues but not more than the first 68 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); deletions of at least the first 62 N-terminal amino acid residues but not more than the first 76 N-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the first 62 N-terminal amino acid residues but not more than the first 92 N-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the first 62 N-terminal amino acid residues but not more than the first 103 N-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the first 68 N-terminal amino acid residues but not more than the first 76 N-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the first 68 N-terminal amino acid residues but not more than the first 92 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); deletions of at least the first 68 N-terminal amino acid residues but not more than the first 103 N-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the first 46 N-terminal amino acid residues but not more than the first 62 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); deletions of at least the first 46 N-terminal amino acid residues but not more than the first 68 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); deletions of at least the first 46 N-terminal amino acid residues but not more than the first 76 N-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); etc. In another embodiment, formulations comprising C-terminal deletion mutants are provided by the present invention. Preferably, the N-terminal amino acid residue of said C-terminal deletion mutants is amino acid residue 1 (Met), 36 (Thr), or 37 (Cys) of Figure 1 (SEQ ID NO: 2). Such formulations comprising mutants include those comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) except for a deletion of at least the last C-terminal amino acid residue (Ser (208)) but no more of the last 55 C-terminal amino acid residues (for example, a deletion of the Glu (154) -Ser residues (298)) of Figure 1 (SEQ ID NO .: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the last C-terminal amino acid residue but not more than the last 65 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the last 10 C-terminal amino acid residues but not more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the last 20 C-terminal amino acid residues but not more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the last 30 C-terminal amino acid residues but no more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the last C-terminal amino acid residues but not more than the last C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the formulation comprises a mutant having a deletion that will include at least the last C-terminal amino acid residues but not more than the last C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). In addition to a formulation comprising a mutant of KGF-2 with the range of C-terminal deletion mutants described above, the present invention is also directed to a formulation having all combinations of the range described above, for example, deletions of at least the last C-terminal amino acid residue but not more than the last 10 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); deletions of at least the last C-terminal amino acid residue but not more than the last 20 C-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the last C-terminal amino acid residue but not more than the last 30 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); deletions of at least the last C-terminal amino acid residue but not more than the last 40 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); deletions of at least the last 10 C-terminal amino acid residues but not more than the last 20 C-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the last 10 C-terminal amino acid residues but not more than the last 30 C-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the last 10 C-terminal amino acid residues but not more than the last 40 C-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2); deletions of at least the last 20 C-terminal amino acid residues but not more than the last 30 C-terminal amino acid residues of Figure 1 (SEQ ID NO .: 2), etc. In yet another embodiment, the KGF-2 polypeptide can be a deletion mutant having amino acids deleted from the N-terminal and C-terminal residues. Such mutants include all combinations of the N-terminal deletion mutants and C-terminal deletion mutants described above. Such mutants include those comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) except for a deletion of at least the first 46 amino acid residues but not more than the first 137 amino acid residues of Figure 1 (SEQ ID NO .: 2) and a deletion of at least the last C-terminal amino acid residue but not more than the last 55 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, a deletion may include at least the first 62, 68, 76, 92, 103 or 122 N-terminal amino acids, but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEO ID NO .: 2 ) and a deletion of at least the last 10, 20, 30, 40 or 50 C-terminal amino acid residues, but not more than the last 55 C-terminal amino acid residues of Figure 1. All combinations of the ranges or ranges previously described.

Substitution Mutants of KGF-2 Useful KGF-2 polypeptides include those that have amino acid substitution. Mature KGF-2 mutant contains 44 charged residues, 32 of which possess a positive charge. Depending on the location of such residues in the three-dimensional structure of the protein, the substitution of one or more of these agglomerated residues, with amino acids that have a negative charge or a neutral charge, can alter the electrostatic interactions of the adjacent residues and can be useful to achieve increased stability and reduced protein aggregation. The aggregation of the proteins can not only result in a loss of activity but can be problematic when preparing pharmaceutical formulations, because they can be immunogenic (Pinckard et al., Clin Exp. Immunol., 2: 331-340 (1967). , Robbins et al., Diabetes 36: 838-845 (1987), Cleland et al., Cri t Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)). Any modification must take into consideration the minimization of the carqa repulsion in the tertiary structure of the protein molecule. Thus, of special interest are substitutions of charged amino acids, with another charge and with neutral or negatively charged amino acids. The latter results in proteins with a reduced positive charge to improve the characteristics of KGF-2. Such improvements include the increased stability and reduced aggregation of the analog compared to the native KGF-2 protein. The replacement of amino acids can also change the selectivity of the binding to cell surface receptors. Ostade et al., Na ture 361: 266-268 (1993), described certain TNF alpha mutations that result in the selective binding of TNF alpha to only one of the two known TNF receptors. The KGF-2 molecules can include one or more substitutions, deletions or additions of amino acids, either from the natural mutation or human manipulation. Examples of some preferred mutations are: Ala (49) Gln, Asn (51) Ala, Ser (54) Val, Ala (63) Pro, Gly (64) Glu, Val (67) hr, Trp (79) Val, Arg (80) Lys, Lys (87) Arg, Tyr (88) rp, Phe (89) and r, Lys (91) Arg, Ser (99) Lys, Lys (102) Gln, Lysl03 ( Glu), Glu (104) Met, As (105) Lys, Pro (107) Asn, Ser (109) Asn, Leu (lll) Met, Thr (114) Arg, Glu (117) Ala, Val (120) lie , Val (123) He, Ala (125) Gly, Ile (126) Val, Asn (127) Glu, Asn (127) Gln, Tyr (130) -OPhe, Met (134) Thr, Lys (136) Glu, Lys (137) Glu, Gly (142) Ala, Ser (143) Lys, Phe (146) Ser, Asn (148) Glu, Lys (151) sn, Leu (152) Phe, Glu ( 154) Gly, Glu (154) sp, Arg (155) Leu, Glu (157) Leu, Gly (160) His, Phe (167) Wing, Asn (168) Lys, Gln (170) Thr, Arg (174) Gly, Tyr (177) Phe, Gly (182) Gln, Ala (185) Val, Ala (185) Leu, Ala (185) He, Arg (187) Gln (190) Lys, Lys (195) Glu, Thr ( 197) Lys, Ser (198) Thr, Arg (194) Glu, Arg (194) Gln, Lys (191) Glu, Lys (191) Gln, Arg (188) Glu, Arg (188) Gln, Lys (183) Glu. By the designation, for example, Ala (49) Gln it is intended that the Ala at position 49 in Figure 1 (SEQ ID NO .: 2) be replaced by Gln. The changes are preferably of a minor nature, such as conservative substitutions of amino acids that do not significantly affect the folding or activity of the protein. Examples of conservative amino acid substitutions known to those skilled in the art are described below: Aromatics: Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar Glutamine Asparagine Basics: Arginine Lysine Histidine acids Aspartic acid Glutamic acid Small Alanine Serine Threonine Methionine Glycine Of course, the number of amino acid substitutions that an expert can make depends on many factors, including those described above. Generally speaking, the number of substitutions for any given KGF-2 polypeptide will be no greater than 50, 40, 30, 20, 10, 5 or 3, depending on the objective. For example, a number of substitutions can be made at the C-terminus of KGF-2 to improve stability. The amino acids in KGF-2 that are essential for function can be identified by methods well known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Sci ence 244: 1081-1085). (1989) The last procedure introduces simple alanine mutations in each residue in the molecule.The resulting mutant molecules are then tested for biological activity such as binding to the receptor or proliferative activity in vi tro e in vi vo. for example, Example 1.) Sites that are critical for ligand-receptor binding can also be determined for structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (See, for example: Smith et al., J. Mol. Bi ol., 224: 899-904 (1992); and de Vos et al. Sci ence, 255: 306-312 (1992)). Other useful KGF polypeptides include polypeptides having serine substitutions for cysteine at amino acid positions 37 and 106 and 150. An unequal number of cysteines means that at least one cysteine residue is available for intermolecular crosslinks or the linkages that can cause the protein adopts an undesirable tertiary structure. The novel KGF-2 proteins having one or more cysteines replaced by serine or, for example, alanine, are generally purified at a higher yield of correctly folded, soluble protein. Although one does not wish to commit to any theory, it is believed that the cysteine residue at position 106 is important for function. This cysteine residue is highly conserved among all the other members of the FGF family.

Therapeutic Uses of the KGF-2 Polypeptide Compositions The polypeptides of the present invention can stimulate the development and proliferation of keratinocyte cells. Accordingly, the compositions of the present invention can be used to stimulate the proliferation of basal epithelial and keratinocyte cells for wound healing purposes, and stimulate the production of the hair follicle and the healing of dermal wounds. These wounds may be superficial in nature or may be deep and involve damage to the dermis and epidermis of the skin. KGF-2 is useful for the treatment of a number of diseases and conditions. For example, KGF-2 is active in vi tro and in vi ve in various models of wound healing. See, U.S. Patent Application No. 08 / 910,875, filed August 13, 1997 and 09 / 023,082 filed February 13, 1998. Individuals to whom KGF-2 is administered can heal their wounds. a normal speed or may have deteriorated healing. When administered to an individual who has no deteriorated healing, KGF-2 is administered to accelerate the normal healing process. When administered to an individual who has deteriorated healing, KGF-2 is administered to make the wound healed which otherwise could heal slowly or not at all. A number of conditions and conditions can result in deterioration of the healed. These conditions and conditions include diabetes (eg type II diabetes mellitus), treatment with steroidal and non-steroidal pharmacological agents, and ischemic blockage or damage. It has been shown that a number of growth factors promote the healing of wounds in individuals with impaired healing. These growth factors include the growth hormone release factor, the platelet derived growth factor, and basic fibroblast growth factors. Thus, the present invention also encompasses the administration of KGF-2 compositions in conjunction with one or more additional growth factors or another agent that promotes wound healing. The compositions of the present invention also promote the healing of anastomotic wounds and other wounds, caused by surgical procedures in individuals who heal their wounds at a normal speed and have deteriorated healing. The compositions of the present invention can also be employed to stimulate the differentiation of cells, for example muscle cells, cells constituting nervous tissue, prostate cells and lung cells. The compositions of the present invention are clinically useful in stimulating the healing of wounds, including surgical wounds, excised wounds, deep wounds involving damage to the dermis and epidermis, wounds to the ocular tissue, wounds to the dental tissue, wounds in the cavity oral, diabetic ulcers, dermal ulcers, ulcers of the ulna, arterial ulcers, venous stasis ulcers, and burns resulting from exposure to heat at extreme temperatures of heat or cold, or exposure to chemicals, in normal individuals and those subject to conditions that induce abnormal healing of wounds, such as uremia, poor nutrition, vitamin deficiencies, obesity, infection, immunosuppression and complications associated with systemic treatment with steroids, radiation therapy, and antineoplastic and antimetabolite drugs. The compositions are also useful for promoting wound healing, associated with ischemia and ischemic damage, for example, chronic venous leg ulcers caused by deterioration of the return and / or insufficiency of the venous circulatory system; to promote skin re-establishment subsequent to skin loss; the increase in epidermal tensile strength and epidermal thickness; and the increased adhesion of skin grafts to a wound bed and to stimulate the re-epithelialization of the wound bed. Other therapeutic uses for KGF-2 polypeptides include, but are not limited to, for example, stimulating the proliferation of epithelial cells and basal keratinocytes for the purpose of treating burns and skin effects such as psoriasis and epidermolysis bullosa. KGF-2 can be used to increase the adherence of skin grafts to a wound bed and stimulate re-epithelialization from the wound bed. KGF-2 can also be used to reduce the side effects of bowel toxicity resulting from treatments with radiation, chemotherapy or viral infections. KGF-2 can be used to treat diseases and conditions of the liver, lung, kidney, breast, pancreas, stomach, small intestine and large intestine. KGF-2 can be used to treat inflammatory bowel diseases, diabetes, thrombocytopenia, hypofibrinogenemia, hypoalbuminemia, hypoglobulinemia, hemorrhagic cystitis, xerostomia, serum keratoconjunctivitis. KGF-2 can be used to stimulate the epithelial cells of the salivary glands, lacrimal glands and the stimulation of the re-epithelialization of the nasal sinuses and the development of the nasal mucosa. A number of other indications that can be treated by the composition of the present invention are described in U.S. Patent Applications Nos. 08 / 910,875 and 09 / 023,082 and are incorporated by reference herein. The present invention is directed to the novel liquid and lyophilized formulations of KGF-2 and the deletion mutants thereof. This invention also relates to formulations of KGF-2 for therapeutic use. The formulations provide superior stability to the active KGF-2 polypeptides and in some cases, potentiate and dramatically increase the wound healing activity of the polypeptides. As used herein, "individual" means an animal, preferably a mammal (such as apes, cows, horses, pigs, boars, sheep, rodents, goats, dogs, cats, chickens, monkeys, rabbits, ferrets, whales and dolphins) and more preferably a human. The KGF-2-? 33 polypeptides used in the formulations of the present invention may or may not have the N-terminal methionine, preferably the polypeptide will lack the N-terminal methionine. The stability of the KGF-2 polypeptide formulations of the present invention is determined by proliferation assays, as described hereinafter.

Keratinocyte Proliferation Assays Dermal keratinocytes are cells in the epidermis of the skin. The development and diffusion of keratinocytes in the skin is an important process in the healing of wounds. A keratinocyte proliferation assay is therefore a valuable indicator of protein activities in stimulating the growth of keratinocytes and consequently, wound healing. Keratinocytes are, however, difficult to develop in vi tro. There are few keratinocyte cell lines. These cell lines have different cellular and genetic defects. In order to avoid complications of this assay due to cellular defects such as loss of key receptors of the growth factor or the dependence of growth factors key to development, the primary dermal keratinocytes are chosen for this test. These primary keratinocytes are obtained from Clonetics, Inc., (San Diego, CA). The bioactivity of the KGF-2 polypeptides can be determined by a cell proliferation assay using murine Baf3 2b cells that have been transfected with the 2iiib fibroblast growth factor receptor (FGFR2iiib). The proliferation of the cells is measured by the incorporation of [Methyl-3H] -thymidine after the cells have been exposed to the protein as described below. The assay is carried out in a 96-well tissue culture plate, with approximately 22,000 Baf3 2b cells in each well. The cells are exposed to different concentrations of a KGF-2 polypeptide in triplicate and "incubated at 37 ° C in a CO 2 incubator for approximately 48 hours." An approximate amount of labeled thymidine-containing cell medium is subsequently added into each well and the incubation is continued for another 5 hours.The cells are then harvested on a fiberglass filter mesh, in the 96-well format, using a cell harvester.The filter meshes are dried and the radioactivity incorporated within each The sample is counted using a flat bed liquid scintillation counter Under these test conditions, cells exposed to KGF-2 show an increased incorporation of radioactivity compared to control cells that have been treated with either an appropriate dilution of the buffer of placebo or simply phosphate-buffered saline, another useful assay for the proliferation of Keratinocytes is with Alamar Blue. Alamar Blue is a viable blue dye that is metabolized by mitochondria when added to culture media. The dye then becomes red in the tissue culture supernatants. The quantities of the red dye can be directly quantified by the difference in reading at the optical densities between 570 nanometers and 600 nm. This reading reflects cellular activities and cell numbers. Normal primary dermal keratinocytes (CC-0255, NHEK-Neo combined) are purchased from Clonetics, Inc. These cells are from passage 2. Keratinocytes are grown in complete keratinocyte growth medium (CC-3001, KGM; Clonetics, Inc.) until these they reach 80% confluence. The cells are trypsinized according to the manufacturer's specification. In summary, the cells are washed twice with Hank's balanced salt solution. 2 to 3 ml of trypsin are added to the cells for approximately 3 to 5 minutes at room temperature. The trypsin neutralization solution is added and the cells are collected. Cells are centrifuged at 600 x g for 5 minutes at room temperature and plated in new flasks at 3,000 cells per square centimeter using pre-heated media. For the proliferation assay, 1,000 to 2,000 keratinocytes are plated per well of the Corning flat bottom 96-well plates in complete medium except for the upper rows. The outer wells are filled with 200 μl of sterile water. This helps keep the temperature and humidity fluctuations of the wells to a minimum. The cells are grown overnight at 37 ° C with 5% C02. The cells are washed twice with basal keratinocyte medium (CC-3101, KBM, Clonetics, Inc.) and 100 μl of KBM is added into each well. They are incubated for 24 hours. The growth factors are diluted in KBM in serial dilution and 100 μl is added to each well. KGM is used as a positive control and KBM is used as a negative control. Six wells are used for each concentration point. It is incubated for two to three days. At the end of the incubation, the cells are washed once with KBM and 100 μl of KBM with 10% v / v of Alamar Blue pre-mixed in the medium is added. It is incubated for 6 to 16 hours until the color of the medium begins to turn red in the positive control of KGM. The D.O. 570 nm less D.O. 600 nm directly by placing the plates in the plate reader.

Construction of Suppression Mutants of KGF-2 Deletion mutants useful for use in the compositions of the present invention can be constructed by the following protocol.

Deletion mutants were constructed from the 5 'end and 3' end of KGF-2 using an optimized KGF-2 construct as a template. The deletions were selected based on the regions of the gene that can negatively affect expression in E. coli. For 5 'deletion the primers listed below were used as the 5' primer. These primers contain the indicated restriction site and an ATG to code for the initiating methionine. The HindIII 3 '208 amino acid primer of KGF-2 (FGF-12) was used for the 3' primer. PCR amplification for 25 rounds was reviewed using standard conditions. The products for the deletion mutant KGF-2 36aa / 208aa were restricted with BspHI for the 5 'site and with HindIII for the 3' site and cloned into pQE60 which has been digested with BspHI and HindIII. All other products were restricted with Ncol for the restriction enzyme 5 'and HindIII for the 3' site, and cloned into pQE60 which had been digested with Ncol and HindIII. For KGF-2 (FGF-12), 3 'HindIII from 36aa / 153aa and 128aa was used as the 3' primer with FGF-12 36aa / 208aa as the 5 'primer. For FGF-12 62aa / 153aa, 3 'HindIII from 128aa was used as the 3' primer with FGF-12 62aa / 208aa as the 5 'primer.

The nomenclature of the resulting clones indicates the first and last amino acid of the polypeptide resulting from the deletion. For example, KGF-2 36aa / 153aa indicates that the first amino acid of the deletion mutant is amino acid 36 and the last amino acid is amino acid 153 of KGF-2. The construction of these KGF-2 deletion mutants is also described in U.S. Patent Applications Nos. 08 / 910,875 and 09 / 023,082 and are incorporated by reference herein. In addition, as indicated below, each mutant has N-terminal Met added to them. However, the KGF-2 suppression polypeptides used in the formulations according to the present invention may or may not have the N-terminal methionine, preferably the polypeptide will be lacking the N-terminal methionine. + Sequences of Suppression Primers: FGF12 36aa / 208aa: 5 'Bsphl GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC (SEQ ID NO .: 3) FGF12 63aa / 208aa: 5 'Ncol GGACAGCCATGGCTGGTCGTCACGTTCG (SEQ ID NO .: 4) FGF12 77aa / 208aa: 5' Ncol GGACAGCCATGGTTCGTTGGCGTAAACTG (SEQ ID NO .: 5) FGF12 93aa / 208aa: 5 'Ncol GGACAGCCATGGAAAAAAACGGTAAAGTTTC (SEQ ID NO .: 6) FGF12 104aa / 208aa: 5"Ncol GGACCCCCATGGAGAACTGCCCGTAGAGC (SEQ ID NO .: 7) FGF12 123aa / 208aa: 5 'Ncol GGACCCCCATGGTCAAAGCCATTAACAGCAAC (SEQ ID NO: 8) FGF12 138aa / 208aa: 5 'Ncol GGACCCCCATGGGGAAACTCTATGGCTCAAAAG (SEQ ID NO .: 9) FGF12 3'HindIII: (Used for all previous deletion clones) CTGCCCAAGCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO .: 10) FGF12 36aa / 153aa: 5 'Bsphl (as described above) 3'HindIII CTGCCCAAGCTTATTACTTCAGCTTACAGTCATTGT (SEQ ID DO NOT. : 11) FGF 12 63aa / 153aa: 5'NcoI and 3'HindIII, as described above.

Construction of the N-terminal Suppression Mutant KGF-2? 33 Construction of KGF-2? 33 in pQE6 To allow for the amplification directed by the Polymerase Chain Reaction and the subcloning of KGF2? 33 into the expression vector of the E. coli protein, pQE6, two nucleotide primers (5952 and 19138) complementary to the desired region of KGF- 2, were synthesized with the following base sequences.

Primer 5952: 5 'GCGGCACATGTCTTACAACCACCTGCAGGGTG3' (SEQ ID NO .: 12) Primer 19138: 5'GGGCCCAAGCTTATGAGTGTACCACCAT3 '(SEQ ID NO: 13) In the case of the N-terminal primer (5952), an AflIII restriction site was incorporated, whereas in the case of the C-terminal primer (19138) a HindIII restriction site was incorporated. Primer 5952 also contains an adjacent ATG sequence and within the structure with the coding region of KGF-2 to allow translation of the fragment cloned in E. coli, whereas primer 19138 contains two stop codons (preferably used in E. coli) adjacent to and within the structure with the coding region of KGF-2 which ensures the correct termination of the translation in E.coli. The Polymerase Chain Reaction was performed using standard conditions well known to those skilled in the art, and the nucleotide sequence for mature KGF-2 (aa 36-208) as a template. The resulting amplicon was restriction digested with AflIII and HindIII and subcloned into the expression vector of the pCE6 protein digested with NcoI / HindIII.

Construction of KGF-2? 33 in pH To allow for the amplification directed by the Polymerase Chain Reaction and the subcloning of KGF2Δ 33 into the E. coli expression vector, pHEl, two oligonucleotide primers (6153 and 6150) corresponding to the desired region of KGF2 were synthesized, with the following sequence of bases.

Primer 6153: 5 'CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG3' (SEQ ID NO: 14) Primer 6150: 5 'CCGGCGGTACCTTATTATGAGTGTACCACCATTGG3' (SEQ ID NO: 15) In the case of the N-terminal primer (6153), an Ndel restriction site was incorporated, whereas in the case of the C-terminal primer (6150) an Asp718 restriction site was incorporated. Primer 6153 also contains an adjacent ATG sequence and within the structure with the coding region of KGF2 to allow translation of the cloned fragment in E. coli, whereas primer 6150 contains two arrest codons (potentially used in E. coli). ) adjacent to and within the structure with the coding region of KGF-2, which ensures the correct termination of the translation in E. coli. The Polymerase Chain Reaction was performed using standard conditions well known to those skilled in the art and the nucleotide sequence for mature KGF2 (aa 36-208) as a template. The resulting amplicon was digested by restriction with Ndel and Asp718 and subcloned into the expression vector of the pHEl protein digested with Ndel / Asp718.

Nucleotide sequence of KGF-? 33 ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTT C CTTTCACCAAA ACT CCTGAAAATCGAAAA AAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGC ATCCTGGAGATAACATCAGTAGAAATCGGAGTTG TTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAA GAAGGGGAAACTC A GGCTCAAAAGAAT AAC AATG CTGTAAGCTGAAGGAGAGGATAGAGG AAATGGATACAAT ACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGG AGAGGACAGAAAACACGAAGGAAAAACACC CTGC CAC C C CAATGGTGGTACACTCATAA (SEQ ID NO .: 16) Amino Acid Sequence of KGF-2? 33 MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSV AND IGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNT YASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMWHS (SEQ ID NO.: 17) B. Construction of a Polynusleotid Sequence of KGF-2? 33 Optimized In order to increase the expression levels of KGF-2? 33 in E. coli, the codons of the complete gene were optimized to fit those most highly used in E. coli. Since the template used for KGF2? 33 was the optimized codon within the N-terminal region, the C-terminal amino acids (84-208) required optimization. First, amino acids 172-208 were optimized at the codon to generate KGF2Δ 33 (sl72-208). This was achieved through an overlap PCR strategy. Oligonucleotides PM07 and PM08 (corresponding to amino acids 172-208) were combined and annealed together by heating them to 70 ° C and allowing them to cool to 37 ° C. The annealed oligonucleotides were then used as a template for a standard PCR reaction, which was directed by primers PM09 and PM10. In a separate PCR reaction following standard conditions well known to those skilled in the art and using KGF2Δ33 as a template, oligonucleotides PM05 (which overlaps with the PstI site within the coding region of KGF2) and PM11 were used to amplify the KGF2 region corresponding to amino acids 84-172. In a third PCR reaction, the product of the first PCR reaction (corresponding to amino acids 172-208 optimized by the codon) and the product of the second PCR reaction (corresponding to amino acids 84-172 not optimized by the codon) were combined and used as template for a standard PCR reaction directed by the oligonucleotides PM05 and PM10. The resulting amplicon was digested with Pstl / HindlII and sub-cloned into pQE6KGF2Δ33, digested with PstI / HindIII, effectively replacing the non-optimized region with the corresponding codon, and creating PQE6KGF2Δ33 (sl72-208). To complete the codon optimization of KGF2, a codon of the synthetic gene optimized for the KGF2 region corresponding to amino acids 84-172 was generated using overlapping oligonucleotides. Four oligonucleotides (PM31, PM32, PM33 and PM34) were combined and seven cycles of the following PCR were performed: 94 ° C, 30 seconds; 46.5 ° C, 30 seconds; and 72 ° C, 30 seconds.

A second PCR reaction directed by primers PM35 and PM36 was then performed following standard procedures. using 1 μl of the first PCR reaction as a template. The resulting gene fragment optimized by the codon was then digested with Pstl / Sall and sub-cloned into PQE6KGF2Δ33 (sl72-208) digested with Pstl / Sall to create a gene that codes for fully optimized KGF2, pQE6KGF2? 33 . To create an alternative expression vector of E. coli protein, KGF2? 33 was amplified by PCR using primers PM102 and PM130 on pQE6KGF2? 33s. The resulting amplicon was digested with Ndel and EcoRV and sub-cloned into the pHEl expression vector which had been digested with Ndel and Asp718 (blunt end) to create pHEl? 33s. Oligonucleotide sequences used in the construction of KGF-2? 33s used in the codon: PM05; CAACCACCTGCAGGGTGACG (SEQ ID NO; 18.) PM07: AACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTG CTCCACGTCGTGGTCAGAAAACCCGTCGTAAAAACACC (SEQ ID NO: 19.) PM08: GGGCCCAAGCTTAAGAGTGTACCACCATTGGCAGAAAGT GAGCAGAGGTGTTTTTACGACGGGTTTTCTGACCACG (SEQ ID NO .; 20) PM09: GCCACATACATTTGTCGACCGTT (SEQ ID NO .: 21) PM10: GGGCCCAAGCTTAAGAGTG (SEQ ID NO .: 22) PM11: GCCACATACATTTGTCGACCGTT (SEQ ID NO .: 23) PM31: CTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCCTTCACCA ATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACCAA A-G (SEQ ID NO. 24) PM32: AGCTTTAACAGCAACAACACCGATTTCAACGGAGGTGATTTC CAGGATGGAGTACGGGCAGTTTTCTTTCTTGGTACCAGAAACTTTAC C (SEQ ID NO. 25) PM33: GGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTGG CTATGAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTAACAA C ( SEQ ID NO. 26) PM3: GTCGACCGTTGTGCTGCCAGTTGAAGGAAGCGTAGGTGTTGT AACCGTTTTCTTCGATACGTTCTTTCAGTTTACAGTCGTTGTTAAATT CTTTGGAACC (SEQ ID NO .: 27) PM35: GCGGCGTCGACCGTTGTGCTGCCAG (SEQ ID NO .: 28) PM36: GCGGCCTGCAGGGTGACGTTCGTTGG (SEQ ID NO .: 29) PMl02: CCGGCGGATCCCATATGTCTTAC AACCACCTGCAGG (SEQ ID NO. : 30) PM130: CGCGCGATATCTTATTAAGAGTGTACCACCATTG (SEQ ID NO 31) Nucleotide sequence of KGF-2? 33 (sl72-208) ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTT CTCCTTCACCAAATACTTCCTGAAAATCGAAAA AAACGGTAAAGTTTCTGGTACCAAGAAAGAAAACTGCCCGTACTCC ATCCTGGAAATCACCTCCGTTGAAATCGGTGTTG TTGCTGTTAAAGCTATCAACTCCAACTACTACCTGGCTATGAACAAG AAAGGT AACTGT CGGT CCAAAGAATT AC AACGACTGTAAACTGAAAGAACGTATCGAAGAAAACGGTTACAACA CCTACGCTTCCTTCAACTGGCAGCACAACGGTCG ACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGT CAGAAAACCCGTCGTAAAAACACCTCTGCTC ACTTTCTGCCAATGGTGGTACA.CTCTTAA (SEQ ID NO .: 32) Amino Acid Sequence of KGF-2? 33 (sl72-208) MSYNHLQGDVRWRKLFS FTKYFLKIEKNGKVSGTKKENCPYS I LE I T SV AND IGWAVKAINSNYYLAMNKKGKLYGSKE FNNDCKLKERIEENGYNT YASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNT SAHFLPMWHS (SEQ ID NO: 33) EXAMPLES EXAMPLE 1 Liquid Formulation of KGF-2 The following ingredients were mixed to create a liquid formulation of KGF-2? 33 which is a liquid that is stored at ** 20 ° C. 2 mg / ml KGF-2? 33 polypeptide, 20 mM sodium acetate, 125 mM sodium chloride, 1 M EDTA, water, pH 6.2 This formulation retained its bioactivity in vi tro for up to 10 months at storage conditions of or below 2 to 8 ° C. The bioactivity at 10 months is shown in Figure 3. This formulation retained all its physico-chemical properties for up to 11 months at storage conditions of or below 2 to 8 ° C. The bioactivity was measured using a cell proliferation assay as follows. BaF3 cells were routinely developed and maintained in RPMI 1640 medium containing 10% NBCS, 10% conditioned medium for WEHI cells, 2 mM glutamine, 600 μg / ml GENETICINE, 1 μl β-mercaptoethanol / 500 ml medium growth, 50 units of penicillin and 50 μg / ml of streptomycin (Ornitz, D., M. et al. (1996) J. Bi ol. Chem. 271: 15292-15297). For cell proliferation assays, BaF3 cells were harvested by centrifugation and washed with Basal medium (this has the same composition as the growth medium, but does not contain WEHI conditioned medium and is supplemented with 1 μg / ml of heparin). After this operation, the cells were resuspended in basal medium and 22,000 cells / 180 μl were plaque / well separated in a 96-well cell culture plate. Appropriate dilutions (10X higher than the required final concentration) of KGF 2 in PBS were made in another 96 well plate, and were added to the cells to a final volume of 200 μl. The cell plates were incubated in an incubator with 5% C02 at 37 ° C for 36 to 40 hours and 0.5 μCi of methyl-3H-thymidine in 50 μl of basal medium was added to each well. The plates were incubated for another 5 hours in the incubator and the cells were harvested by filtration on a glass fiber filter using a Tomtec 96 harvester. The incorporated thymidine was counted on a Wallac β plate scintillation counter.

EXAMPLE 2 Lyophilized Formulation of KGF-2 The following ingredients were mixed to create a lyophilized formulation of KGF-2? 33. 10 mg / ml KGF-2? 33, 10 inM sodium citrate, 20 mM sodium chloride, 1 mM EDTA, 7% sucrose w / v, water (removed after lyophilization) pH 6.2 This formulation retained its bioactivity in vi tro for up to 9 months at storage conditions of or below 45 ° C. Bioactivity at 9 months is shown in Figure 4. Bioactivity was measured using the cell proliferation assay detailed in Example 1. Reverse phase HPLC showed that the formulation retained its physico-chemical properties for up to 8 months at temperatures of or below 45 ° C and 75% relative humidity.

EXAMPLE 3 KGF-2 in a Thick Formulation The following ingredients were mixed to create a thick formulation of KGF-2? 33. 2 mg / ml KGF-2? 33, 10 mM sodium citrate, 20 mM sodium chloride, 1 mM EDTA, 7% sucrose w / v, 1.25% carboxymethylcellulose, water pH 6.2.

This formulation is prepared by adding the KGF-2? 33 polypeptide to the carboxymethyl cellulose solution. The viscosity of the resulting formulation was about 250 cps as determined by the rotating needle viscometer. The KGF-2 polypeptide retained the bioactivity in the presence of carboxymethylcellulose. The bioactivity of the formulation was evaluated using the cell proliferation assay detailed in Example 1.

EXAMPLE 4 KGF-2 in a Gel Formulation The following ingredients were mixed to create a gel formulation of KGF-2? 33. 2 mg / ml KGF-2? 33, 10 mM sodium citrate, 20 mM sodium chloride, 1 mM EDTA, 7% sucrose w / v, 16% Pluronic F127, water pH 6.2.

KGF-2? 33 was added to a Pluronic solution at about 2 ° C to about 8 ° C. The viscosity of the resulting formulation was about 50 cps at 20 ° C and solid at about 37 ° C. KGF-2 retained the bioactivity in the presence of Pluronic F127 as measured by the cell proliferation assay detailed in Example 1, EXAMPLE 5 Activation of KGF-2 by Monothioglycerol KGF-2? 33 protein reserve formulations (0.1 to 2.0 mg / ml) were prepared with or without monothioglycerol (MTG). Protein formulations were diluted in 1 x phosphate-buffered saline (PBS) at pH 7.2 to achieve the concentrations required for use in cell proliferation assays.

Cell culture BaF32b cells were routinely developed and maintained in RPMI 1640 medium containing 10% NBCS, 10% WEHI cell conditioned medium, 2 mM glutamine, 600 μg / ml GENETICINE, 1 μl β-mercaptoethanol / 500 ml medium growth, 50 units of penicillin and 50 μg / ml of streptomycin (Ornitz, D., M. et al. (1996) J. Bi ol. Chem. 271: 15292-15297).

Cell Proliferation Assays For cell proliferation assays, BaF32b cells were harvested by centrifugation and washed with Basal medium (this has the same composition as the growth medium, but does not contain WEHI conditioned media and is supplemented with 1 μg / ml heparin). After this operation the cells were resuspended in basal medium and plated 22,000 cells / 180 μl per well in a 96-well cell culture plate. Appropriate dilutions (10 X greater than the required final concentration) of KGF-2 in PBS were made in another 96-well plate, and were added to the cells to a final volume of 200 μl. The cell plates were incubated in an incubator with 5% CO_ at 37 ° C for 36 to 40 hours, and 0.5 μCi of methyl- H-thymidine in 50 μl of basal medium was added to each well. The plates were incubated for another 5 hours in the incubator and the cells were harvested by filtration on a glass fiber filter using a Tomtec Harvester 96 harvester. Incorporated thymidine was counted in a Wallac β scintillation counter.

Results A. Effect of MTG concentration on the activity of KGF-2 The cell proliferation assay was carried out with KGF-2 exposed to different concentrations of monothioglycerol (MTG). The control samples did not contain excipient. With MTG, the stimulation of KGF-2 activity was observed with various concentrations of MTG as shown in Figure 5. The increase in activity was between 10-150% of the control, depending on the concentration of MTG used. This improvement in cell proliferation activity was not observed with other members of this growth factor family. From these observations, it was concluded that the stimulation of the activity of KGF-2 by MTG was very specific.

Conclusions Monothioglycerol appears to specifically stimulate cell proliferation activity in vitro of KGF-2.

EXAMPLE 6 Gel Formulation of KGF-2 with Citrate The following ingredients were mixed to create a formulation of KGF-2 as a liquid at room temperature, and which subsequently gels after application to the skin. mM sodium citrate, 125 mM sodium chloride, 1 mM disodium EDTA, 17% Pluronic 127, pH 6.0, water.

EXAMPLE 7 Gel Formulation of KGF-2 with Acetate The following ingredients were mixed to create a formulation of KGF-2 that can gel after application to the skin. mM sodium acetate, 125 mM sodium chloride, 1 mM disodium EDTA, 17% Pluronic 127, pH 6.0, water.

EXAMPLE 8 Liquid Formulations of KGF-2 The suitability of sodium citrate as a buffer in which to maintain KGF-2? 33 was evaluated in four separate formulations, and at three separate pHs: pH 5.0, pH 5.5 and pH 6.0.

Formulations A. KGF-2? 33 1 or 2 mg / ml sodium citrate 20 ntM, 10. 125 mM sodium chloride, 1 mM disodium EDTA, Water.

B. As in "A" above, also including 1% glycerol. C. As in "A" above, also including 0.05% methionine. D. As in "A" above, also including 1% monothioglycerol.

The concentration of KGF-2? 33 was 1 and 2 mg / ml in all the above formulations.

Formulation of Lyophilization KGF-2? 33 was lyophilized in the presence of one of three volume agents: mannitol, sucrose and trehalose.

Formulations: A. 10 mM sodium citrate, 20 mM sodium chloride, 1 mM disodium EDTA and 4% mannitol, pH 6.0.

B. 10 mM sodium citrate, 20 mM sodium chloride, 1 mM disodium EDTA, and 7% sucrose, pH 6.0. C. 10 mM sodium citrate, 20 mM sodium chloride, 1 mM disodium EDTA, and 8% trehalose, pH 6.0.

The concentration of the KGF-2 polypeptide was 3 mg / ml and 8 mg / ml. The evaluation parameters were RP-HPLC, SDS-PAGE, appearance, before and after reconstitution with water. 3. Lyophilization of 10 mg / ml of KGF-2 It was evaluated whether or not the formulation will allow lyophilization of the protein at 10 mg / ml, as well as the subsequent stability of the protein after reconstitution.

Formulations: mM sodium citrate, 20 mM sodium chloride, 1 mM disodium EDTA, and 4% mannitol, pH 6.0. The lyophilized products were reconstituted with water or water containing 15 monothioglycerol.

- ' It will be clear that the invention can be practiced differently than as described in particular in the description and in the previous examples. Numerous modifications and variations of the present invention are possible in the light of the foregoing teachings and, therefore, are within the scope of the appended claims. The full description of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are incorporated therein.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (11)

REININDICATIONS Having described the invention as above, the content of the following claims is claimed as property:

1. A pharmaceutical composition, characterized in that it comprises: a) a KGF-2 polypeptide in a concentration range of about 0.02 to about 40 mg / ml (w / v); b) a buffer having a buffer capacity of between about pH 5.0 and pH 8.0 at a concentration range of about 5 mM to about 50 mM; and c) a pharmaceutically acceptable diluent for bringing the composition to a designated volume; or a reaction product thereof.

2. The pharmaceutical composition according to claim 1, further characterized in that it comprises: d) a chelating agent at a concentration range of about 0.1 mM to about 10 mM; and e) sodium chloride at a concentration range of about 0.01 mM to about 150 mM.

3. The pharmaceutical composition according to claim 1, characterized in that it further comprises one of: f) about 0.5% up to about 2% w / v glycerol, g) about 0.1% up to about 1% w / v methionine, oh) about 0.1% up to about 2% w / v of monothioglycerol.

4. The pharmaceutical composition according to claim 1, characterized in that the KGF-2 polypeptide is present in a concentration range of about 0.05 to about 30 mg / ml (w / v).

5. The pharmaceutical composition according to claim 4, characterized in that the KGF-2 polypeptide is present in a concentration range of about 0.1 to about 20 mg / ml (w / v).

6. The pharmaceutical composition according to the _. claim 5, characterized in that the KGF-2 polypeptide is present in a concentration range of about 0.2 to 4 mg / ml.

7. The pharmaceutical composition according to claim 1, characterized in that the KGF-2 polypeptide is KGF-2-? 33.

8. The pharmaceutical composition according to claim 1, characterized in that the diluent is water.

9. The pharmaceutical composition according to claim 2, characterized in that the chelating agent is EDTA at a concentration of approximately 1 mM, and the NaCl is present at a concentration of approximately 125 mM.

10. The pharmaceutical composition according to claim 1, characterized in that the pH is from about pH 5.5 to about pH 6.5. 11. The pharmaceutical composition according to claim 10, characterized in that the pH is about pH 6.2. 12. The pharmaceutical composition according to claim 1, characterized in that the buffer is selected from the group consisting of phosphoric, acetic, aconitic, citric, glutaric, malic, succinic, carbonic acid and an alkaline or alkaline earth metal salt thereof. 13. The pharmaceutical composition according to claim 12, characterized in that the buffer is a phosphate, acetate or citrate salt. 14. The pharmaceutical composition according to claim 13, characterized in that the buffer is a citrate salt. 15. The pharmaceutical composition according to claim 1, characterized in that the buffer is present in a concentration range of about 5 mM to about 30 mM. 16. The pharmaceutical composition according to claim 15, characterized in that the buffer is a citrate salt present in a concentration of about 10 mM to about 20 mM. 17. The pharmaceutical composition according to claim 1, characterized in that it further comprises a stabilizing amount of one or more of (a) an antioxidant or (b) a thiol compound. 18. The pharmaceutical composition according to claim 1, characterized in that the composition is maintained at a temperature of or below -20 ° C. 19. The pharmaceutical composition according to claim 1, characterized in that it comprises: a) 2 mg / ml of KGF-2? 33 (w / v) polypeptide; b) 20 mM sodium acetate; c) 125 mM NaCl; d) 1 mM EDTA; and e) water as a diluent, or a reaction product thereof. 20. The pharmaceutical composition according to claim 19, characterized in that the KGF-2? 33 polypeptide is selected from the group consisting of the KGF-? 33 polypeptide having an N-terminal methionine, the KGF-2? 33 polypeptide lacking a N-terminal methionine, and a mixture thereof. 21. A pharmaceutical composition, characterized in that it comprises: a) a KGF-2 polypeptide in a concentration range of about 0.02 to about 40 mg / ml (w / v); b) a buffer having a buffer capacity of about pH 5.0 and about pH 8.0, at a concentration range of about 5 mM to about 50 mM; c) a volume agent; and d) a pharmaceutically acceptable diluent for bringing the composition to a designated volume; or a reaction product thereof. 22. The pharmaceutical composition according to claim 21, characterized in that the volume agent is selected from the group consisting of sucrose, glycine, mannitol, trehalose, and mixtures thereof. 23. The pharmaceutical composition according to claim 21, characterized in that it comprises: e) a chelating agent at a concentration range of about 0.1 mM to about 10 mM; and f) NaCl at a concentration range of about 0.01 mM to about 125 mM. 24. The pharmaceutical composition according to claim 22, characterized in that the volume agent is sucrose or a mixture of sucrose and glycine. 25. The pharmaceutical composition according to claim 22, characterized in that the volume agent is present in a concentration of about 2% to about 10% w / v. 26. The pharmaceutical composition according to claim 22, characterized in that the volume agent is 5% mannitol, 7% sucrose, 8% trehalose, or 2% glycine + 0.5% sucrose. 27. The pharmaceutical composition according to claim 21, characterized in that the pH is about pH 6.2. 28. The pharmaceutical composition according to claim 21, characterized in that the diluent is water. 29. The pharmaceutical composition according to claim 21, characterized in that the buffer is selected from the group consisting of phosphoric, acetic, aconitic, citric, glutaric, malic, succinic, carbonic acid and an alkaline or alkaline earth metal salt thereof. 30. The pharmaceutical composition according to claim 29, characterized in that the buffer is a phosphate or citrate salt. 31. The pharmaceutical composition according to claim 30, characterized in that the buffer is a citrate salt. 32. The pharmaceutical composition according to claim 28, characterized in that more than 90% of the water is removed by lyophilization. 33. The pharmaceutical composition according to claim 32, characterized in that it is reconstituted with an amount of sterile water, effective to maintain the isotonic conditions of about 290 Osm. 34. The pharmaceutical composition according to claim 21, characterized in that the KGF polypeptide is KGF-2? 33. 35. The pharmaceutical composition according to claim 34, characterized in that the KGF-2? 33 polypeptide is selected from the group consisting of the KGF-2? 33 polypeptide having an N-terminal methionine, the KGF-2? 33 polypeptide lacks a N-terminal methionine, and a mixture thereof. 36. The pharmaceutical composition according to claim 21, characterized in that the buffer is added in a concentration of about 5 mM to about 50 mM. 37. The pharmaceutical composition according to claim 36, characterized in that the buffer is citrate at a concentration of approximately 10 mM. 38. The pharmaceutical composition according to claim 21, characterized in that it further includes a stabilizing amount of one or more of (g) an antioxidant, or (h) a thiol compound. 39. The pharmaceutical composition according to claim 32, characterized in that the composition is reconstituted in sterile water containing a stabilizing amount of an antioxidant comprising: a) about 0.01% up to about 2% w / v monothioglycerol, b) about 0.01% up about 2% ascorbic acid, c) about 0.01% to about 2% w / v of methionine or d) combinations thereof. 40. A pharmaceutical composition, characterized in that it comprises: a} a KGF-2 polypeptide in a concentration range of about 0.02 to about 40 mg / ml (w / v); b) citric acid or a pharmaceutically acceptable salt thereof, at a concentration range of about 5 mM to about 20 mM; c) NaCl at a concentration range of about 0.01 mM to about 125 mM, d) EDTA as a concentration range of about 0.1 mM to about 10 mM and e) one or more of sucrose, mannitol, glycine or trehalose at a range of concentration of about 2% w / v to about 15% w / v; and f) water. 41. The pharmaceutical composition according to claim 40, characterized in that the KGF-2 polypeptide is present at a concentration of about 2 mg / ml, about 4 mg / ml, or about 10 mg / ml. 42. The pharmaceutical composition according to claim 40, characterized in that more than 90% of the water is removed by lyophilization. 43. The pharmaceutical composition according to claim 1, characterized in that it further comprises a thickening agent in an amount effective to bring the viscosity to about 50 to about 10,000 cps. 44. The pharmaceutical composition according to claim 43, characterized in that the thickening agent is present in an amount effective to raise the viscosity to about 50 to about 1,000 cps. 45. The pharmaceutical composition according to claim 44, characterized in that the thickening agent is in an amount effective to raise the viscosity to about 200 to about 300 cps. 46. The pharmaceutical composition according to claim 21, characterized in that it further comprises a thickening agent in an amount effective to raise the viscosity to about 50 to about 10,000 cps. 47. The pharmaceutical composition according to claim 43, characterized in that the thickening agent is present in a concentration of 0 to 5% (w / v). 48. The pharmaceutical composition according to claim 43, characterized in that the thickening agent is an etherified water soluble cellulose or a high molecular weight polymer of acrylic acid crosslinked with allylucrose or an allyl ether of pentaerythritol. 49. The pharmaceutical composition according to claim 48, characterized in that the etherified cellulose is an alkylcellulose, hydroxyalkylcellulose, carboxyalkylcellulose or alkylhydroxyalkylcellulose. 50. The pharmaceutical composition according to claim 43, characterized in that the etherified cellulose is methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose. 51. The pharmaceutical composition according to claim 49, characterized in that the etherified cellulose derivative has a molecular weight of about 50,000 to about 700,000 and is present in a concentration of about 0 to about 20% by weight. 52. The pharmaceutical composition according to claim 51, characterized in that the etherified cellulose derivative has a molecular weight of about 80,000 to about 240,000 and is present in a concentration of about 2% to about 8% by weight. 53. The pharmaceutical composition according to claim 46, characterized 11! The buffer is citrate at a concentration of about 10 mM to about 50 mM. 54. The pharmaceutical composition according to claim 53, characterized in that the buffer is citrate in a concentration of about 10 mM to about 20 mM. 55. The pharmaceutical composition according to claim 53, characterized in that the volume agent is sucrose in a concentration from about 0.01% to about 5% sucrose. 56. The pharmaceutical composition according to claim 55, characterized in that the thickening agent is added directly to a liquid formulation and afterwards it is lyophilized. 57. The pharmaceutical composition according to claim 55, characterized in that the thickening agent is added to a lyophilized formulation by reconstituting said formulation by the addition of a suitable diluent having a thickening agent dissolved therein. 58. A thick solution composition of the KGF-2 polypeptide, characterized in that it is formed by mixing: a) a topically effective amount of a KGF-2 polypeptide; b) about 10 mM to about 500 mM sodium citrate buffer; c) about 0.01 to about 150 mM NaCl; d) 1 mM EDTA; e) about 0.1 to about 7% sucrose; f) about 0.75 to about 1.5% (w / w) of carboxymethylcellulose, or about 0.5 to about 1.5% of hydroxypropylmethylcellulose or about 0.25 to about 0.75% of hydroxyethylcellulose or about 0 to 1% of carbomer or any combination thereof. 59. The composition according to claim 1, further characterized in that it comprises a gelling agent in an amount effective to raise the viscosity to about 0.1 to about 10,000 cps at room temperature. 60. The composition according to claim 21, characterized in that it further comprises a gelling agent in an amount effective to raise the viscosity to about 0.1 to about 10,000 cps at room temperature. 61. The composition according to claim 59, characterized in that the gel-forming agent is a water-soluble polymer capable of forming a viscous aqueous solution, or a non-water-soluble, water-swellable polymer capable of forming a viscous solution. 62. The composition according to claim 61, characterized in that the gel-forming agent is a high molecular weight polymer selected from the group consisting of vinyl polymer, polyoxyethylene-polyoxypropylene copolymer, polysaccharide, protein, poly (ethylene oxide), acrylamide polymer or a salt thereof. 63. The composition according to claim 62, characterized in that the gel-forming agent is (1) a vinyl polymer selected from the group consisting of polyacrylic acid, polymethacrylic acid, polyvinylpyrrolidone, polyvinyl alcohol, and salts and esters thereof; (2) a polysaccharide selected from the group consisting of a cellulose derivative, a glycosaminoglycan, agar, pectin, alginic acid, dextran, α-amylose, amylopectin, chitosan, and salt esters thereof. 64. The composition according to claim 62, characterized in that the gel-forming agent is a glycosaminoglycan selected from the group consisting of hyaluronic acid, chondroitin, chondroitin-4-sulfate, heparan sulfate, heparin and salts and esters thereof. 65. The composition according to claim 64, characterized in that the glycosaminoglycan is present in combination with collagen, gelatin or fibronectin. 66. The composition according to claim 62, characterized in that the gel-forming agent is an acrylamide polymer selected from the group consisting of a polyacrylamide or a polymethacrylamide. 67. the composition according to claim 62, characterized in that the gel-forming agent is a polyoxyethylene-polyoxypropylene block copolymer. 68. The composition according to claim 67, characterized in that it comprises about 10 to about 60% by weight of a polyoxyethylene-polyoxypropylene block copolymer having an average molecular weight of about 500 to 50,000. 69. The composition according to claim 68, characterized in that it comprises about 14 to about 18% by weight of a polyoxyethylene-polyoxypropylene block copolymer having a molecular weight in the range of 1,000 to 15,000. 70. The composition according to claim 69, characterized in that the gel-forming agent is a polyoxyethylene-polyoxypropylene block copolymer and is Pluronic F108, Pluronic F127, or Poloxamer 407. 71. The composition according to claim 1, characterized in that the KGF-2 polypeptide is present in a concentration of about 0.01 mg / ml to about 10 mg / ml. 72. The composition according to claim 59, characterized in that the composition is formed by mixing: a) a KGF-2 polypeptide, at a calculated final concentration of 0.01 mg / ml to about 10 mg / ml; b) an effective amount of a buffering agent; c) about 10% to about 60% by weight of a polyoxyethylene-polyoxypropylene block copolymer having an average molecular weight of about 500 to 50,000; and d) a pharmaceutically acceptable diluent, preferably water. 73. The composition according to claim 72, characterized in that the polyoxyethylene-polyoxypropylene block copolymer is present at a concentration of about 14% up to about 18%. 74. A gel formulation of KGF-2, characterized in that it comprises: a) a pharmaceutically acceptable amount of KGF-2 polypeptide; b) about 10 mM to about 500 mM sodium citrate; c) about 0.01 mM to about 150 mM NaCl; d) approximately 1 mM EDTA; e) about 0.1% to about 7% sucrose; f) about 14% to about 18% Pluronic F127; and g) about pH 6.2 75. A gel formulation of KGF-2, characterized in that it comprises: a) a KGF-2 polypeptide at a concentration range from about 0.01 mg / ml to about 10 mg / ml (w / v), b) sodium citrate at a concentration range of about 5 mM to about 20 mM; c) about 10% to about 25% (w / v), of Pluronic 127 or Poloxamer 407; and d) water until the capacity. 76. The gel formulation according to claim 75, characterized in that it further comprises: a) EDTA at a concentration range of about 0.1 mM to about 10 mM; b) NaCl at a concentration range of about 0.01 mM to about 125 mM. 77. The pharmaceutical composition according to claim 1, characterized in that the KGF-2 polypeptide is an N-terminal deletion selected from the group consisting of Ala (63) -Ser (208) (KGF-2? 28) and Ser (69 ) -Ser (208) (KGF-2? 33). 78. The pharmaceutical composition according to claim 77, characterized in that the KGF-2 polypeptide has an N-ter-methionine, lacks an N-terminal methionine, or is a mixture thereof. 79. The pharmaceutical composition according to claim 1, characterized in that the KGF-2 polypeptide is an i or N-terminal or C-terminal deletion mutant selected from the group consisting of Ala (39) -Ser (208); Pro (47) -Se (208); Val (77) - Ser (208); Glu (93) - Ser (208); Glu (104) ~ Ser (208); Val (123) -Ser (208); Gly (138) -Ser (208); Met (l), Thr (36); and Cys (37) - Lys (153). 15 80. The pharmaceutical composition of COHJ-OGÍ-I UQÜ characterized in that the KGF-2 polypeptide has an N-terminal methionine, lacks an N-terminal methionine, or is an 20 mixture thereof.

11. The pharmaceutical composition of -.a r? Iv? N.uicaciop 7, characterized in that the KGF-2? 33 polypeptide is selected from the group Which consists of the KGF-2? 33 polypeptide having an N-terminal methionine, KGF-2? 33 polypeptide lacking an N-terminal methionine, and a mixture thereof.