MXPA01012387A - 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 formulations of KGF-2 for therapeutic use, for example, to promote or accelerate wound healing.

Description

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

Previous Technique The fibroblast growth factor family has emerged as a large family of growth factors involved in the growth and regeneration of soft tissue. 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 broad, similar mitogenic spectrum, for example, they promote REF 134558 ..., -y * ~ á. * ~ *? h 'i ii? ^ f-tiiiiiTtT'f- ^ lrf ^ - "** - ** -« "- - > Ai ^ proliferation of a variety of cells of mesodermal and neuroectodermal origin and / or promote angiogenesis. KGF was originally identified as a member of the FGF family by sequence homology or factor purification and cloning. Keratinocyte growth factor (KGF) was isolated as a mitogen from a cultured murine keratinocyte line (Rubin, J.S. et al., Proc. Nati, Acad. Sci. USA, 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 adult kidney, colon and ileum, but not in brain or lung (Finch, P., et al., Science 245: 752-755 (1989)). KGF shows conserved regions within the FGF family of proteins. KGF binds to the FGF-2 receptor with high affinity. The deteriorated healing of wounds is a significant source of morbidity and can result in complications such as dehiscence, anastomotic rupture and unhealed wounds. In normal individuals, wound healing is achieved without complications. In contrast, wound healing is associated with various conditions such as diabetes, infection, immunosuppression, obesity and malnutrition (Cruse, PJ and Foord, R., Arch. Surg. 107: 206 (1973); Schrock, TR et al. ., Ann. Surg., 177: 513 (1973), Poole, GU, Jr., Surgery 91: 631 (1985), Irvin, GL et al., Am. Surg. 51: 418 (1985)). The repair of wounds is the result of complex interactions and complex biological processes. Three phases have been described in the normal healing of wounds: the acute inflammatory phase, the synthesis of the extracellular matrix and the collagen, and the remodeling (Peacock, E.E., Jr., Wound Repair, 2a. 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 the growth and regeneration of soft tissue in pharmaceutical compositions that (1) are stable over prolonged periods of storage, (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 substitution mutants thereof (referred to herein as KGF-2 polypeptides). This invention further relates to the use of such formulations of the KGF-2 polypeptides to promote or accelerate the growth or regeneration of soft tissue, for example in the healing of wounds, or in the treatment of mucocitis or inflammatory bowel disease. The - preferred formulations of the present invention employ novel mutant forms of KGF-2, and in one embodiment employ a deletion mutant referred to herein as KGF2-? 33. The co-ingredients employed in the formulations (1) provide storage stability to the KGF-2 polypeptide, (2) further increase the activity of the soft tissue healing of the therapeutic composition, and / or (3) provide the KGF-2 polypeptide in a 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 approximately pH 8.0. Useful buffers include phosphate, acetate, aconite, succinate, malate, carbonate and citrate, with 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, citrate being preferred. A third aspect of the invention relates to a formulation comprising a KGF-2 polypeptide and a thiol-containing compound, preferably monothioglycerol, 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. sodium and preservatives such as m-cresol, phenol, chlorobutane, chlorobutanol, benzyl alcohol, methylparabens and propylparabens. Antimicrobial preservatives can decrease the stability of KGF-2 formulations. Surprisingly, a combination of methylparaben and propylparaben was found to be suitable for use in the KGF-2 formulations. The formulations of the present invention may also have a top layer of nitrogen atmosphere over the space top 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. The sequencing was performed using a 373 Automatic DNA sequencer (Applied Biosystems, Inc.). The accuracy of the sequencing is predicted as 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 epidermal, primary, normal 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 bioactivity for KGF-2? 33 in liquid formulation, stability at 10 months. Figure 4 shows the results of the bioactivity for the lyophilized preparation of KGF-2? 33, stability at 9 months. Figure 5 shows the effect of monothiolglycerol 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 activity of KGF-2, in the keratinocyte proliferation assay described below and binds to the isoforms of the FGF 1 receptor. -iiib and 2-iiib. The present invention is directed to the pharmaceutical and veterinary formulations of KGF-2 polypeptides. The 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 includes fragments, derivatives and analogs of the polypeptide of Figure 1 (SEQ ID NO: 2) or that encoded by the deposited cDNA, which retain 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 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 physico-chemical properties and substantial biological activity of the KGF-2 polypeptides in a given period of time. The formulations according to the present invention include the gel forms, thick solution, solution and lyophilized. 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 phosphoric, acetic, aconitic, citric, glutaric, malic, succinic and carbon acids. An alkaline or alkaline earth metal salt of one of the aforementioned acids is typically employed. 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-2β3 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 from about 10 mM to about 20 mM. The formulation may also include sodium chloride, glycine, sucrose or mannitol, or combinations thereof, as a tonifier at a concentration of about 0 mM to about 150 mM, preferably 10 to about 150 mM, most preferably at about 125 mM. , and a metal chelating agent, such as EDTA, at a concentration of about 0 mM to about 10 mM, most preferably at about 1 mM. In addition, a liquid formulation of the present invention can 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-containing 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, the storage conditions are at 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 polypeptide formulation. 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 4) optionally one or more of the following: a) sodium chloride, glycine, sucrose or mannitol or combinations thereof, as a toning agent, b) a chelating agent, c) a stabilizing amount of an antioxidant, and d) a stabilizer amount of a protein stabilizer. The KGF-2 polypeptide is preferably kept 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 sodium bisulfate, cysteine, sodium sulfite, 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 may be employed include ethylenediaminetetraacetic acid (EDTA), or diethylenetriaminpentaacetic 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. Additionally, the formulations of the present invention may include one or more preservatives, such as benzyl alcohol, preferably at a concentration of about 0.5% to about 1.5%, most preferably at a concentration of about 0.9%; r z > > t chlorobutanol, preferably at a concentration of about 0.01% to about 1%, most preferably about 0.5%; methylparaben, preferably at a concentration of from about 0.1% to about 0.2%, more preferably at about 0.18%; propylparaben, preferably at a concentration of from about 0.01% to about 0.05%, more preferably about 0.02%; m-cresol, preferably at a concentration of from about 0.1% to about 1%, more preferably about 0.3%; and / or phenol, preferably at a concentration of from about 0.1% to about 1%, more preferably at about 0.5%. Particularly preferred is methylparaben and propylparaben used together; with methylparaben at a concentration of about 0.1% to about 0.2% and propylparaben at a concentration of about 0.10% to about 0.05%. More preferred is a combination of methylparaben and propylparaben, with methylparaben at a concentration of 0.18% and propylparaben at a concentration of 0.02%. 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 (w / v), 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, phosphate and carbonic acids. An alkaline or alkaline earth metal salt of one of the aforementioned acids is typically employed. 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 solution formulation are from about pH 5.0 to about pH 8.0, preferably pH 5.5 to pH 6.5, and most preferably about pH 6.2 Sodium acetate or sodium citrate are the preferred buffering agents, with Sodium citrate which is the most preferred. To the above solution are also preferably added: 4) a chelating agent, such as EDTA at a concentration range of about 0.1 mM to about 10 mM, more preferably to about 1 mM; 5) a tonifier, such as sodium chloride, glycine, sucrose or mannitol, or combinations thereof at a concentration of about 0.01 mM to about 150 mM and more preferably to about 125 mM. Optionally, a liquid formulation can also include a protein stabilizing amount of a compound selected from the group consisting of: a) about 0.5% up to about 2% w / v glycerol, b) about 0.1% up to about 1% w / v of methionine, or c) approximately 0.1% hast aax 2% w / v 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 about 0.02 to about 40 mg / ml (w / v), more preferably about 0.1 to about 20 mg / ml, and most preferably about 0.2 to 4 mg / ml. 2) 10 mM sodium citrate or 20 mM sodium acetate; 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) 125 mM sodium chloride; 4) 1 mM EDTA; and 5) water as a diluent, wherein the solution is at about pH 6.2 and is stored at about -20 ° C. More preferably, the solution formulation comprises a composition formed by mixing: t? ylk? aáJuL * ». 1) about 1.0 mg / ml of a KGF-2 polypeptide; 2) 20 mM citrate, pH 5-5.5; and 3) 0.01% polysorbate 80. The solution also preferably includes either about 7% sucrose or a combination of 2% glycine and 0.5% sucrose. Alternatively, the solution formulation comprises a composition formed by mixing: 1) about 1.0 mg / ml of a polypeptide KGF-2; 2) 20 mM citrate, pH 5-5.5; 3) 1 mM EDTA; and 4) 0.01% polysorbate 80. The solution also preferably includes either about 7% sucrose or a combination of 2% glycine and 0.5% sucrose. The present inventors have discovered that KGF-2 polypeptides are easily oxidized, aggregated and precipitated from the solution. Although the oxidation of KGF-2 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 biological activity. Additionally, according to the pH of the AÜLJÍ solution approaches the pH for KGF-2, the protein will precipitate from 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 the 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 approximately pH 6.0-6.2 provides a liquid formulation that reduces aggregation of the KGF-2 polypeptide and increases stability, the liquid formulation of the 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 the polypeptides KGF-2 comprising: a KGF-2 polypeptide and a buffer having a buffer capacity of between about pH 5.0 and about pH 8.0, more Ú.A * .iybrl¿t? and r - mj k preferably pH 5.5 to pH 6.5, most preferably pH 6.2. Useful buffers include buffers derived from phosphoric, aconitic, citric, glutaric, malic, succinic and carbon acids. An alkaline or alkaline earth metal salt of one of the aforementioned acids is typically employed. More preferably, the buffer will be phosphate 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-2β3 in water. The preferable concentration is from about 5 mM to about 50 mM and more preferably about 10 mM. Most preferably, the citrate buffer will be added at a concentration of approximately 10 mM. Also preferably included in the formulation is sodium chloride as a tonifier at a concentration of about 0 mM to about 150 mM, most preferably about 20 mM, and a metal chelating agent, such as EDTA, at a concentration of about 0 mM to about 10 mM, most preferably to about 1 mM. In addition, volume / cryoprotective agents such as sucrose, glycine, mannitol, trehalose or other bulking agents i.y ~ ia.imJ ** - 1. ** & ~ ^ & .- < Pharmaceutically acceptable IS & amp; amp; amp; 3 are included in the formulation.

The amount of volume agent used will be such that the solution is isotonic and is in a 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 the 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 i? hiiílii ll tk.y?,?? and, B? á? * i £ i.í * JL kyi- - • - "- - - i? tjam sodium citrate, at a concentration range of approximately 5 mM to about 20 mM, 2) sodium chloride at a concentration range of about 0 mM to about 125 mM, 3) EDTA at a concentration range of about 0 mM to about 10 mM, 4) one or more of sucrose, mannitol , glycine or trehalose or mixtures thereof at a concentration range of about 2% w / v to about 15% w / v; and 5) 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.The lyophilized formulations of the KGF-2 polypeptide are reconstituted in sterile water to maintain the conditions Isotonic islets of approximately 290 mOsm. The KGF-2 polypeptides can be reconstituted in sterile water, optionally containing a stabilizing amount of antioxidants comprising: a) about 0.01% to about 2% w / v monothioglycerol, b) about 0.01% up to about 2% w / o v of ascorbic acid, c) about 0.01% to about 2% w / v of methionine or d) combinations thereof. The present invention includes lyophilization cycles that produce a stable formulation of the KGF-2 polypeptide. The lyophilization cycle is designed to keep the KGF-2 polypeptide product below its collapse temperature during the primary drying phase. In addition, the moisture content is directed 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, of KGF-2, according to the present invention was determined as follows: Another exemplary lyophilization cycle for the lyophilization formulation of KGF-2 according to the 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 physiochemical properties for up to 8 months at temperatures of or below 45 ° C andkí L < t * - »-» * -S .. * A »g | m" »JJ jgtiU i ^ j- ^ aiíi 75% relative humidity. The 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 having an increased viscosity may be beneficial for topical applications where controlled release, adherence to the shape of a wound or avoidance of bleeding may be important. Such thick 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 a pharmaceutical composition of KGF-2. The thickening agent could raise the viscosity of up to about 50 to about 10,000 A ^ -J -. «. Jttfaufc * - »» »»? Rf »Aa» centipoises (cps), more preferably about 50 to about 1,000 cps and most preferably about 200 to about 300 cps. The 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 water soluble carbomers and carbomer (high molecular weight polymers of crosslinked acrylic acid with either allylucrose or allylic ethers of pentaerythritol). Examples of etherified cellulose are well known in the art (listed in the United States Pharmacopeia) 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 from about 0 to about 20% by weight of a cellulose derivative having a molecular weight of from about 50,000 to about 700,000. In a preferred embodiment, the cellulose derivative is present at from about 2% to about 8% by weight, and has a molecular weight in the range of about 80,000. up to approximately 240,000. Preferred cellulose derivatives are hydroxypropylmethylcellulose, methylcellulose, carboxymethylcellulose, and hydroxyethylcellulose. When the thickening agents are added to the injectable formulations, detailed above, the salts and buffering agents can 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 M citrate, more preferably about 10 mM up to - about 50 mM citrate and most preferably about 10 mM to about 20 mM citrate.

Additionally, 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, most preferably water, having a thickening agent dissolved therein. Such thick formulations could be administered by spray.

An example of a solution of the preferred, thick KGF-2 polypeptide according to the present invention comprises a product formed by mixing: 1) a topically effective amount of a KGF polypeptide, preferably KGF-2Δ 33; 2) about 10 mM to about 500 mM of the 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 or a combination of about 2.0% glycine and about 0.5% 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 1% of carbomer or any combination thereof. The pH of such a formulation is most preferably pH 6.2. 2) Geling Agents: Yet another aspect of the present invention is directed to gel formulations for KGF-2 polypeptides. The gelling agents can be added to the 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 the avoidance of bleeding may be important. Such gel formulations are employed for topical uses such as wound healing, to treat skin disorders or any other use that could be treated via the topical application of the pharmaceutical composition of KGF-2. Gel formulations for KGF-2 polypeptides according to the present invention comprise: 1) a topically effective amount of a KGF polypeptide; 2) a shock absorber; 3) a pharmaceutically acceptable diluent, preferably water; Y 4) a high molecular weight gel-forming compound. The viscosity of the gel formulations of the present invention may be in the range of about 1 to about 10,000 cps at room temperature, more preferably about 20 to about 100 cps at room temperature. The 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, water-insoluble polymers (eg, collagen) that can also form a viscous solution and that gel on contact with the skin. High molecular weight, gel-forming, useful compounds 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 fully incorporated by reference herein. l? faaA * - * - '^ áa ^ ¿M-.jfe ^ a-gt n..jMi? fa 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 (α-amylose or amylopectin) and chitosan. Useful glucoamiglucans include hyaluronic acid, chondroitin, chondroitin 4-sulfate, heparan sulfate and heparin. The glucosamiglucans can be used to improve the healing of wounds 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 commercially designated as PLURONICS (BASF) or POLAXAMERS (BASF). In a preferred embodiment, the gel of the present invention may 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 can comprising about 14 to about 18% by weight of the 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 greater potential for use in topical drug delivery systems, because they show 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 the mammal and is heated by the body temperature, the viscosity increases as the solution gels. Pluronic gels can be used for the controlled distribution 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 at which the polypeptides are released into the wound and thereby allows prolonged contact between the polypeptides and the site of i ii l. AM &? Arí £ yá * á Í. J The wound. The benefits of using such gel formulations include maintaining wound moisture and having a pharmaceutical composition that conforms to the shape of the wound or to another site 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 the mixture: 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 0.1% to about 7% sucrose or about 2.0% glycine and about 0.5% sucrose; 6) about 14% to about 18% Pluronic F127; and 7) water, wherein the formulation is at a pH of about pH 6.2. yi ** 4í. > 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 more preferably about 0.2 mg / ml; 2) sodium citrate at a concentration range of about 5 mM to about 20 mM; 3) about 10% to about 25% (w / v), preferably about 15 to about 25, and most preferably about 16% Pluronic 127 or Poloxamer 407; 4) about 6.7% to about 7.3% sucrose, preferably about 7% sucrose or about 2.0% glycine and about 0.5% sucrose; and 5) water until the capacity. The gel formulation optionally further includes one or more of the following: 6) EDTA at a concentration in the range of about 0.1 mM to about 10 mM. 7) sodium chloride at a concentration range of about 0.01 mM to about 125 mM. The preferred pH ranges for the gel formulation are 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% up to about 2% w / v glycerol, b) about 0.1% up to about 1% w / v methionine, c) about 0.1% up 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% up to about 0.05% w / v polysorbate 20, h) arginine, preferably at a concentration of about 0.5% up to about 2.5%, most preferably about 1.7%, i) heparin or a heparin analogue (negatively charged), j) dextran sulfate, preferably at a concentration of about 0.5% and 0.05%, k) cyclodextrins or sulfated cyclodextrins, 1) anionic or polyanionic species [analogs of heparin], m) lysine, preferably at a concentration of 10%, n) hydroxypropyl-β-cyclodextrin, preferably at a concentration of about 2 to about 10%, or) β-cyclodextrin, sulfated, preferably at a concentration of about 0.1% and 10%, more preferably about 1%, op) combinations thereof.

Administration of KGF-2 polypeptides Formulations of the KGF-2 polypeptide of the present invention can employ suitable pharmaceutical 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 dose of the pharmaceutical compositions of the present invention will be decided by the treating 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 another agent is employed, if any; age, body weight, health Íá.? ., i? rl? d $ ká ia general, sex and diet of the patient; the time of administration, the route of administration, and the rate of excretion of the composition; the duration of the treatment; drugs (such as a chemotherapeutic agent) used in combination or coincidentally 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 Sciences (latest edition), Mack Publishing Company, Easton, PA. The "effective amount" of KGF-2 for purposes hereof (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 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 treatment and / or .. * > «« AifcifcBaM rjjm «t. ^ ,. tfc > "3B" «> . . ^. sS.i-íA prophylaxis of the specific indication. In most cases, the dose of KGF-2 is about 1 μg / kg to about 30 mg / kg of body weight daily, taking into account the routes of administration, symptoms, etc. However, the dose can be as low as 0.001 μg / kg. For example, in the specific case of the doses for topical administration, these are preferably administered from about 0.01 μg to 9 mg per cm2. In the case of intranasal and intraocular administration, 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 administered parenterally 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 therapeutic discretion. If administered continuously, the KGF-2 polypeptide is typically administered at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour, either through 1 to 4 injections. per day or by continuous subcutaneous infusions, for example, using a minipump. A Intravenous bag solution or a bottled solution can also be employed. A course of treatment with the KGF-2 polypeptide to affect the fibrinolytic system seems to be optimal if it is continued for longer than a certain minimum number of days, 7 days in the case of the mice. The length of treatment necessary to observe changes and the interval of 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 (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, for example, one that is non-toxic to patients at the doses and concentrations used, and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds 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 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 Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, PA. The KGF-2 polypeptides can also be administered to the eye to treat damage to the lacrimal gland, disorders and pathologies in animals and humans such as a liquid, drops, or a thick liquid, or 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 the breast epithelium, in animals and humans, as liquid droplets or in the form of a spray. 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 shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with blood of the patient. 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 Pharmaceutical Sciences (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 or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counter ions 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 7, most preferably about pH 6.2. It will be understood that the use of some of the excipients, carriers, or stabilizers will result in the formation of KGF-2 salts. The KGF-2 that is going to be used for therapeutic administration may be sterile. Sterility is easily achieved by filtration through sterile filtration membranes (e.g., 0.2 micron 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 a 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 vials are filled with 1 ml of 1% aqueous KGF-2 solution (p / v) sterilized by 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 to the patient can be adjusted to achieve the regular blood levels at low, as measured by RIA (radioimmunoassay), 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., Biopolymers 22: 547-556 (1983)), poly- (2-hydroxyethyl methacrylate) (R. Langer et al., J Biomed, Mater. Res. 15: 167-277 (1981), and R. Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R. Langer et al., í £ '.ír. ^ * lát¿íyM * < * ji¡ * ki¡-- Sfeaamfc »» Id.) or poly-D- (-) -3-hydroxybutyric acid (European Patent EP-133,988). Sustained-release KGF-2 compositions also include liposomally entrapped KGF-2. Liposomes containing KGF-2 are prepared by methods known per se: German Patent DE-3, 218, 121; Epstein et al., Proc. Nati Acad. Sci. USA. 82: 3688-3692 (1985); Hwang et al., Proc. Nati Acad. Sci. USA. 77: 4030-4034 (1980); European Patent 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 European Patent 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% of cholesterol, the selected proportion 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 or biological products, whose notice reflects approval by the manufacturing agency, use or sale for administration human 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 epidermal 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 United States Patent Application No 08 / 910,875 and can bind to the 1-iiib 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 clone of the KGF-2 cDNA was deposited as ATCC Deposit No. 75977 on December 16, 1994 in the North American Collection of Species Crops (American Type Culture Collection), Patent Deposit, 10801 University BIvd., Manassas, VA 20110 -2209. In addition, a cDNA encoding KGF-2Δ33 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 "analog", when referring to the polypeptide of Figure 1 (SEQ ID NO: 2) or that encoded by the deposited cDNA, mean a polypeptide that retains essentially the same function or biological activity as such a polypeptide. In this way, 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 analogue 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 an amino acid residue conserved or non-conserved (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 a compound to increase the half-life of the polypeptide (eg, polyethylene glycol), or (iv) one in which additional amino acids are fused to the polypeptide 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 the like are considered within the scope of those skilled in the art from the teachings herein.

. JbeygpJB -t.Jt -M.t..AdMi? tjjaßBk¿ ^^ - ^^ > ^ * J ???: íta 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 joined by peptidyl bonds. The word "polypeptide" is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are described 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 effect of the structure or function of the protein. If such differences 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 carries out 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 "asylated polypeptide" is meant a polypeptide removed from its native environment. In this way, 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 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 comparison of the amino acid sequence and its substituted sequence of preservative amino acids of a polypeptide to the sequence of a second polypeptide. By "% similarity" for two polypeptides is meant a similarity rating produced by comparing the amino acid sequences of the two polypeptides, using the Bestfit program (Wisconsin Sequential Analysis Package, Version 8 for 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 Applied Mathematics 2: 482-489, 1981) to find the best segment of similarity between the two sequences. For a polypeptide having at least one amino acid sequence, for example, 95% "identical" to a reference amino acid sequence of a KGF-2 polypeptide, it is implied that the amino acid sequence of the polypeptide is identical to the reference sequence, except that the polypeptide sequence can include up to 5 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 positions or carboxyl-terminal of the reference amino acid sequence or at any site between those terminal positions, interposed either individually between the residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether or not any particular polypeptide is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the amino acid sequence shown in Figure 1 [SEQ ID NO. DO NOT. 2) or to 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 Sequential Analysis, Version 8 for 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, for example, 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. l¡yJyriyy.iíSÁt &.iíy «L». The proteins of the invention can be of natural origin, recombinantly produced, or can be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: 5 Structures and Molecular Principles, WH Freeman &Co. ., NY, and Hunkapiller, M. Et al., Nature 310: 105-111 (1984)). For example, a peptide corresponding to a fragment of the KGF-2 polypeptide of the invention can be synthesized by the use of a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a - replacement or addition to the sequence of the KGF-2 polypeptide. Non-classical amino acids include, but are not limited to, the D isomers of the common amino acids, 2,4-5-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid,? -Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulin, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine , ß-alanine, fluoro-amino acids, designer amino acids such as ß-methylamino acids, Carnetylamino acids, α-methylamino acids, and amino acid analogs in general. In addition, the amino acid may be D 5 (dextrorotatory) or L (levorotatory).

Variants of non-natural origin can be produced using mutagenesis techniques known in the art, which include, but are not limited to, oligonucleotide-mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (see, for example, Cárter et al., Nucí Acids Res. 13: 4331 (1986), and Zoller et al., Nucí Acids Res. 10: 6487 (1982)), cassette mutagenesis (see, for example, Wells et al., Gene 34: 315 (1985)), restriction selection mutagenesis (see, for example, Wells et al., Philos. Trans.R. Soc. London SerA 317: 415 (1986)). The invention further encompasses KGF-2 polypeptides that are differentially modified during or after translation, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to an antibody molecule or another cellular ligand, etc. Any of the numerous chemical modifications can be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin; etc.

The additional post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of the N-terminal or C-terminal ends, coupling of the chemical moieties to the main chain of amino acids, chemical modifications of the N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of expression in the prokaryotic host cell. The polypeptides can also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity tag to allow detection and isolation of the protein. Also provided by the invention are the chemically modified derivatives of KGF-2 which may provide additional advantages such as increased solubility, stability and increased circulation time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical portions for derivatization can be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol and the like. The polypeptides can be modified at random positions within the molecule, or at predetermined positions within the molecule, and can include one, two, three, or more linked chemical moieties. The polymer can be of any molecular weight, and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicates that in polyethylene glycol preparations, some molecules will weigh more, some less, than the established molecular weight) for ease of handling and the manufacture. Other sizes may be used, depending on the desired therapeutic profile (for example, the duration of the desired sustained release, the effects, if any, on biological activity, ease of handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, polyethylene glycol can have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000 , 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000 , 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000 or 100,000 kDa.

J ,, j ¿¿hajfc- «B. < As noted above, polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56: 59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18: 2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10: 638-646 (1999), the descriptions of each of which are incorporated by reference herein. The polyethylene glycol molecules (or other chemical moieties) must be linked to the protein with consideration of the effects on the functional or antigenic domains of the protein. There are a number of linking methods available to those skilled in the art, for example, European Patent EP-0, 401, 384, incorporated by reference herein (coupling of PEG to G-CSF), see also Malik et al. ., Exp. Hematol. 20: 1028-1035 (1992) (pegylation report of GM-CSF using tresyl chloride). For example, polyethylene glycol can be covalently linked through the amino acid residues via a reactive group, such as an amino group or free carboxy. The reactive groups are those to which an activated polyethylene glycol molecule can be linked. The amino acid residues that have a free amino group can include lysine residues and residues of N-terminal amino acids; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. The sulfhydryl groups can also be used as a reactive group for the linking of the polyethylene glycol molecules. Preferred for therapeutic purposes is the linkage to an amino group, such as coupling to the N-terminus or to the lysine group. As suggested above, polyethylene glycol can be bound to proteins via the link to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a protein via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine. One or more reaction chemistries can be used to link the polyethylene glycol to the specific amino acid residues (eg, lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residues (eg. example, lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein. Chemically modified proteins at the N-terminus can be specifically desired. Using polyethylene glycol as an illustration of the present composition, it can be selected, from a variety t? Á &.é. ????? ? út. Silah of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to the protein (or peptide) molecules in the reaction mixture, the type of pegylation reaction to be performed, and the method of obtaining the N-terminally pegylated protein, selected. The method of obtaining the N-terminally pegylated preparation (for example, the separation of this portion from other onopegylated portions, if necessary) can be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. The - selective proteins, chemically modified at the end N, can be carried out by reductive alkylation which exploits the differential reactivity of the different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, the substantially selective derivatization of the N-terminal protein with a polymer-containing carbonyl group is achieved. As indicated above, the pegylation of the proteins of the invention can be achieved by any number of means. For example, polyethylene glycol can be linked to the protein either directly or through an intervening linker. Systems without a linker for binding of polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992); Francis et al., Intern. J. Of Hematol. 68: 1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the descriptions of each of which are incorporated by reference herein. A system for linking polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs three-fold MPEG, which is produced by modifying monomethoxy polyethylene glycol (MPEG) using tresyl chloride (C1S02CH2CF3). After the reaction of the protein with the Threefold MPEG, the polyethylene glycol is directly linked to the amino groups of the protein. Thus, the invention includes the protein-polyethylene glycol conjugates produced by the reaction of the proteins of the invention with a polyethylene glycol molecule having the 2,2,2-trifluoroethanesulfonyl group. Polyethylene glycol can also be bound to proteins using a number of different linkers of intervention. For example, U.S. Patent No. 5,612,460, the entire disclosure of which is incorporated by reference herein, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is linked to the protein by a linker, they can also be produced by the reaction of the proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2, 4, 5-trichlorophenylcarbonate , MPEG-p-nitrophenol carbonate, and various derivatives of MPEG-succinate. A further number of polyethylene glycol derivatives and reaction chemistries for linking polyethylene glycol to proteins are described in WO 98/32466, the full disclosure of which is incorporated by reference herein. Protein products Pegylates produced using the reaction chemistries described herein are included within the scope of the invention. The number of polyethylene glycol moieties linked to each protein of the invention (e.g., the degree of substitution) may also vary. For example, the pegylated proteins of the invention can be linked on average to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution is within the ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19 or 18-20 portions of polyethylene glycol per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Cri t. Rev. Thera, Drug Carrier Sys. 9: 249-304 (1992).

Suppression Mutants of KGF-2 Native KGF-2 is relatively unstable in the aqueous state and undergoes chemical and physical degradation which results in 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 (the numbering begins with the first amino acid in the (Met) protein: Thr (residue 36) - Ser (residue 208) Gly (41) - Ser (208 ) Cys (37) --Ser (208) Gln (42) --Ser (208) Gln (38) --Ser (208) Asp (43) --Ser (208) Ala (39) --Ser (208) ) Met (44) --Ser (208) Leu (40) --Ser (208) Val (45) --Ser (208) Ser (46) --Ser (208) Pro (47) --Ser (208) ) Glu (48) --Ser (208) Wing (49) --Ser (208) Thr (50) --Ser (208) Asn (51) --Ser (208) Ser (52) --Ser (208) ) Ser (53) --Ser (208) Ser (54) --Ser (208) Ser (55) --Ser (208) Ser (56 - Ser (208) Met (1 Thr (36 or Cys (37) --- His (200) Phe (57 - Ser (208) Met (1 Thr (36 or Cys (37) --- Ala (199) Ser (59 --Ser (208) Met (1 Thr (36 or Cys (37) --- Ser (198) Ser (62 --Ser (208) Met (1 Thr (36 or Cys (37) --- Thr (197) Ala (63 - Ser (208) Met (1 Thr (36 or Cys (37) --- Asn (196) - Gly (64 - Ser (208) Met (1 Thr (36 o Cys (37) --- Lys (195) Arg (65 - Ser (208) Met (1 Thr (36 or Cys (37) - -Arg (194) Val (67 - Ser (208) Met (1 Thr (36 or Cys (37) - -Arg (193) Ser (69 - Ser (208) Met (1 Thr (36 or Cys (37) - -Thr (192) Val (77 --Ser (208) Met (1 Thr (36 or Cys (37) - -Lys (191) Arg (80 - Ser (208) Met (1 Thr (36 or Cys (37) - -Arg (188) Met (1) Thr (36) or Cys (37) --His (207) Met (1), Thr (36), or Cys (37 - Arg (187) Met (1) Thr (36), or Cys (37) --Val (206) Met (1), Thr (36), or Cys (37 --Lys (183) Met (1) Thr (36), or Cys (37) --Val (205) Met (1) Thr (36), or Cys (37) --Met (204) Met (1) Thr (36), or Cys (37) - Pro (203) Met (1) Thr (36), or Cys (37) - Leu (202) Met (1), Thr (36), or Cys (37) -Phe (201) 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 (1), Thr (36), or Cys (37) -Lys (153). Also included by the present invention are deletion mutants that have deleted amino acids - from the N-terminus and the C-terminus. Such mutants include all combinations of N-terminal deletion mutants and C-terminal deletion mutants described above, for example, Ala (39) -His (200), Met (44) ) --Arg (193), Ala (63) --Lys (153), Ser (69) --Lys (153), etc. These 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 the 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 (e.g. you."*. Ufe ». • - .fJts. ^^ ji ^, ^ á ^^ -? ^^? ^ Áli? Í-iá.J 'minus Met (l) - Gln (38)) but not more than the first 147 amino acid residues N-terminals 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 no 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 no more than the first 137 residues of - 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 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-ternal 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 residues of - N-terminal amino acids but not more than the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID DO NOT. 2) . In addition to a formulation comprising a mutant of KGF-2 with the ranges 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 DO NOT. 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 DO NOT. 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. itía á, * tíí * Xá.yi. .. Hfc.- ..

In another embodiment, the formulations comprising the C-terminal deletion mutants are provided by the present invention. Preferably, the N-terminal amino acid residue of the 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 not more than the last 55 C-terminal amino acid residues (eg, a deletion of amino acid residues Glu (154) - be (208)) 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 the Figure üh áA? * áú * t. s & s &J 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 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 40 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 50 C-terminal amino acid residues but not more than the last 55 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 ranges of C-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 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 - DO NOT. 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 the amino acids deleted from the N-terminal and C-terminal residues. Such mutants include all combinations of the N-terminal deletion mutants and the C-terminal deletion mutants described above. Such mutants include those comprising the sequence of amino acids shown in Figure 1 (SEQ ID No. 2) except for a deletion of 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) 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 (SEQ ID NO 2) and a suppression 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 described above are also included .

Substitution Mutants KGF -2 Useful KGF-2 polypeptides include those that have amino acid substitution. Native mature KGF-2 contains 44 charged residues, 32 of which carry 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 residues grouped with the ll, a:? ' i? y.i.í £ zá? rJ, and, .ikty¡y? ~. and .. ..Lam-iM - > .-.-..to. I .I ^ JI M and - amino acids that have a negative charge or a neutral charge, can alter the electrostatic interactions of adjacent residues and can be useful to achieve increased stability and reduced aggregation of the protein. The aggregation of the proteins can not only result in a loss of activity but also be problematic when preparing pharmaceutical formulations, because these can be immunogenic (Pinckard et al., Clin Exp. Immunol., 2: 331-340). (1967), Robbins et al., Diabetes 36: 838-845 (1987), Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)). - Any modification should give consideration to minimize the load repulsion in the tertiary structure of the protein molecule. Thus, of special interest are the substitutions of the amino acids loaded with another charge, and with the 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, as 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., Nature 361: 266-268 (1993), described certain mutations of T? F-alpha that give as a result 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) Thr, Trp (79) Val, Arg (80) Lys, Lys (87) Arg, Tyr (88) Trp, Phe (89) Tyr, Lys (91) Arg, Ser (99) Lys, Lys (102) Gln, Lys (103) (Glu), Gly (104) Met, Asn (105) Lys, Pro (107) Asn, Ser (109) Asn, Leu (111) Met, Thr (114) Arg, Glu (117) Ala, - Val (120) He, Val (123) He, Ala (125) Gly, He (126) Val, Asn (127) Glu, Asn (127) Gln, Tyr (130) Phe, Met (134) Thr, Lys (136) Glu, Lys (137) Glu, Gly (142) Ala, Ser (143) Lys, Phe (146) Ser, Asn (148) Glu, Lys (151) Asn, Leu (152) Phe, Glu ( 154) Gly, Glu (154) Asp, Arg (155) Leu, Glu (157) Leu, Gly (160) His, Phe (167) Ala, 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. For the designation, for example, Ala (49) gln it is intended that the Ala at position 49 of Figure 1 (SEQ ID NO 2) be replaced by Gln.

The changes are preferably of a minor nature, such as conservative amino acid substitutions 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 as described below: Aromatics: phenylalanine tryptophan tyrosine Hydrophobic leucine isoleucine valine Polar: glutamine asparagine Basic: arginine lysine histidine Acids: aspartic acid glutamic acid Small alanine serine threonine methionine glycine te?? i'tttl |. ... -Ln.i.av._. And, Of course, the number of amino acid substitutions that one skilled in the art would 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 that can be made at the C-terminus of KGF-2 to improve stability. The amino acids in KGF-2 that are essential for the function can be identified by the methods well - known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 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 activity. proliferative in vi tro or in vivo (See for example, Example 1.) Sites that are critical for the ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling. for example: Smith et al., J. Mol. Biol., 224: 899-904 (1992), and de Vos et al., Science 255: 306-312 (1992)).

Other useful KGF polypeptides include polypeptides that have substitutions of serine by cysteine at the amino acid positions 37 and 106 and 150. A non-uniform number of cysteine means that at least one cysteine residue is available for crosslinks or intermolecular bonds that can cause the protein to adopt an undesirable tertiary structure. The new KGF-2 proteins having one or more cysteine replaced by serine or for example alanine are generally purified at a higher yield of the correctly folded, soluble protein. Although you do not want to be committed 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. In addition, KGF-2 polypeptides are described in PCT / US95 / 01790, filed on February 14, 1995 and U.S. Patent Applications Nos. 08 / 461,195, filed June 5, 1995, 08 / 696,135, filed on August 13, 1996, 60 / 023,852, filed on August 13, 1996, 60 / 039,045, filed on February 28, 1997, 08 / 862,432, filed on May 23, 1997; 60 / 055,561, filed August 13, 1997, 08 / 910,875, filed on August 13, 1997, 09 / 023,082, filed on February 13, 1998, 09 / 345,373, filed July 1, 1999, 60 / 142,343, filed on July 2, 1999, 60 / 143,648, filed July 14, 1999, 60 / 144,024, filed July 15, 1999, 60 / 148,628, filed on August 12, 1999, 60 / 149,935, filed on September 24, 1999, 60 / 163,375, filed on November 3, 1999, 60 / 171,677, filed on December 22, 1999 and 60 / 198,322, filed on April 19, 2000, the descriptions of the which are incorporated by reference herein.

Therapeutic Uses of the KGF-2 Polypeptide Compositions The polypeptides of the present invention can stimulate the growth and proliferation of keratinocyte cells. Accordingly, the compositions of the present invention can be employed to stimulate the proliferation of epithelial cells and basal keratinocytes for wound healing purposes, and to stimulate the proliferation 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 treating a number of diseases and conditions. For example, KGF-2 is active in vi tro and in vivo in various models of wound healing. See, United States Patent Applications Nos. 08 / 910,875, filed on August 13, 1997 and 09 / 023,082, filed on February 13, 1998. The individual to whom KGF-2 is administered may heal his wounds at a normal speed or may have impaired 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 facilitate the healing of wounds that might otherwise heal slowly or not at all. A number of afflictions and conditions may result - the deterioration of the healed. These afflictions and conditions include diabetes (for example, Type II diabetes mellitus), treatment with steroids and non-steroidal pharmacological agents, and blockage or ischemic damage. A number of growth factors have been shown to promote healing of wounds in individuals with impaired healing. These growth factors include the growth hormone release factor, the platelet derived growth factor, and the growth factors of basic fibroblasts. Thus, the present invention also encompasses the administration of the 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 and other wounds caused by surgical procedures in individuals, which 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 that constitute nervous tissue, prostate cells, and lung cells. The compositions of the present invention are clinically useful in the stimulation of wound healing, including surgical wounds, excision wounds, deep wounds involving damage to the dermis and epidermis, ocular tissue wounds, dental tissue wounds, wounds of the oral cavity, diabetic ulcers, skin ulcers, ulcer ulcers, 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 wound healing, such as uremia, malnutrition, vitamin deficiencies, obesity, infection, immunosuppression, and complications associated with systemic steroid therapy, radiation therapy, and antineoplastic and antimetabolite drugs. The compositions are also useful for promoting healing of wounds associated with ischemia and ischemic damage, for example, chronic venous leg ulcers caused by a deterioration of the return of the venous circulatory system and / or insufficiency thereof.; for the promotion of dermal recovery subsequent to skin loss; increase in the tensile strength of the epidermis and epidermal thickness; and increase adhesion of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. Other therapeutic uses for KGF-2 polypeptides include, but are not limited to, for example, stimulating the proliferation of basal epithelial cells and keratinocytes for the purpose of treating burns and skin defects such as psoriasis and epidermolysis bullosa. KGF-2 can be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. KGF-2 can also be used to reduce the side effects of bowel toxicity, which results from radiation, chemotherapy treatments 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, dry keratoconjunctivitis. KGF-2 can be used to stimulate the epithelial cells of the salivary glands, lacrimal glands and stimulation of the re-epithelialization of the 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 new liquid and lyophilized formulations of KGF-2 of the deletion mutants thereof. This invention also relates to formulations of KGF-2 for therapeutic use. The formulations pre 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, by "individual or individual" is meant an animal, preferably a mammal (such as apes, cows, horses, pigs, bears, sheep, rodents, goats, dogs, cats, chickens, monkeys, rabbits, ferrets, whales, and dolphins), and more preferably a human. The KGF-2α3 polypeptide 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. Other therapeutic uses of KGF-2 are described in U.S. Patent Applications Nos. 60 / 074,585, filed February 13, 1998, 60 / 114,484, filed December 30, 1998, and 09 / 248,998, filed. on February 12, 1999, the descriptions of which are incorporated by reference herein.

Keratinocyte Proliferation Assays Dermal keratinocytes are cells in the epidermis of the skin. The development and dispersion of keratinocytes in the skin is an important process in the healing of wounds. A keratinocyte proliferation assay is therefore a valuable indicator of the activities of the protein in stimulating the development of keratinocytes and, consequently, of 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 the complications of this assay for cellular defects such as the loss of the key growth factor receptors, or the dependence of growth factors key to development, the primary dermal keratinocytes are chosen for this assay. These primary keratinocytes are obtained from Clonetics, Inc. (San Diego, CA). The bioactivity of KGF-2 polypeptides can be determined by a cell proliferation assay using murine Baf3 2b cells that have been transfected with the fibroblast growth factor receptor 2iiib (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. Subsequently, an approximate amount of the cell medium containing the The marked thymidine, in 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 in each sample is counted using a flat bed liquid scintillation counter. Under these assay conditions, cells exposed to KGF-2 show increased incorporation of radioactivity compared to control cells that have been treated either with an appropriate dilution of the placebo buffer or simply with the phosphate buffered saline. Another useful keratinocyte proliferation assay is with Alamar Blue. Blue Alamar is a viable blue dye that is metabolized by mitochondria when added to culture media. The dye then becomes red in the supernatants of the tissue culture. The quantities of the red dye can be directly quantified by the difference in reading at the optical densities between 570 nm and 600 nm. This reading reflects cellular activities and the number of cells. Normal primary dermal keratinocytes (CC-0255, NHEK-Neo combined) are purchased from Clonetics, Inc. These cells are passage 2. Keratinocytes are developed in complete growth medium of keratinocytes (CC-3001, KGM, Clonetics, Inc.) until 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 3-5 minutes at room temperature. The trypsin neutralization solution is added and the cells are harvested. The cells are centrifuged at 600 xg for 5 minutes at room temperature and placed in new flasks at 3,000 cells per square centimeter using preheated 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 external 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 to 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. HE They are incubated for two to three days. At the end of the incubation, the cells are washed once with KBM and 100 μl of KBM are added with 10% v / v of Alamar Blue, pre-mixed in the medium. It is incubated for 6 to 16 hours until the color of the medium begins to turn red in the positive control with KGM. The optical density at 570 nm less optical density 600 nm is measured directly by placing the plates in the plate reader.

Construction of the Suppression Mutants KGF-2 Deletion mutants useful for use in the compositions of the present invention can be constructed by the following protocol. The deletion mutants were constructed from the 5 'end and the 3' end of the KGF-2 gene using a KGF-2 construct optimized 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 3'HindIII primer of 208 amino acids of KGF-2 (FGF-12) was used for the 3 'primer. PCR amplification for 25 rounds was performed 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 products were restricted with Ncol for the 5 'restriction enzyme and with HindIII for the 3' site, and cloned into pQE60 which had been digested with Ncol and HindIII. For KGF-2 (FGF-12), 36aa / l53aa and 128aa 3 'HindIII was used as the 3' primer with FGF-12 36aa / 208aa as the 5 'primer. For FGF-12, 62aa / l53aa, 128aa was used 3 'HindIII 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 acids 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 it. However, the KGF-2 suppression polypeptides used in the formulations according to the present invention may or may not have the N-terminal methionine, < L »?? & - * - • - * - < VA * -JLJ-. t, ßkJ m *? »Ut * r & amp; < *. ^ MMar ^ n-JtA «- ^ aáß ^ t preferably the polypeptide will lack 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 NO.11) FGF12 63aa / l53aa: 5 'Ncol 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 amplification directed by the Polymerase Chain Reaction and subcloning of KGF2Δ33 into the E. coli protein expression vector, pQE6, two oligonucleotide primers (5952 and 19138) complementary to the desired region of KGF2 were synthesized, with the following base sequence .

Primer 5952: 5 'GCGGCACATGTCTTACAACCACCTGCAGGGTG 3' (SEQ ID NO 12) Primer 19138: 5 'GGGCCCAAGCTTATGAGTGTACCACCAT 3' (SEQ ID NO.13) In the case of the N-terminal primer (5952), an AflII 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 ATG sequence adjacent and in structure with the coding region of KGF2 to allow translation of the 'i ^ Ay.jí & * A ^. ~ fragment cloned in E. coli, while primer 19138 contains two stop codons (preferably used in adjacent E. coli and in structure with the coding region of KGF2 which ensures the correct translational termination 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. was digested by restriction by AfHI and HindIII and subcloned into the expression vector of pCE6 protein digested with NcoI / HindIII.

Construction of KGF -2 A33 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 'CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG 3' (SEQ ID NO.14) Primer 6150: 5 'CCGGCGGTACCTTATTATGAGTGTACCACCATTGG 3' (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 ATG sequence adjacent and in structure with the coding region of KGF2, to allow translation of the cloned fragment in E. coli, while primer 6150 contains two stop codons (preferably used - in E. coli) adjacent and in structure with the coding region of KGF-2 that ensures the correct translational termination 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 digested by restriction with Ndel and Asp718 and subcloned into the pHEl protein expression vector digested with Ndel / Asp718.

Nucleotide sequence of KGF -2? 33 ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCTTTCACCAAATACTTCCTGAAAATCGAAAA AAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAG CATCCTGGAGATAACATCAGTAGAAATCGGAGTTG TTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAA GAAGGGGAAACTCTATGGCTCAAAAGAATTTAAC AATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAAT ACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGG AGAGGACAGAAAACACGAAGGAAAAACACCTCTCGCTCACTTTCTT CCAATGGTGGTACACTCATAA (SEQ ID NO.

Amino acid sequence of KGF-2? 33 MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMWHS (SEQ ID NO. 71) B. Construction of a KGF-2? 33 Optimized Polynucleotide Sequence In order to increase the expression levels of KGF2? 33 in E. coli, the codons of the complete gene were optimized to fit those most highly used in E. coli. As the template used to generate the KGF2? 33 was the codon optimized within the N-terminal region, the C-terminal amino acids (84-208) required optimization. First, amino acids 172-208 were optimized by codon to generate KGF2Δ 33 (sl72-208). This was achieved through the PCR overlap strategy. The 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 overlap with the PstI site within the coding region of KGF2) and PMll 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 PCR reaction (corresponding to amino acids 84-172 not optimized by the codon) were combined and used as a template for a standard PCR reaction directed by the oligonucleotides PM05 and PM10. The amplicon .? aaft .i í ?? Üti. N i yjlá k.i *? S? The resulting was digested with PstI / HindIII and subcloned into PQE6KGF2? 33 digested with PstI / HindIII, effectively replacing the corresponding codon-optimized region, and creating pQE6KGF2? 33 (sl72-208). To complete the codon optimization of KGF2, a synthetic gene codon 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 PCRs 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 template. The optimized gene fragment of the resulting codon was then digested with PstI / SalI and subcloned into pQE6KGF2Δ33 (sl72-208) digested with PstI / SalI to create a gene coding for fully optimized KGF2, pQE6KGF2Δ33. To create an alternative E. coli protein expression vector, KGF2? 33S was amplified by PCR using primers PM102 and PM130 on pQE6KGF2? 33s. The resulting amplicon was digested with Ndel and EcoRV and subcloned into the pHEl expression vector which had Has been digested with Ndel and Asp718 (blunt end) to create pH? 33s. The oligonucleotide sequences used in the construction of KGF-2? 33s optimized at 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) PMll: GCCACATACATTTGTCGACCGTT (SEQ ID NO. 23) PM31 : CTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCCTTCACC AAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACCA AG (SEQ ID NO. 24) PM32: AGCTTTAACAGCAACAACACCGATTTCAACGGAGGTGATTTC CAGGATGGAGTACGGGCAGTTTTCTTTCTTGGTACCAGAAACTTTA CC (SEQ ID NO. 25) PM33: GGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTG GCTATGAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTAAC AAC (SEQ ID NO. 26) PM3: GTCGACCGTTGTGCTGCCAGTTGAAGGAAGCGTAGGTGTTGT AACCGTTTTCTTCGATACGTTCTTTCAGTTTACAGTCGTTGTTAAAT TCTTTGGAACC (SEQ ID NO. 27) PM35: GCGGCGTCGACCGTTGTGCTGCCAG (SEQ ID NO: 28) PM36: GCGGCCTGCAGGGTGACGTTCGTTGG (SEQ ID NO: 29) PMl02: CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG (SEQ ID NO: 30) PM130: CGCGCGATATCTTATTAAGAGTGTACCACCATTG (SEQ ID No. 31) Nucleotide sequence of KGF-2? 33 (sl72-208): ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGT TCTCCTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGT TTCTGGTACCAAGAAAGAAAACTGCCCGTACTCCATCCTGGAAATC ACCTCCGTTGAAATCGGTGTTGTTGCTGTTAAAGCTATCAACTCCA - ACTACTACCTGGCTATGAACAAGAAAGGTAAACTGTACGGTTCCAA AGAATTTAACAACGACTGTAAACTGAAAGAACGTATCGAAGAAAA CGGTTACAACACCTACGCTTCCTTCAACTGGCAGCACAACGGTCGA CAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGTC AGAAAACCCGTCGTAAAAACACCTCTGCTCACTTTCTGCCAATGGT GGTACACTCTTAA (SEQ ID NO.12) Amino Acid Sequence of KGF -2? 33 (S172-208) MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITS VEIGWAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN TYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMWHS (SEQ ID NO.

Examples EXAMPLE 1 Liquid Formulation of KGF-2 The following ingredients were mixed to create a liquid KGF-2? 33 formulation stored at -20 ° C. 2 mg / ml KGF-2? 33 polypeptide, 20 mM sodium acetate, 125 mM sodium chloride, 1 mM EDTA, Water, pH 6.2.

This formulation retained its bioactivity in vi tro for up to 10 months at storage conditions at or below 2 to 8 ° C. The 10-month bioactivity is shown in Figure 3. This formulation retained all its physico-chemical properties for up to 11 months at or below storage conditions. 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 WEHI cells, 2 mM glutamine, 600 μg / ml GENETICINE, 1 μl β-mercaptoethanol / 500 ml of growth medium, 50 units of penicillin and 50 μg / ml of streptomycin (Ornitz, D., M. et al. (1996) J. Biol. Chem. 271: 15292-15297). For cell proliferation assays, BaF3 cells were harvested by centrifugation and washed with the basal medium (this has the same composition as the growth medium, but does not contain the WEHI conditioned medium and is supplemented with 1 μg / ml heparin) . After this operation, the cells were resuspended in the basal medium and 22,000 cells / 180 μl were seeded in well / well in the 96-well cell culture box. Appropriate dilutions (10 X greater than the required final concentration) of KGF2 in PBS were made in another 96-well plate and added to the cells to a final volume of 200 μl. The cell plates were incubated in an incubator at 37 ° C, 5% C02 for 36-40 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. Incorporated thymidine was counted on a Wallace scintillation counter.

Example 2 Lyophilized Formulation of KGF-2 The following ingredients were mixed to create the lyophilized formulation of KGF-2? 33. 10 mg / ml KGF-2? 33, 10 mM sodium citrate, 20 mM sodium chloride, 1 mM EDTA, 7% w / v sucrose, water (removed after lyophilization) - pH 6.2.

This formulation retained its bioactivity in vi tro for up to 9 months at storage conditions at or below 45 ° C. The 9-month bioactivity is shown in Figure 4. Bioactivity was measured using the cell proliferation assay detailed in Example 1. Reverse phase HPLC demonstrated that the formulation retained its physicochemical properties for up to 8 months at temperatures of or below 45 ° C and 75% relative humidity.

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

This formulation was 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 the gel formulation of KGF-2? 33. 2 mg / ml KGF-2? 33, 10 mM sodium citrate, 20 mM sodium chloride, 1 mM EDTA, 7% w / v sucrose, 16% Pluronic F127, water, pH 6.2.

KGF-2? 33 is 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 The following reserve formulations of KGF-2 protein 33 (0.1 to 2.0 mg / ml) with or without monothioglycerol (MTG) were prepared. The 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 of growth medium, 50 units of penicillin and 50 μg / ml of streptomycin (Ornitz, D., M. et al. (1996) J. Biol. Chem. 271: 15292-15297).

Cell Proliferation Assays For cell proliferation assays, BaF32b cells were harvested by centrifugation and washed with the Basal medium (this has the same composition as the growth medium, but does not contain the WEHI conditioned medium and is supplemented with 1 μg / ml of heparin). After this operation, the cells were resuspended in the basal medium and 22,000 cells / 180 μl were plated / well in a 96-well cell culture plate. The 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 at 37 ° C with 5% C02 for 36-40 hours and 0.5 μCi of methyl-3H-thymidine in 50 μl of basal medium were 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.

Resulted TO . 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 and is shown in Figure 5. The increase in activity was between 10-150% of the control depending on the concentration of MTG used. This increase 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 with KGF-2.

Example 6 Formulation of KGF-2 in Gel with Ci trata The following ingredients were mixed to create a KGF-2 formulation in a liquid form at room temperature, and which subsequently gels after application to the skin. 20 mM sodium citrate, 125 mM sodium chloride, 1 mM disodium EDTA, 17% Pluronic 127, pH 6.0, water.

Example 7 Formulation of KGF-2 in Gel with Acetate The following ingredients were mixed to create the formulation of KGF-2 which can gel after application to the skin. 20 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 20 mM sodium citrate, 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. 2. 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 M sodium chloride, 1 mM disodium EDTA and 7% sucrose, pH 6.0. C. 10 mM sodium citrate, 20 M 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 KGF -2 at 10 mg / ml It was evaluated whether or not the formulation would allow lyophilization of the protein at 10 mg / ml, as well as the Subsequent stability of the protein after reconstitution.

Formulation: 10 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 1% monothioglycerol.

Example 9 Stability of the Thickening Agent The following formulations are prepared according to the methods of the previous examples. Configurations 1 and 2 are KGF-2 lyophilized alone. Configurations 3 and 4 include the thickening agent as part of the liotorta; and configurations 5-8 include the thickening agent as part of the liquid diluent. Í? Tl ?? é-Jr ** bAS? - * l > l - ** l? a2h .. 5ata ^ S ^^? fJÉ -, -, atajl Example 10 The use of KGF-2 in the treatment of chronic wounds is expected to involve multiple topical applications of the drug. The lyophilized configuration requires a separate bottle of pharmaceutical product to be reconstituted by application due to the absence of any preservative. This results in a significant amount of waste of the drug, as well as a preparation of the most intense work product to be performed in each dosage. Ideally, the commercial configuration of KGF-2 could involve a simple KGF-2 bottle that could be used for multiple applications. To investigate the feasibility of this configuration, the compatibility of KGF-2 with preservatives was examined. The preservatives were selected from a list of products approved by the FDA. The five candidate curators selected were based on the frequency in the Physician's Desk Reference (PDR) as well as previous work published with biopharmaceutical products. Since the formulation and dosage form have not been finalized, strong emphasis on compatibility with elastomeric closure or stability at pH 6.2 was not initially placed. The concentrations were selected based on the values of the literature as well as in the guidelines of the FDS / USP and BP (British Pharmacopoeia). The following table presents the five candidate curators examined in this study. 1. "Parabens" is defined in this example as 0.18% methylparaben and 0.02% propylparaben.

In order to use a conservator, two criteria must be met. First, the compatibility and f 113 Stability with the pharmaceutical product must be established. Second, microbial effectiveness must be established (by USP < 51 >). The purpose of this study was to examine the short-term compatibility of KGF-2 with these preservatives.

MATERIALS AND METHODS Formulations All chemical products were purchased from Spectrum and were USP / NF grade or equivalent. KGF-2 was diafiltered in a base formulation buffer containing one of the following: 0.9% benzyl alcohol, 0.5% chlorobutanol, 0.5% phenol, 0.3% m-cresol, or 0.18% methylparaben + 0.02% of propylparaben ("parabens"). The diafiltration was performed in a stirred Amicon cell with a 10 kD Biomax membrane. All processing was carried out at 2-8 ° C. A total of 5 buffer exchanges were made for each diafiltration. The recoveries during the diafiltration were > 90% KGF-2 was diluted to 1.0 mg / ml and filled (1.0 ml) in 2 ml Schott Purform flasks, and sealed with a Diakyo D-7771 serum stopper and an aluminum engargolado seal. The bottles were stored at -80 ° C, 2-8 ° C, and 25 ° C / 60% relative humidity. The formulations that containing precipitation were filtered on a 0.2 μm filter before further analysis.

Appearance The visual inspection was performed using the fluorescent light of front lighting against a black and white background under normal amplification.

SDS-PAGE SDS-PAGE was performed under non-reducing conditions using a 16% Tris-glycine Novex gel. A total of 2 μg of KGF-2 was loaded per band. The gels were run under constant voltage (125 V) for approximately 2 hours.

The staining was performed using a Daiichi silver staining kit according to the manufacturer's instructions.

Bioactivity The bioactivity of KGF-2 is determined using a murine lymphocyte cell line, Baf3 2b, which has been stably transfected with the fibroblast growth factor receptor 2iiib. The activity is based on cell proliferation as measured by the incorporation of [methyl-3 H] thymidine after exposure to KGF-2. -ri fei Differential Scanning Calorimetry (DSC) The DSC thermograms of each formulation were obtained on a Sciences Nano DSC calorimeter (model 5100). The scans were performed from 5 ° C to 80 ° C at a rate of 1 ° C / minute in the direction of heating and cooling. The melting temperature (Tm) was measured as the apex of the thermal transition. No signal was observed in the direction of cooling in any of the samples, indicating that the unfolding was irreversible.

RP-HPLC (concentration, purity, oxidized percent) RP-HPLC (reverse phase high resolution liquid chromatography) was performed on a Waters 2690 Alliance equipment equipped with a Waters 996 photodiode array detector. A Waters column was used. Delta-Pak C18 (2.1 x 150 mm, 5 μm, 300 A) for separation using a linear gradient of solvent A (0.1% TFA in water) to solvent B (0.07% TFA in acetonitrile). The concentration was determined by correlating the total peak area of an unknown curve to a standard using an internal reference standard of known concentration. A typical chromatogram of the standard is shown below. Two major peaks that are separate and have been previously identified as the intact KGF-2 peak and an oxidized form of KGF-2. The oxidized percent is reported as an area percentage of the total area. Percent purity is the percentage area attributed to the sum of the intact and oxidized form.

Resulted Chlorobutanol and parabens show promising compatibility with KGF-2 at 2-8 ° C. Benzyl alcohol, m-cresol and phenol cause rapid aggregation of KGF-2 when stored at 2-8 ° C. When stored at 25 ° C, all formulations look poor after one week of storage. KGF-2 precipitates the solution when subjected to de-freezing / thawing conditions in the presence of m-cresol and phenol. All tested preservatives have a slight destabilizing effect on the thermal stability of KGF-2. The melting temperature, as determined by DSC, shows a drop of approximately 5 ° C in the presence of all the preservatives. Of the tested preservatives, parabens have the least impact. The specific activity of KGF-2 is not affected by the presence of any of the preservatives tested. Although the total activity of the formulations stored at 25 ° C does not decrease after six weeks of storage due to the precipitation of KGF-2, the soluble protein in The formulations of chlorobutanol, parabens and benzyl alcohol, kept its specific activity. At storage at 2-8 ° C, all formulations remain within 10% of their initial concentrations as determined by RP-HPLC. When stored at 25 ° C, formulations containing m-cresol or phenol showed the fastest rate of precipitation. Of the formulations tested, those containing parabens or chlorobutanol retained most of KGF-2 in solution during the course of short-term stability. Benzyl alcohol causes rapid oxidation of KGF-2, followed by continuous degradation of the oxidized form. This is shown by a high initial level of oxidation (formed during diafiltration), followed by a slow transition to additional oxidized forms. When stored at 2-8 ° C, the remaining formulations have comparable oxidation rates of approximately 4% per month. When stored at 25 ° C, the oxidation rates increase to 30-40% per month. Of the formulations tested, the one that contains parabens has the slowest average oxidation rate. The purity is determined as the sum of the main and oxidized form of KGF-2 as a percentage of the total chromatographic area. Formulations containing benzyl alcohol or phenol show a purity initially low, followed by an elevation and then a gradual loss of purity. This corresponds to an acute increase to zero time of a chromatographic peak that seems to correspond to the soluble aggregate. As the precipitate continues to form, this peak disappears without any subsequent increase in any other peak area. When stored at 25 ° C, the formulation containing the parabens retains the highest purity. SDS-PAGE shows a slight increase in the dimer band in the formulation containing benzyl alcohol at time zero as well as at six weeks of storage. They are observed a light grated in the band and increased levels of the dimer in the formulations stored at 25 ° C.

Conclusions KGF-2 is incompatible with benzyl alcohol, with m-cresol and with phenol under the conditions examined. In these formulations, aggregation followed by precipitation was the primary pathway for degradation. Of the conservatives tested, methyl / propylparaben has the least impact of the short-term stability of KGF-2.

Example 11 KGF-2 in a lyophilized formulation The following ingredients were mixed to create a pre-mix formulation of KGF-2? 33. 3. 3 mg / ml KGF-2? 33, 10 mM sodium citrate, 20 mM sodium chloride 1 mM EDTA, 2% w / v glycine 0.5% w / v sucrose, water (removed after lyophilization) pH 6.2 The formulation was subsequently lyophilized according to the second cycle of lyophilization described above.

This formulation retained its bioactivity in vi tro for up to 12 months at storage conditions at or below 25 ° C. Í? ^ R ^ ÉÍÍL y.

Example 12 Topical formulation of KGF-2 The following ingredients were mixed to create a KGF-2 formulation for topical application. 1. 0 mg / ml KGF-2? 33, 0.46% hydroxyethylcellulose (HEC) 7% sucrose 20 mM sodium citrate 20 mM sodium chloride 1 mM EDTA pH 6.2 It will be clear that the invention can be practiced in another way than is particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. The complete description of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or others) I A? Jy j. J3jt, iJt.LEÍ.% ,, M¡É * »? . - - -I- «rfaj Afc jd2úb * and documents) cited herein are incorporated by reference herein. LIST OF SEQUENCES < 110 > Human Genome Sciences, Inc. Gentz, Remer L. Chopra, Arvind Kaushal, Parveen Spitznagel, Thomas Unsworth, Edward Khan, Fazal < 120 > Formulations of Factor 2 Growth 10 of Keratinocytes < 130 > 1488.103PC05 < 140 > to be assigned < 141 > With the present < 150 > US 60 / 137,448 < 151 > 1999-06-02 < 150 > US 60 / 160,913 < 151 > 1999-10-22 20 < 160 > 33 < 170 > Patentln Ver. 2.0 25 < 210 > 1 < 211 > 627 < 212 > DNA < 213 > Homo sapiens 30 < 220 > < 221 > CDS < 222 > (1) . (624) < 400 > 1 35 atg tgg aaa tgg ata ctg here cat tgt gcc tea gcc ttt ccc falls ctg 48 Met Trp Lys Trp lie Leu Thr His Cys Wing Wing Wing Phe Pro His Leu 1 5 10 15 ccc ggc tgc tgc tgc tgc tgc ttt ttg ttg ctg ttc ttg gtg tct tcc 96 40 ro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 gtc ect gtc acc tgc ca gcc ctt ggt cag gac atg gtg tea cea gag 144 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 45 35 40 45 gcc acc aac tct tct tcc tcc tcc tccctct ect tcc age gcg gga 192 Wing Thr Asn Being Being Being Being Phe Being Being Pro Pro Being Wing Gly 50 55 60 l? ^? ÁryíaÁ? A. a ^^ r ^ .rA, .. and ..... *., «..., ... ^ ... ^ agg cat gtg cgg age tac aat falls ctt ca gga gat gtc cgc tgg aga 240 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 aag cta ttc tct tctc acc aag tac ttt etc aag att gag aag aac ggg 288 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys lie Glu Lys Asn Gly 85 90 95 aag gtc age ggg acc aag aag gag aac tgc ccg tac age ate ctg gag 336 10 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser lie Leu Glu 100 105 110 ata here tea gta gaa ate gga gtt gtt gcc gtc aaa gcc att aac age 384 lie Thr Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser 15 115 120 125 aac tat tac tta gcc atg aac aag aag ggg aaa etc tat ggc tea aaa 432 Asn Tyr Tyr Leu Wing Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 20 gaa ttt aac aat gac tgt aag ctg aag gag agg ata gag gaa aat gga 480 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly 145 150 155 160 tac aat tat tat gca tea ttt aac tgg cag cat aat ggg agg caa atg 52 8 25 Tyr Asn Thr Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 tat gtg gca ttg aat gga aaa gga gct cea agg aga gga cag aaa here 576 Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 30 180 185 190 cga agg aaa aat accctct gct falls ttt ctt cea atg gtg gta falls tea 624 Arg Arg Lys Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 195 200 205 35 tag 627 < 210 > 2 40 < 211 > 208 < 212 > PRT < 213 > Homo sapiens < 400 > 2 45 Met Trp Lys Trp He Leu Thr His Cys Wing Ser Wing Phe Pro His Leu 1 - 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20. .25 30 50 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Wing Tnr Asn Being Ser Being Being Phe Being Pro Pro Being Wing Gly 50 55 60 5 Arg HIS Val Arg Being Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 10 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu 100 105 110 He Thr Ser Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser 15 H5 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 20 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Wing Being Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 25 Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 30 195 2Q0 205 < 210 > 3 < 211 > 35 < 212 > DNA -3 > -5 '< 213 > Homo sapiens < 400 > 3 ggaccctcat gacctgccag gctctgggtc aggac 35 40 < 210 > 4 < 211 > 28 < 212 > DNA < 213 > Homo sapiens 45 < 400 > 4 ggacagccat ggctggtcgt cacgttcg 28 < 210 > 5"50 < 211 > 29 A • A?, YES * A? * »^ JfcA gfa ^ J ^ M» l «ME? < Mt? ^ * C? .M «J.» ». ^ IcA., ÍMAia ^ McJt-lt) ^« - »! ?? kc? To-il.- -rifata J * 3 < 212 > DNA < 213 > Homo sapiens < 400 > 5 ggacagccat ggttcgttgg cgtaaactg 29 < 210 > 6 < 211 > 31 < 212 > DNA < 213 > Homo sapiens < 400 > 6 ggacagccat ggaaaaaaac ggtaaagttt c 31 < 210 > 7 < 211 > 29 < 212 > DNA < 213 > Homo sapiens < 400 > 7 ggacccccat ggagaactgc ccgtagagc 29 < 210 > 8 < 211 > 32 < 212 > DNA < 213 > Homo sapiens < 400 > 8 ggacccccat ggtcaaagcc attaacagca ac 32 < 210 > 9 < 211 > 33 < 212 > DNA < 213 > Homo sapiens < 400 > 9 ggacccccat ggggaaactc tatggctcaa aag 33 < 210 > 10 < 211 > 37 < 212 > DNA < 213 > Homo sapiens < 400 > 10 ctgcceaagc ttattatgag tgtaccacca ttggaag 37 < 210 > 11 < 211 > 36 < 212 > A D N < 213 > Homo sapiens < 400 > 11 ctgcccaagc ttattacttc agcttacagt cattgt 36 < 210 > 12 < 211 > 32 < 212 > DNA < 213 > Homo sapiens < 400 > 12 gcggcacatg tcttacaacc acctgcaggg tg 32 < 210 > 13 < 2 H > 28 < 212 > A D N < 213 > Homo sapiens < 400 > 13 gggcccaagc ttatgagtgt accaccat 28 < 210 > 14 < 211 > 36 < 2? 2 > DNA < 213 > Homo sapiens < 400 > 14 ccggcggatc ccatatgtct tacaaccacc tgeagg 36 < 210 > 15 < 211 > 35 < 212 > DNA < 213 > Homo sapiens < 400 > 15 ccggcggtac cttattatga gtgtaccacc attgg 35 < 210 > 16 < 211 > 426 < 212 > DNA < 213 > Homo sapiens < 400 > 16 accacctgca atgtcttaca cgttggcgta gggtgacgtt tttcaccaaa aactgttctc 60 aaatcgaaaa tacttcctga aaaeggtaaa gtttetggga ccaagaagga gaactgcccg 120 tacagcatcc tggagataac atcagtagaa ateggagttg ttgccgtcaa agecattaac 180 ageaactatt aettagecat gaacaagaag gggaaactct atggctcaaa agaatttaac 240 agctgaagga aatgactgta gaggatagag gaaaatggat acaataecta tgcatcattt 300 aactggcagc ataatgggag gcaaatgtat gtggcattga atggaaaagg agetecaagg 360 10 agaggacaga aaacacgaag gaaaaacacc tctgctcact ttcttccaat ggtggtacac 420 tcataa 426 15 < 210 > 17 < 211 > 141 < 212 > PRT < 213 > Homo sapiens 20 < 400 > 17 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 25 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 30 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 35 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 40 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys 115 120 125 45 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 50 < 210 > 18 < 211 > twenty < 212 > DNA < 213 > Homo sapiens < 400 > 18 caaccacctg cagggtgacg 20 < 210 > 19 < 211 > 78 < 212 > DNA < 213 > Homo sapiens < 400 > 19 aacggtcgac aaatgtatgt ggcactgaac ggtaaaggtg ctccacgtcg tggtcagaaa 60 acccgtcgta aaaacacc 78 < 210 > 20 < 211 > 76 < 212 > DNA < 213 > Homo sapiens < 400 > 20 gggcccaagc ttaagagtgt accaccattg gcagaaagtg agcagaggtg tttttacgac 60 gggttttctg accacg 76 < 210 > 21 < 211 > 23 < 212 > DNA < 213 > Homo sapiens < 400 > 21 gccacataca tttgtcgacc gtt 23 < 210 > 22 < 211 > 19 < 212 > DNA < 213 > Homo sapiens < 400 > 22 gggcccaagc ttaagagtg 19 < 210 > 23 < 211 > 23 < 212 > DNA < 213 > Homo sapiens < 400 > 23 gccacataca tttgtcgacc gtt 23 < 210 > 24 5 < 211 > 90 < 212 > DNA < 213 > Homo sapiens < 400 > 24 '"ctgcagggtg acgttcgttg gcgtaaactg ttctccttca ccaaatactt cctgaaaatc 60 gaaaaaaacg gtaaagtttc tggtaccaag 90 15 < 210 > 25 < 211 > 90 < 212 > DNA < 213 > Homo sapiens 0 < 400 > 25 agetttaaca gcaacaacac cgatttcaac ggaggtgatt tccaggatgg agtacgggca 60 gttttctttc ttggtaccag aaactttacc 90 25 < 210 > 26 < 211 > 90 < 212 > DNA < 213 > Homo sapiens 30 < 400 > 26 ggtgttgttg ctgttaaagc tatcaactcc aactactacc tggctatgaa caagaaaggt 60 aaactgtacg gttccaaaga atttaacaac 90 35 < 210 > 27 < 211 > 100 < 212 > DNA < 213 > Homo sapiens 40 < 400 > 27 gtcgaccgtt gtgctgccag ttgaaggaag cgtaggtgtt gtaaccgttt tettegatac 60 gttctttcag tttacagtcg ttgttaaatt ctttggaacc 100 45 < 210 > 28 < 211 > 25 < 212 > DNA -30 < 213 > Homo sapiens ? Jh - * AJ «fc.Aa« to «^ < 400 > 28 gcggcgtcga ccgttgtgct gccag 25 5 < 210 > 29 < 211 > 26 < 212 > DNA < 213 > Homo sapiens 10 < 400 > 29 gcggcctgca gggtgacgtt cgttgg 26 < 210 > 30 15 < 211 > 36 < 1 > DNA < 213 > Homo sapiens < 400 > 30 20 ccggcggatc ccatatgtct tacaaccacc tgeagg 36 < 210 > 31 < 211 > 34 25 < 212 > DNA < 213 > Homo sapiens < 400 > 31 cgcgcgatat cttattaaga gtgtaccacc attg 34 30 < 210 > 32 < 211 > 426 < 212 > DNA 35 < 213 > Homo sapiens < 400 > 32 atgtcttaca accacctgca gggtgacgtt cgttggcgta aactgttctc cttcaccaaa 60 40 tacttcctga aaatcgaaaa aaaeggtaaa gtttctggta ccaagaaaga aaactgcccg 120 tactccatcc tggaaatcac ctccgttgaa atcggtgttg ttgctgttaa agetatcaac 180 tccaactact acctggctat gaacaagaaa ggtaaactgt acggttccaa agaatttaac 240 45 aacgactgta aactgaaaga acgtatcgaa gaaaaeggtt acaacaccta cgcttccttc 300 aactggcagc acaacggtcg acaaatgtat gtggcactga acggtaaagg tgctccacgt 360 50 cgtggtcaga aaacccgtcg taaaaacacc tctgctcact ttctgccaat ggtggtacac 420 i? ? .? I am I. tcttaa 426 < 210 > 33 < 211 > 141 < 212 > PRT < 213 > Homo sapiens < 400 > 33 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser He Leu Glu He Thr Ser 35 40 45 Val Glu He Gly Val Val Ala Val Lys Ala He Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg He Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Wing Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Wing 100 105 110 Leu Asn Gly Lys Gly Wing Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Wing His Phe Leu Pro Met Val Val His Ser 130 135 140 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (78)

1. CLAIMS 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 about pH 5.0 to about 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; and (d) a preservative selected from the group consisting of m-cresol, chlorobutanol, and a mixture of methylparaben and propylparaben; or a reaction product thereof. 2. The pharmaceutical composition according to claim 1, characterized in that it further comprises one or more of: (a) a chelating agent at a concentration range of from about 0 mM to about 10 mM; and (b) a tonifier at a concentration range of from about 0 mM to about 150 mM. 3. The pharmaceutical composition according to claim 2, characterized in that the tonifier is selected from the group consisting of sodium chloride, glycine, sucrose, mannitol, and mixtures thereof. 4. The pharmaceutical composition according to claim 1, further characterized in that it comprises one of: 1. about 0.5% to about 2% w / v glycerol, 2. about 0.1% to about 1% w / v methionine, or 3. about 0.1% to about 2% w / v of monothioglycerol. 5. 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). 6. The pharmaceutical composition according to claim 5, characterized in that the KGF-2 polypeptide is present in a concentration range of about 0.1 to about 20 mg / ml (w / v). 7. The pharmaceutical composition according to claim 6, characterized in that the KGF-2 polypeptide is present in a concentration range of about 0.2 to 4 mg / ml. • The pharmaceutical composition according to claim 1, characterized in that the KGF-2 polypeptide is KGF-2-? 33. 9. The pharmaceutical composition according to claim 1, characterized in that the diluent is water. 10. The pharmaceutical composition according to claim 2, characterized in that the chelating agent is EDTA at a concentration of about 1 mM, and the toning agent is present at a concentration of about 125 mM. 11. The pharmaceutical composition according to claim 1, characterized in that the pH is from about pH 5.5 to about pH 6.5. 12. The pharmaceutical composition according to claim 11, characterized in that the pH is about pH 6.0. The pharmaceutical composition according to claim 1, characterized in that the buffer is selected from the group consisting of phosphonic, acetic, aconitic, citric, glutaric, malic, succinic, carbonic acid, and an alkali metal or alkaline earth metal salt of the same. 14. The pharmaceutical composition according to claim 13, characterized in that the buffer is a phosphate, acetate or citrate salt. 15. The pharmaceutical composition according to claim 13, characterized in that the buffer is a citrate salt. 16. 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. 17. The pharmaceutical composition according to claim 16, characterized in that the buffer is a citrate salt present in a concentration of about 10 mM to about 20 mM. 18. 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. 19. The pharmaceutical composition according to claim 1, characterized in that the composition is maintained at a temperature of or below -20 ° C. 20. The pharmaceutical composition according to claim 1, characterized in that the KGF-2? 33 polypeptide is selected from the group consisting of a KGF-2? 33 polypeptide having an N-terminal methionine, the polypeptide KGF-2? 33 lacks an N-terminal methionine, and a mixture thereof. 21. The pharmaceutical composition according to claim 1, characterized in that it also comprises a bulking agent. 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 22, characterized in that the volume agent is sucrose or a mixture of sucrose and glycine. 24. The pharmaceutical composition according to claim 2, characterized in that it also comprises a bulking agent. 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 phosphonic, acetic, aconitic, citric, glutaric, malic, succinic, carbonic acids, and an alkali metal or alkaline earth metal salt of - the same. 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 approximately 290 mOsm. **? kiu »? The pharmaceutical composition according to claim 21, characterized in that the KGF-2 polypeptide is KGF-2-? 33. 35. The pharmaceutical composition according to claim 34, characterized in that the polypeptide KGF-2? 33 is selected from the group consisting of the KGF-2? 33 polypeptide having an N-terminal methionine, the KGF-2? 33 polypeptide lacking an 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% to about 2% w / v monothioglycerol, b) about 0.01% to about 2% w / v ascorbic acid, c) about 0.01% to about 2% w / v methionine or d) combinations thereof. 40. The pharmaceutical composition according to claim 1, 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. 41. The pharmaceutical composition according to claim 40, characterized in that the thickening agent is present in an amount effective to raise the viscosity to about 50 to about 1,000 cps. 42. The pharmaceutical composition according to claim 41, characterized in that the thickening agent is in an effective amount to raise the viscosity to about 200 to about 300 cps. 43. The pharmaceutical composition according to claim 40, characterized in that the thickening agent is present in a concentration of 0 to 5% (w / w). 44. The pharmaceutical composition according to claim 40, characterized in that the thickening agent is an etherified cellulose soluble in water or a high molecular weight polymer of acrylic acid crosslinked with allylucrose or an allyl ether of pentaerythritol. 45. The pharmaceutical composition according to claim 44, characterized in that the etherified cellulose is an alkylcellulose, hydroxyalkylcellulose, carboxyalkylcellulose or alkylhydroxyalkylcellulose. 46. The pharmaceutical composition according to claim 40, characterized in that the etherified cellulose is methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose. 47. The pharmaceutical composition according to claim 46, 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. 48. The pharmaceutical composition according to claim 47, 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. 49. The pharmaceutical composition according to claim 42, characterized in that the buffer is ^^. ? kfaffl citrate at a concentration of about 10 mM to about 50 mM. 50. The pharmaceutical composition according to claim 49, characterized in that the buffer is citrate in a concentration of about 10 mM to about 20 mM citrate. 51. The pharmaceutical composition according to claim 49, characterized in that the volume agent is sucrose in a concentration of about 0.01% to about 5% sucrose. 52. The pharmaceutical composition according to claim 51, characterized in that the thickening agent is directly added to a liquid formulation and thereafter lyophilized. 53. The pharmaceutical composition according to claim 51, characterized in that the thickening agent is added to a lyophilized formulation by reconstituting the formulation by the addition of a suitable diluent having a thickening agent dissolved therein. 54. 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. 55. The composition according to claim 1, characterized in that it further comprises a gelling agent in an amount effective to raise the viscosity from about 0.1 to about 10,000 cps at room temperature. 56. The composition according to claim 21, characterized in that it further comprises a gelling agent in an amount effective to raise the viscosity from about 0.1 to about 10,000 cps at room temperature. 57. The composition according to claim 55, characterized in that the gel-forming agent is a water-soluble polymer capable of forming a viscous aqueous solution, or a water-swellable, water-swellable polymer capable of forming a solution viscose. 58. The composition according to claim 57, 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. 59. The composition according to claim 58, characterized in that the gel-forming agent is (1) a vinyl polymer selected from the group it consists of polyacrylic acid, polymethacrylic acid, polyvinylpyrrolidone, polyvinyl alcohol, and salts and esters thereof; or (2) a polysaccharide selected from the group consisting of a cellulose derivative, a glycosaminoglycan, agar, pectin, alginic acid, dextran, α-amylose, amylopectin, chitosan, and salts and esters thereof. 60. The composition according to claim 58, characterized in that the gel-forming agent is a glycosaminoglycan selected from the group consisting of hyaluronic acid, chondroitin, 4-chondroitin sulfate, heparan sulfate, heparin and salts and esters of the same. 61. The composition according to claim 60, characterized in that the glycosaminoglycan is present in combination with collagen, gelatin, or fibronectin. 62. The composition according to claim 58, characterized in that the gel-forming agent is an acrylamide polymer selected from the group consisting of a polyacrylamide or a polymethacrylamide. 63. The composition according to claim 58, characterized in that the gel-forming agent is a polyoxyethylene-polyoxypropylene block copolymer. 64. The composition according to claim 63, 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. 65. The composition according to claim 64, 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. 66. 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. 67. 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). 68. The pharmaceutical composition according to claim 67, characterized in that the KGF-2 polypeptide has an N-terminal methionine, lacks an N-terminal methionine, or is a mixture thereof. 69 The pharmaceutical composition according to claim 1, characterized in that the KGF-2 polypeptide is an N-terminal or C-terminal deletion mutant selected from the group consisting of Ala (39) -Ser (208); Pro (47) - Ser (208); Val (77) - Ser (208); Glu (93) - Ser (208); Glu (104) - Ser (208); Val (123) -Ser (208); Gly (138) - Ser (208); Met (1), Thr (36); and Cys (37) - Lys (153). 70. The pharmaceutical composition according to claim 69, characterized in that the KGF-2 polypeptide has an N-terminal methionine, lacks an N-terminal methionine, or is a mixture thereof. 71. The pharmaceutical composition according to claim 1, characterized in that it further comprises one of: (a) lysine; (b) hydroxypropyl-β-cyclodextrin; and (c) sulfated β-cyclodextrin; or combinations thereof. 72. The pharmaceutical composition according to claim 1, characterized in that the preservative is a mixture of methylparaben and propylparaben. 73. The pharmaceutical composition according to claim 72, characterized in that the composition comprises 0.18% methylparaben and 0.02% propylparaben. > * * * - * - »- '* -»'. * »*» • "" »» tfH «fc < te * «-» 74. A pharmaceutical composition, characterized in that it comprises: (a) approximately 1.0 mg / ml KGF-2; (b) 20 mM citrate, pH 5-5.5; and (c) 0.01% polysorbate 80. 75. The pharmaceutical composition according to claim 74, further characterized in that it comprises 1 mM EDTA. 76. A pharmaceutical composition, characterized in that it comprises: (a) approximately 3.3 mg / ml KGF-2; (b) 10 mM sodium citrate; (c) 20 mM sodium chloride; (d) 1 mM EDTA; (e) 2% w / v glycine; (f) 0.5% w / v sucrose; (g) water; and (h) pH about 6.2; or a reaction product thereof. 77. The pharmaceutical composition according to claim 77, characterized in that more than 90% of water is removed by lyophilization. 78. A pharmaceutical composition, characterized in that it comprises: (a) about 1.0 mg / ml KGF-2; ? 1 L. (b) 0.46% hydroxyethylcellulose; (c) 7% sucrose; (d) 20 mM sodium citrate; (e) 20 mM sodium chloride; (f) 1 mM EDTA; and (g) pH about 6.2; or the reaction products thereof