KR20020010920A - Keratinocyte growth factor-2 formulations - Google Patents

Abstract

The present invention relates to liquid and lyophilized preparations of keratinocyte growth factor (KGF-2) and derivatives thereof. The present invention also relates to a formulation of KGF-2 for therapeutic use, for example, to promote or accelerate wound healing.

Description

[0001] KERATINOCYTE GROWTH FACTOR-2 FORMULATIONS [0002]

Fibroblast growth factor family was found as a large family of growth factors related to soft tissue growth and regeneration. This includes several members that share varying degrees of homology at the current protein level, with one exception being seen to have a similar broad myotene range, that is, they have different cell differentiation of mesodermal and neuroectodermal origin ≪ / RTI > and / or promotes angiogenesis.

KGF was first identified as a member of the FGF family by sequence homology or by factoring and cloning. Keratinocyte growth factor (KGF) was isolated as mitogen from cultured murine keratinocyte lines (Rubin, JS et al . , Proc. Natl. Acad. Sci. USA 86: 802-806 (1989). Unlike other members of the FGF family, KGF promotes epithelial cell growth with little activity against cells derived from hepatic insufficiency. The keratinocyte growth factor is produced by fibroblasts derived from skin and fetal lungs (Rubin et al. (1989)]. The keratinocyte growth factor mRNA was expressed in adult kidney, colon and iliac, but not in brain or lung (Finch, PW et al., Science 245: 752-755 (1989)). KGF represents a conserved region within the FGF protein family. KGF binds with high affinity for the FGF-2 receptor.

Healing of damaged wounds is an important source of morbidity, and can lead to complications such as degenerative, mucinous, and unhealed wounds. In normal individuals, wound healing does not cause complications. In contrast, impaired healing is accompanied by some symptoms 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 97: 631 (1985); Irvin, GL et al . , Am. Surg. 51: 418 (1985)).

Scar repair is the result of complex interactions and biological processes. Normal wound healing has been described in three steps: an acute inflammatory phase, an exogenous matrix and collagen synthesis and reconstitution (Peacock, E. E., Jr., Wound Repair (2nd Ed.), Philadelphia WB Saunders (1984)). This process involves the interaction of keratinocytes, fibroblasts and inflammatory cells at the wound site.

(1) stable for extended storage periods, (2) increase the pharmacological activity or efficacy of the polypeptide, and / or (3) facilitate the application or administration of the polypeptide in therapeutic regimens. Lt; RTI ID = 0.0 > and / or < / RTI >

The present invention relates to liquid preparations and lyophilized preparations of keratinocyte growth factor-2 (KGF-2) and derivatives thereof. The present invention also relates to KGF-2 preparations, in particular topical preparations and injectable preparations which can be used for therapeutic purposes in conditions requiring soft tissue growth and regeneration.

Figures 1A-1C show the cDNA of KGF-2 and the corresponding deduced amino acid sequence. The first 35 or 36 amino acid residues represent the putative leader sequence (underlined). Amino acids are indicated by the standard single letter abbreviation. The inaccuracy of sequencing is a common problem encountered when attempting to determine the polynucleotide sequence. Sequence determinations were performed using a 373 automated DNA sequencer (Applied Biosystems, Inc.). The accuracy of sequencing is expected to be more than 97% accurate (SEQ ID NOS: 1 and 2).

Figures 2A-2C show that the KGF-2 polypeptide of the present invention stimulates normal primary epidermal keratinocyte proliferation. Figure 2A shows the promotion of normal epithelial keratinocyte proliferation by KGF-2. Figure 2B shows the promotion of normal primary epidermal keratinocyte proliferation by KGF-2? 33. Figure 2C shows the promotion of normal primary epidermal keratinocyte proliferation by KGF-2? 28.

Figure 3 shows the viability results for the KGF-2 [Delta] 33 liquid formulation, 10 month stability.

Figure 4 shows the viability results for KGF-2? 33 lyophilized formulation, 9 month stability.

Figure 5 shows the effect of monothiol glycerol on KGF-2 viability.

DETAILED DESCRIPTION OF THE INVENTION

KGF-2 promotes the proliferation of epithelial keratinocytes, but does not promote the proliferation of insect tissue cells such as fibroblasts. Thus, " a polypeptide having KGF-2 protein-like activity " includes a polypeptide which exhibits KGF-2 activity in a keratinocyte proliferation assay described below and binds to 1-iiib and 2-iiib which are in the same FGF receptor form.

The present invention relates to pharmaceutical and veterinary preparations of KGF-2 polypeptides. KGF-2 polypeptide refers to a polypeptide encoded by the polypeptide of Figure 1 (SEQ ID NO: 2) or the deposited cDNA for reference herein and includes fragments, derivatives and analogs of the polypeptide of Figure 1 (SEQ ID NO: 2) Or polypeptides encoded by the deposited cDNA, which possess essentially the same biological function as the parent polypeptide. The polypeptides used in the present invention may include recombinant polypeptides, natural or synthetic polypeptides, preferably recombinant polypeptides.

The KGF-2 polypeptide was found to be poor in activity and stability at pH below 4.5 or at about pH 8.0. The present inventors have confirmed that the KGF-2 polypeptide is oxidized and settled. Formulating these polypeptides for therapeutic use has been challenging in many respects. In order to maintain physiochemical properties and biological activity, the KGF-2 polypeptide may be combined with an antioxidant such as an oxygen scavenger compound and / or a protein stabilizer such as a thiol-containing compound and / or a metal chelating agent, Can be formulated with EDTA. As used herein, the term " stabilization " means that the physiochemical properties and substantial biological activity of the KGF-2 polypeptide are maintained for a given period of time.

The formulations of the present invention include gels, thickened solutions, solutions, and lyophilized forms. The term " pharmaceutical composition " or " composition "

Injectable preparation

Liquid formulation

A first aspect of the invention is directed to a liquid formulation of a KGF-2 polypeptide comprising a KGF-2 polypeptide and a pharmaceutically acceptable salt thereof, between about pH 5.0 and about pH 8.0, more preferably between pH 5.5 and pH 6.5, and a buffer having a buffering capacity at pH 6.2. Useful buffering agents include buffers derived from phosphate, acetic acid, aconitic acid, citric acid, glutaric acid, malic acid, succinic acid and carboxylic acid. Commonly used are the alkali or alkaline earth salts of one of the abovementioned acids. A more preferred buffer is acetate or citrate, and the most preferred buffer is citrate. For example, the formulation comprises a composition formed by admixing a buffered amount of citric acid or a pharmaceutically acceptable salt thereof with KGF-2? 33 in water. Alternatively, the preparation may comprise a composition formed by admixing a buffered amount of acetic acid, or a pharmaceutically acceptable salt thereof, with KGF-2? 33 in water. A preferred buffer concentration is from about 5 mM to about 50 mM. The most preferred concentration of acetate buffer will be about 20 mM, and the most preferred concentration of citrate buffer will be from about 10 mM to about 20 mM. The formulation may also contain NaCl, glycine, sucrose or mannitol or a mixture thereof as a tonicifier at a concentration of about 0 mM to about 150 mM, preferably 10 mM to about 150 mM, most preferably about 125 mM And includes a metal chelating agent, for example EDTA, at a concentration of from about 0 mM to about 10 mM, most preferably at about 1 mM. The liquid preparations of the present invention may also contain one or more stabilizing amounts of antioxidants (e.g., ascorbate) and / or (b) protein stabilizing amounts of thiol-compounds (e.g., monothiol glycerol (MTG) . Without wishing to be bound by theory, it is believed that thiol compounds such as MTG act to protect the free sulfhydryl groups present in the KGF-polypeptide. Storage conditions for the liquid formulation are generally about 2 < 0 > C to about 8 < 0 > C. Alternatively, the storage conditions may be -20 占 폚 or lower. The most preferred storage condition is about -20 < 0 > C. Maintaining the KGF-2 liquid formulation in the frozen state limits the amount of oxidation to the polypeptide to be produced with a stable polypeptide preparation.

Liquid formulations

(1) a therapeutically effective amount of a KGF-2 polypeptide;

(2) an effective amount of a buffer having a buffering capacity of between about pH 5.0 and about pH 8.0; And

(3) a pharmaceutically acceptable carrier;

Lt; / RTI >

(4) As required

(a) isotonic agents such as NaCl, glycine, sucrose or mannitol or a combination thereof,

(b) a chelating agent,

(c) stabilizing amount of antioxidant

(d) a stabilizing amount of a protein stabilizing agent

≪ / RTI >

The KGF-2 polypeptide is preferably liberated in solution.

The compositions of the present invention are prepared by mixing the above ingredients together, preferably in a concentration to weight ratio as referred to herein.

Antioxidants that can be used in the liquid preparation include sodium bisulfate, cysteine, sodium sulfite, ascorbic acid, tocopherol, and butylated hydroxyanisole. Stabilizers that can be used in the liquid preparation include thiols, for example, cysteine, methionine and thioglycerol. Examples of chelating agents which can be used include ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DPTA), but EDTA is preferably used.

Antioxidants or thiols may increase the stability of the KGF-2 polypeptide. This makes it possible to produce drugs that can be stored for a longer period of time.

In addition, the formulations of the present invention preferably contain at least one preservative at a concentration of from about 0.5% to about 1.5%, most preferably about 0.9%, for example, benzyl alcohol; Preferably from about 0.01% to about 1%, most preferably about 0.5%, of chlorobutanol; Preferably from about 0.1% to about 0.2%, most preferably 0.18% methyl paraben; M-cresol, preferably at a concentration of from about 0.01% to about 0.05%, most preferably at least about 0.02% propyl paraben; preferably from about 0.1% to about 1%, most preferably at about 0.3%; And / or preferably from about 0.1% to about 1%, most preferably about 0.5%, of phenol. Especially preferred is the use of methylparaben and propylparaben together; Methylparaben is used in a concentration of about 0.1% to about 0.2%, and propylparaben is used in a concentration of about 0.10% to about 0.05%. Most preferred is a combination of methyl paraben and propyl paraben, methyl paraben is used in a concentration of 0.18%, and propyl paraben is used in a concentration of 0.02%.

A more preferred liquid composition comprises

(W / v), more preferably about 0.05 to about 30 mg / ml (w / v), even more preferably about 0.1 to about 20 mg / (w / v), even more preferably about 10 mg / ml (w / v), most preferably about 0.2-4 mg / ml;

(2) a buffer having a buffering capacity of between about pH 5.0 and about pH 8.0 with a concentration range of from about 5 mM to about 50 mM, preferably from about 5 mM to about 30 mM;

(3) a pharmaceutically acceptable diluent, preferably water

.

Buffering agents useful for the formulations of the present invention include buffers derived from acetic acid, aconitic acid, citric acid, glutaric acid, malic acid, succinic acid, phosphate, and carboxylic acid. Alkali or alkaline earth salts of one of these acids are generally used. Acetate and citrate buffers, such as acetic acid or a pharmaceutically acceptable salt thereof, are preferred. A preferred pH range for the solution preparation is 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 is the preferred buffer, and sodium citrate is the most preferred buffer.

To the solution was added

(4) a chelating agent, such as EDTA, wherein the concentration range is from about 0.1 mM to about 10 mM, more preferably about 1 mM;

(5) an isotonic agent such as NaCl, glycine, sucrose or mannitol, or a combination thereof, wherein the concentration range is from about 0.01 mM to about 150 mM, more preferably about 125 mM

Is further added.

If desired, the liquid formulation may comprise

(a) from about 0.5% to about 2% w / v glycerol;

(b) from about 0.1% to about 1% w / v methionine; or

(c) from about 0.1% to about 2% w / v monothioglycerol

As a protein stabilizing amount.

A preferred embodiment of this aspect of the invention

(1) the concentration ranges from about 0.02 to about 40 mg / ml (w / v), more preferably from about 0.1 to about 20 mg / ml (w / v), and most preferably from about 0.2 to 4 mg / KGF-2 polypeptide;

(2) 10 mM sodium citrate or 20 mM sodium acetate;

(3) 125 mM NaCl;

(4) 1 mM EDTA; And

(5) Water as a diluent

≪ / RTI >

More preferably, the solution formulation comprises

(1) about 0.2 to about 4 mg / ml KGF-2 polypeptide;

(2) 20 mM sodium acetate;

(3) 125 mM NaCl;

(4) 1 mM EDTA; And

(5) Water as a diluent

≪ / RTI >

Most preferably, the solution formulation comprises

(1) about 1.0 mg / ml KGF-2 polypeptide;

(2) 20 mM citrate, pH 5-5.5; And

(3) 0.01% polysorbate 80

≪ / RTI >

In addition, the solution comprises about 7% sucrose or a combination of 2% glycine and 0.5% sucrose.

Alternatively, the solution formulation may comprise

(1) about 1.0 mg / ml KGF-2 polypeptide;

(2) 20 mM citrate, pH 5-5.5;

(3) 1 mM EDTA; And

(4) 0.01% polysorbate 80

≪ / RTI >

In addition, the solution comprises about 7% sucrose or a combination of 2% glycine and 0.5% sucrose.

The present inventors have confirmed that the KGF-2 polypeptide easily oxidizes, aggregates, and precipitates out of solution. Oxidation of KGF-2 does not destroy the biological activity, and limiting the degree of oxidation of the product can result in a more stable product. The present inventors have observed that when the liquid preparation is present at too low a pH, the KGF-2 polypeptide loses its biological activity. Also, as the pH of the solution approaches the pI of KGF-2, the protein will precipitate out of solution. Thus, the present inventors have confirmed that the liquid formulation should be maintained within the range of about pH 6.0 to about pH 7.0, and that the pH of about 6.2 is the optimum pH for stabilizing the KGF-2 polypeptide. Also, surprisingly, the present inventors have also found that the citrate buffer stabilizes the KGF-2 polypeptide in particular.

The use of a citrate buffer at about pH 6.0-6.2 provides a liquid formulation with reduced aggregation of KGF-2 polypeptide and improved stability, but the liquid polypeptide formulation may still undergo oxidation and precipitation processes of the KGF-2 polypeptide have. Thus, the present inventors have developed a lyophilized preparation as described below.

The lyophilized preparation

A second aspect of the invention is directed to a lyophilized formulation of a KGF-2 polypeptide comprising a KGF-2 polypeptide and a pharmaceutically acceptable salt thereof, at a pH of from about 5.0 to about pH 8.0, more preferably at a pH of from 5.5 to pH 6.5, and a buffer having a buffering capacity of pH 6.2. Useful buffers include buffers derived from phosphate, aconitic acid, citric acid, glutaric acid, malic acid, succinic acid and carboxylic acid. Alkali or alkaline earth salts of one of the above acids are generally used. More preferably, the buffer is phosphate or citrate, most preferably citrate. For example, the formulation may comprise a composition formed by mixing KGF-2? 33 in water with a buffered amount of citric acid or a pharmaceutically acceptable salt thereof. A preferred concentration of the buffer is from about 5 mM to about 50 mM, more preferably about 10 mM. Most preferably the concentration of cysteate buffer will be about 10 mM. Also included within the formulation are NaCl from about 0 mM to about 150 mM, most preferably about 20 mM as isotonizing agent, a metal chelating agent such as EDTA at about 0 mM to about 10 mM, and most preferably at about 1 < RTI ID = mM. < / RTI > Bulk / cold protectants such as sucrose, glycine, mannitol, trehalose or other pharmaceutically acceptable bulking agents may also be included in the formulation. The amount of bulking agent used is the amount of isotonicity of the solution, which is from about 2% to about 10% w / v. Preferred concentrations are: 5% mannitol, 7% sucrose, 8% trehalose, or 2% glycine + 0.5% sucrose. It is more preferred to use a sucrose or sucrose / glycine mixture. The lyophilized formulation of the present invention may also comprise one or more of (a) a stabilizing amount of an antioxidant, such as ascorbate, or (b) a stabilizing amount of a thiol-compound, such as monothioglycerol . Storage conditions of the lyophilized preparation are generally about 2 < 0 > C to about 25 < 0 > C. More preferably, the storage conditions are less than about 2 < 0 > C to about 8 < 0 > C.

The KGF-2 polypeptide is lyophilized in an initial solution at a protein concentration of about 0.02 mg / ml to about 10 mg / ml.

The initial freeze-dried solution (in addition to the KGF-2 polypeptide)

(1) an effective amount of citric acid or a pharmaceutically acceptable salt thereof, wherein the concentration range is from about 5 mM to about 20 mM, preferably sodium citrate;

(2) NaCl in a concentration range of from about 0 mM to about 125 mM;

(3) EDTA with a concentration range of from about 0 mM to about 10 mM;

(4) one or more sucrose, mannitol, glycine, or trehalose, or mixtures thereof, having a concentration range of from about 2% w / v to about 15% w / v;

(5) Water

.

A preferred pH range for lyophilized buffer is about 5.5 to about 8.0, preferably about pH 6.2.

More preferably, the lyophilized buffer comprises 10 mM sodium citrate, 20 mM sodium chloride, 1 mM 2 Sodium EDTA (pH 6.2) and 7% sucrose.

The lyophilized KGF-2 polypeptide preparation is reconstituted in sterile water to maintain an isotopic condition of about 290 mOsm. A) from about 0.01% to about 2% w / v monothioglycerol, b) from about 0.01% to about 2% w / v ascorbic acid, c) from about 0.01% About 0.01% to about 2% w / v methionine, or d) a combination thereof.

The present invention includes a lyophilization cycle yielding a stable KGF-2 polypeptide preparation. The lyophilization cycle is designed to maintain the KGF-2 polypeptide below the collapse temperature of the KGF-2 polypeptide during the primary drying step. Also, the wet content is preferably less than 5%, more preferably less than 2%. Such a protocol must be determined for any particular protein according to individual criteria. An exemplary lyophilization cycle for a lyophilized formulation containing KGF-2 sucrose according to the present invention was determined as follows:

Temperature (℃) Pressure (mTorr) Time (minutes) 5 (keep) Atmospheric pressure 60 5 to -45 (descent) Atmospheric pressure 120 -45 (keep) Atmospheric pressure 120 -45 (keep) 75 to 100 60 -45 to -20 (rise) 75 to 100 125 -20 (keep) 75 to 100 2100 -20 to +25 (rise) 75 to 100 225 -25 (keep) 75 to 100 1020

Another exemplary lyophilization cycle for KGF-2 lyophilized formulations according to the present invention was determined as follows:

Temperature (℃) Pressure (mTorr) Time (minutes) 5 (keep) Atmospheric pressure 60 5 to -45 (descent) Atmospheric pressure 120 -45 (keep) Atmospheric pressure 120 -45 (keep) 75 to 100 180 -45 to -30 (rise) 30 120 -30 (keep) 30 4200 -20 to +25 (rise) 100 60 -25 (keep) 100 960

The lyophilized formulations of the present invention provide products that have unexpected increased stability. Indeed, the lyophilized KGF-2 formulations of the present invention are biologically stable for at least 9 months at a temperature of less than 45 ° C (FIG. 4). As can be seen from the reversed phase HPLC, the lyophilized KGF-2 formulations of the present invention maintained their physico-chemical properties until August at a temperature of less than 45 ° C and 75% relative humidity. It is very unusual for proteins to maintain their activity over a long period at such a high temperature.

Thickened formulations and gel formulations

A third aspect of the invention relates to thickened or gel formulations of KGF-2 polypeptides.

1) Thickener

Thickening agents may be added to the liquid formulation described above to increase the viscosity of the resulting formulation. Formulations with increased viscosity may be useful for topical administration where it may be important to avoid controlled release, attachment to the shape of the wound or run-off. Such thickened formulations are used for topical application, such as wound healing, or any other application that can be treated by topical administration of a KGF-2 pharmaceutical composition, to treat skin disorders.

The thickener should increase the viscosity from about 50 to about 10,000 centipoise (cps), more preferably from about 50 to about 1,000 cps and most preferably from about 200 to about 300 cps. The viscosity was measured by a rotary method using a spindle viscometer. The most preferred concentration of the thickener is 0 to 5% (w / w). The thickened solution will always remain liquid.

Examples of suitable thickeners include, but are not limited to, water-soluble etherified celluloses and carbomers (high molecular weight polymers of acrylic acid cross-linked with allyl esters of allyl sucrose or pentaerythritol). Examples of the etherified celluloses are known in the art (described in USP), and examples thereof include alkylcelluloses, hydroxyalkylcelluloses, and alkylhydroxyalkylcelluloses (e.g., methylcellulose, hydroxyethylcellulose, hydroxy Propylcellulose, hydroxypropylmethylcellulose and the like). In a further embodiment, the topical or incising gel may comprise from about 0 to about 20 weight percent 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 from about 2 to about 8 wt%, and the molecular weight range is from about 80,000 to about 240,000. Preferred cellulose derivatives are hydroxypropylmethylcellulose, methylcellulose, carboxymethylcellulose, and hydroxyethylcellulose.

When thickening agents are added to the injectable formulation, the salts and buffers described above are added to or removed from the formulation for optimal stability. For example, the citrate concentration can be increased. A preferred concentration of citrate is, for example, about 10 mM to 500 mM citrate, more preferably about 10 mM to about 50 mM citrate, and most preferably about 10 mM to about 20 mM citrate. In addition, the amount of sucrose may be reduced to within the range of about 0% to about 5% sucrose in the lyophilized formulation.

The thickening agent may be added directly to the liquid preparation according to the present invention and then lyophilized. Alternatively, the lyophilized formulations of the present invention may be reconstituted by the addition of a suitable diluent, most preferably water in which the thickener is dissolved. These thickened preparations can be administered by spraying.

An example of a preferred thickened KGF-2 polypeptide solution according to the present invention comprises

(1) a locally effective amount of a KGF-2 polypeptide, preferably KGF-2? 33;

(2) from about 10 mM to about 50 mM sodium citrate buffer;

(3) about 0.01 to about 150 mM NaCl;

(4) from about 0.75 to about 1.27 mM, preferably about 1 mM EDTA;

(5) from 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) carboxymethylcellulose or about 0.5 to about 1.5% hydroxypropylmethylcellulose or about 0.25 to about 0.75% hydroxyethylcellulose or about 0 to 1% ≪ / RTI >

And the like.

The pH of these formulations is most preferably pH 6.2.

2) Gelling agent

Another aspect of the invention relates to a gel formulation of a KGF-2 polypeptide. Gelling agents are added to the injectable formulations of the present invention to provide a liquid that is liquid at room temperature and solidifies when applied to the skin surface (about 37 DEG C). Such formulations may be useful for topical administration where it may be important to avoid controlled release, attachment to the shape of the wound or spillage. Such gel preparations are used for topical applications such as wound healing to treat skin disorders or any other application that can be treated by topical administration of KGF-2 pharmaceutical compositions.

The gel preparation for KGF-2 polypeptide according to the present invention

(1) a locally effective amount of a KGF polypeptide;

(2) buffering agents;

(3) a pharmaceutically acceptable diluent, preferably water; And

(4) Gel-forming high molecular weight compounds

.

The viscosity of the gel formulations of the present invention may range from about 1 to about 10,000 cps at room temperature, and most preferably from about 20 to about 100 cps at room temperature. The viscosity was measured by a rotary method using a spindle viscometer.

The gel-forming high molecular weight compounds used in the present invention generally comprise a water-soluble, water-swellable polymer capable of forming a viscous aqueous solution or water-soluble polymer or viscous solution and subsequently forming a gel upon contact with the skin For example, collagen).

Useful gel-forming high molecular weight compounds can be selected from vinyl polymers, polyoxyethylene-polyoxypropylene copolymers, polysaccharides, proteins, poly (ethylene oxide), acrylamide polymers and their derivatives and salts. Other compounds that can be used to prepare pharmaceutical gel preparations for use in wound healing can be found in U.S. Patent No. 5,427,778, which is incorporated herein by reference in its entirety.

Useful vinyl polymers (or substituted polyethylenes) include polyacrylic acid, polymethacrylic acid, polyvinyl pyrrolidone and polyvinyl alcohol. Useful polysaccharides include cellulose derivatives, glycosaminoglycans, agar, pectin, alginic acid, dextran, starch (alpha-amylose or amylopectin) and chitosan. Useful glycosaminoglycans include hyaluronic acid, chondroitin, chondroitin-4-sulfate, heparan sulfate and heparin. Glycosaminoglycan can be used with any other gel-forming polymer, for example, with any other gel-forming polymer, such as collagen, gelatin, fibronectin, to enhance wound healing. The acrylamide polymer may be a polyacrylamide or polymethacrylamide polymer.

Preferred high molecular weight gel-forming compounds are polyoxyethylene-polyoxypropylene block copolymers, in particular those block copolymers sold under the tradename Fluronics (BASF) or Pollockamer (BASF).

In one preferred embodiment, the gel of the present invention may comprise from about 10 to about 60 weight percent of a polyoxyethylene-polyoxypropylene block copolymer having an average molecular weight of about 500 to 50,000. In one preferred embodiment, the gel of the present invention may comprise from about 14 to about 18% of the block copolymer having a molecular weight range of from 1,000 to 15,000. Preferred block copolymers of the present invention are Pluronic F108 and Pluronic F127.

Polyoxyethylene-polyoxypropylene block copolymers (pluronic or poloxamer) have great potential for use in topical drug delivery systems because they exhibit opposite thermal gelation behavior and have good drug release properties And low toxicity. The gel is formed when the solution is warmed. Therefore, when the gel is brought into contact with an aqueous solution of low viscosity at room temperature or a mammalian body, the gel is heated by body temperature to increase viscosity as a solution gel. Pluronic gels can be used, for example, for controlled delivery of KGF-2 polypeptides to other sites where wound and topical delivery are desirable. The KGF-2 polypeptide can be combined with pluronic in the liquid state and can be applied to the wound. Gelling occurs and the rate at which the polypeptide is released into the wound is effectively reduced, allowing for an extended period of time between the polypeptide and the wound site. An advantage of using such a gel preparation is that it moisturizes the wound site and possesses a pharmaceutical compound that is formable at the wound site or other site where the compound can be applied.

Preferred gel formulations of KGF-2 polypeptides according to the present invention include citrate buffers and pluronic. The formulation may contain a certain amount of citric acid or a pharmaceutically acceptable salt thereof.

In addition, the gel preparation according to the present invention may contain a chelating agent, a stabilizing amount of an antioxidant or a thiol. The gel preparation may contain a high molecular weight compound, such as pluronic or water soluble etherified cellulose, in an amount to form a gel. In the gel preparation according to the present invention, it is preferred that the KGF-2 polypeptide has a concentration of about 0.01 mg / ml to about 10 mg / ml.

Preferably, the gel formulation comprises

(1) a KGF-2 polypeptide, preferably KGF-2? 33, wherein the final calculated concentration is from 0.01 mg / ml to about 10 mg / ml;

(2) an effective amount of a buffering agent;

(3) a polyoxyethylene-polyoxypropylene block copolymer having an average molecular weight of from about 10% to about 60%, more preferably from about 14% to about 18%, and from about 500 to 50,000;

(4) a pharmaceutically acceptable diluent, preferably water

.

Other preferred gel formulations include

(1) a pharmaceutically active amount of a KGF-2 polypeptide;

(2) from about 10 mM to about 500 mM sodium citrate;

(3) from about 0.01 mM to about 150 mM NaCl;

(4) about 1 mM EDTA

(5) from 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 pH of the formulation is about pH 6.2.

Most preferably,

(1) a KGF having a concentration range of from about 0.01 mg / ml to about 10 mg / ml (w / v), more preferably from about 0.1 mg / ml to about 3 mg / ml, and most preferably about 0.2 mg / -2 < / RTI > polypeptide, preferably KGF-2? 33;

(2) a sodium citrate having a concentration range of from about 5 mM to about 20 mM;

(3) pluronic 127 or poloxamer 407 from about 10% to about 25% (w / v), preferably from about 15% to about 25%, most preferably about 16%;

(4) from about 6.7% to about 7.3% sucrose, preferably about 7% sucrose or about 2.0% glycine and about 0.5% sucrose; And

(5) An appropriate amount of water

.

The gel preparation may optionally contain

(6) EDTA wherein the concentration range is from about 0.1 mM to about 10 mM;

(7) NaCl with a concentration range of from about 0.01 mM to about 125 mM

≪ / RTI > The preferred pH range of the gel formulation is from about pH 5.0 to about pH 8.0, preferably pH 6.2, and the resulting gel formulation should be isotonic.

3) Additional stabilizers:

All of the formulations of the present invention described above may be advantageously employed with antioxidants, metal chelating agents, thiol-containing compounds and other common stabilizers. Examples of such stabilizers include

(a) from about 0.5% to about 2% w / v glycerol;

(b) from about 0.1% to about 1% w / v methionine;

(c) from about 0.1% to about 2% w / v monothioglycerol;

(d) from about 1 mM to about 10 mM EDTA;

(e) from about 0.01% to about 2% w / v ascorbic acid;

(f) 0.003% to about 0.02% w / v polysorbate 80;

(g) 0.001% to about 0.02% w / v polysorbate 20;

(h) arginine, preferably arginine, at a concentration of about 0.5% to about 2.5%, most preferably about 1.7%;

(i) heparin or a heparin analog (negatively charged);

(j) dextran sulfate, preferably dextran sulfate at a concentration of about 0.5% and 0.05%;

(k) cyclodextrin or sulfated cyclodextrin;

(l) anionic or polyanionic species [heparin analogue];

(m) lysine, preferably lysine at a concentration of 10%;

(n) hydroxypropyl-δ-cyclodextrin, preferably hydroxypropyl-δ-cyclodextrin having a concentration of from about 2 to about 10%;

(o) sulfated-delta-cyclodextrin, preferably sulfated-delta-cyclodextrin at a concentration of about 0.1% and 10%, most preferably about 1%; or

(p) < / RTI >

But is not limited thereto.

Administration of KGF-2 polypeptide

The KGF-2 polypeptide preparation of the present invention may employ a suitable pharmaceutical diluent 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 should conform to the route of administration. Preferably, the pharmaceutical composition will be formulated according to the present invention as described above. Water is the preferred diluent.

The polypeptide having KGF-2 activity may be administered with the pharmaceutical composition together with one or more pharmaceutically acceptable carriers. When administered to humans, it will be appreciated that the total daily dose of the pharmaceutical compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective dosage level for any particular patient will depend upon the type and extent of the response desired to be obtained; A specific composition including whether or not other antigens are present, if necessary; Age, weight, general health status, gender and diet; The time of administration, the route of administration and the secretion time of the composition; Duration of treatment; Drugs (e. G., Chemotherapeutic agents) used or co-used with a particular composition; And similar factors well known in the medical arts. Suitable formulations known in the art can be found in Remington ' s Pharmaceutical Sciences (final), Mack Publishing Company, Easton, PA. Thus, an " effective amount " of KGF-2 (including an effective amount of KGF-2) for the purposes herein is determined through this consideration.

The pharmaceutical composition of the present invention can be administered in a convenient manner, examples of which include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarticular, subcutaneous, intranasal Administration, inhalation administration, intravenous administration or intradermal administration. Parenteral and topical delivery is the preferred route of administration. As used herein, the term " parenteral " means a route of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is administered in an amount effective to treat and / or prevent a particular condition. In most cases, the daily dosage of KGF-2 is from about 1 [mu] g / kg to about 30 mg / kg, taking into account the route of administration, signs, and the like. However, the dose may be as low as 0.001 [mu] g / kg. For example, in a particular case of topical administration, it is preferred to administer from about 0.01 [mu] g to 9 mg per cm < ^{2 &} gt ;. When administered intranasally and intravenously, it is preferable to administer about 0.001 / / ml to about 10 mg / ml, more preferably about 0.05 mg / ml to about 4 mg / ml.

As a general suggestion, the total pharmacologically effective amount of KGF-2 polypeptide administered parenterally is about 1 [mu] g / kg / day to 10 mg / kg / day, although this is a cautious treatment. When administered sequentially, the KGF-2 polypeptide is typically administered at a dose of about 1 μg / kg / hour to about 50 μg / kg / hour by one to four injections per day or continuous subcutaneous infusion, Lt; / RTI > / kg / hour. An intravenous bag solution or bottle solution may also be used.

The course of KGF-2 polypeptide treatment that affects the fibrinolysis system appears to be optimal if it is longer than a certain minimum of 7 days for mice. The duration of treatment required to observe the change and the interval with treatment for the response that occurs will depend on the desired effect.

In one embodiment of the parenteral administration, the KGF-2 polypeptide is administered at a desired purity with a pharmaceutically acceptable carrier, i.e., a carrier that is not toxic to the receptor at the dose and concentration employed and is compatible with the other ingredients of the formulation By injection into an injectable unit dosage form (solution, suspension or emulsion). For example, the agent preferably does not comprise an oxidizing agent and other compounds known to be detrimental to the polypeptide.

Generally, the formulations are prepared by uniformly and thoroughly contacting the KGF-2 polypeptide with a liquid carrier or a finely divided solid carrier or both. Then, if necessary, the product is shaped into the desired formulation. The carrier is preferably a parenteral carrier, more preferably a solution in which the blood of the receptor is isomerized. Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles, such as fixed oils and ethyl oleate, as well as liposomes, are also useful herein. Suitable formulations known in the art can be found in Remington ' s Pharmaceutical Sciences (final), Mack Publishing Company, Easton, PA.

In addition, the KGF-2 polypeptide can be administered to the eye as a liquid, a drip or a thickened liquid, a gel, to treat laryngeal injuries, diseases and lesions in animals and humans.

In addition, the KGF-2 polypeptide can be administered to the nasal mucosa in the form of a liquid drop or spray to treat diseases and disorders of the nasal mucosa and epithelium of animals and humans.

Generally, the formulations are prepared by uniformly and thoroughly contacting the KGF-2 polypeptide with a liquid carrier or a finely divided solid carrier or both. Then, if necessary, the product is shaped into the desired formulation. The carrier is preferably a parenteral carrier, more preferably a solution in which the blood of the receptor is isomerized. Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles, such as fixed oils and ethyl oleate, as well as liposomes, are also useful herein. Suitable formulations known in the art can be found in Remington ' s Pharmaceutical Sciences (final), Mack Publishing Company, Easton, PA.

In addition, the carrier may contain small amounts of suitable additives, such as materials that enhance isotacticity and chemical stability. Such materials are non-toxic to the receptor at the dosages and concentrations employed, examples of which include buffers (e.g., phosphate, citrate, succinate, acetic acid and other organic acids or salts thereof); Antioxidants (such as ascorbic acid); Low molecular weight (less than 10 residues) polypeptides, such as polyarginine or tripeptides; Proteins (e.g., serum albumin, gelatin or immunoglobulins); Hydrophilic polymers (e.g., polyvinylpyrrolidone); Amino acids (e.g., glycine, glutamic acid, aspartic acid or arginine); Celluloses or derivatives thereof, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or texturins; Chelating agents such as EDTA; Sugar alcohols (e.g., mannitol or sorbitol); Counter ion such as sodium; And / or a surfactant (e.g., polysorbate, poloxamer, or PEG).

KGF-2 generally has a pH of from about 5 to about 8, preferably from about 6 to about 7, and most preferably from about pH 6.2 to about 0.01 to 50 mg / ml, preferably from 0.01 to 10 0.0 > mg / ml. < / RTI > It will be appreciated that the use of the excipients, carriers or stabilizers described above will produce KGF-2 salts.

KGF-2 used for therapeutic administration can be sterilized. Sterilization is readily accomplished by filtration through a sterile filter membrane (e. G., A 0.2 micron membrane). The therapeutic KGF-2 composition may be placed in an intravenous solution bag or vial having a container having a sterile access port, e.g., a stopper pierceable with a hypodermic needle.

Usually, KGF-2 will be stored in unit dose containers or in multi-dose containers, for example as a solution in a sealed ampoule or vial, or as a lyophilized preparation for reconstitution. As an example of a lyophilized preparation, a 3-ml vial is filled with 1 ml of sterile-filtered 1% (w / v) aqueous KGF-2 solution and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting lyophilized KGF-2 with water for injection, which may optionally contain one or more antioxidants.

In addition, administration can be configured in a patient-specific manner to provide a predetermined concentration of KGF-2 activity in the blood, e.g., as measured by the RIA technique. Thus, administration to a patient can be adjusted so that about 50 to 1000 ng / ml, preferably 150 to 500 ng / ml, is routinely administered via the blood level, as measured by RIA.

In addition, KGF-2 can be suitably administered by a sustained-release system. Suitable examples of the sustained-release composition include a molded article, for example a semipermeable polymer matrix in the form of a film or microcapsule. Examples of the sustained-release matrix include polylactide (U.S. Patent No. 3,773,939, EP No. 58,481), copolymer 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., Id.) Or poly-D - (-) - 3-hydroxybutyric acid (EP No. 133,988). In addition, the sustained release KGF-2 composition comprises KGF-2 captured by liposomes. Liposomes containing KGF-2 are prepared by per se known methods: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP 52322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application No. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545 and EP 102,324. Generally, the liposomes are in the form of small (about 200-800 ANGSTROM) single-lamellae, with a liquid content of greater than about 30 mol% cholesterol and the rate selected for optimal KGF-2 therapy is adjusted.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more pharmaceutical compositions of the invention. Such containers may be provided with a warning in the form prescribed by a governmental agency regulating the manufacture, use, or sale of a drug or biological product, and some warnings may be issued by government agencies to manufacture, use or sale approval for human administration Reflect. The polypeptides, agonists and antagonists of the present invention may also be used in combination with other therapeutic compounds.

When investigating the biological activity and stability of KGF-2 polypeptides produced according to the formulation of the present invention by the present inventors, the use of monothioglycerol can stabilize the KGF-2 polypeptide, and the KGF-2 polypeptide As a reinforcing agent for < RTI ID = 0.0 > a < / RTI > The optimum concentration range for the enhancing effect of monothioglycerol is 0.1% to 2% w / v.

KGF-2 polypeptide

KGF-2 promotes the proliferation of epithelial and epithelial keratinocytes, but does not promote the proliferation of insect tissue cells such as fibroblasts. Thus, a " polypeptide having KGF-2 protein-like activity " is described herein below and exhibits KGF-2 activity in the keratinocyte proliferation assay described in US application Ser. No. 08 / 910,875, including FGF receptor kinase 1-iiib and 2-iiib ≪ / RTI > A " polypeptide having KGF-2 protein-like activity " exhibits substantially similar activity compared to the KGF-2 protein (i.e., the candidate The polypeptide exhibits greater activity or exhibits an activity of at most 10-fold, preferably at most about 2-fold less than the reference KGF-2 protein.

The KGF-2 polypeptide used in the formulation of the present invention may or may not have N-terminal methionine, but preferably the polypeptide is free of N-terminal methionine.

The KGF-2 cDNA clone was deposited on December 16, 1994 with ATCC accession number 75977 to the American Type Culture Collection (ATCC), Manassas University, Vaughan 10801, Virginia, 20110-2209, The cDNA encoding KGF-2? 33 inserted in vector pHE4-5 was deposited with ATCC on Jan. 9, 1998, and received ATCC Accession No. 209575.

The terms " fragment, " " derivative " and " analogue " used herein to refer to polypeptides encoded by the polypeptides of Figure 1 (SEQ ID NO: 2) or the deposited cDNAs refer to the same biological function or activity as those polypeptides ≪ / RTI > Thus, analogs include pro-proteins that are capable of being activated by cleavage of the pro-protein moiety to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, and a recombinant polypeptide is preferred.

Fragments, derivatives or analogs of the polypeptides of Figure 1 (SEQ ID NO: 2) or polypeptides encoded by the deposited cNDA are useful for (i) conserving or non-conserved amino acid residues of one or more amino acid residues, preferably conserved amino acid residues (Ii) one or more amino acid residues comprise a substituent, or (iii) the mature polypeptide is substituted with another compound, For example, a compound that increases the half-life of the polypeptide (e. G., Polyethylene glycol), or (iv) a mature polypeptide has additional amino acids, such as a leader or secretory sequence, or a mature polypeptide, May be fused. Such fragments, derivatives and analogs are considered to be within the purview of those skilled in the art from the teachings herein.

The terms " peptide " and " oligopeptide ", as used herein, are synonymous (as commonly recognized), and each term is intended to include a chain of amino acids, It can be used interchangeably. As used herein, the term " polypeptide " means a chain containing at least 10 amino acid residues. All oligopeptide and polypeptide structures or sequences herein are described from left to right, with the orientation being the carboxy terminus from the amino terminus.

It will be understood in the art that some amino acid sequences of KGF-2 polypeptides can be altered without significantly affecting the structure or function of the protein. When considering these sequence differences, one has to remember the critical part of the protein that determines the activity of the protein. Generally, residues that form a tertiary structure can be substituted, but residues that perform similar functions are used. In other cases, when the change occurs in a noncritical region of the protein, the type of the residue need not be considered.

The polypeptides of the invention are preferably in isolated form. &Quot; Isolated polypeptide " means a polypeptide that has been removed from its natural environment. Thus, polypeptides produced in and / or contained within the recombinant host cells are considered isolated for the purposes of the present invention. The term also refers to polypeptides that are partially or substantially purified from recombinant host cells or natural sources.

The pharmaceutical composition of the present invention comprises not less than 90%, not less than 95%, not less than 96%, not less than 97%, not more than 98% of the polypeptide of SEQ ID NO: 2 as well as the KGF-2 polypeptide (particularly mature polypeptide) (More preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) and deletion mutants thereof, Also included is a portion of such a polypeptide, wherein the portion of the polypeptide (eg, a deletion mutant described below) generally contains at least 30 amino acids, more preferably at least 50 amino acids.

As is known in the art, " similarity " between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with its conservative amino acid substituted sequence and the sequence of the second polypeptide.

The "% similarity" of the two polypeptides was determined using the Bestfit program [Wisconsin Sequencing Package for Unix (Version 8) by Genetics Computer Group, Inc., 575 University Park, Madison Science Drive 53711 Wisconsin, USA) Quot; means the similarity value obtained by comparing the amino acid sequence of the polypeptide. Bestfit finds optimal similarity fragments between two sequences using the local homology algorithm described in Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981).

For example, a polypeptide having an " identical " amino acid sequence of greater than 95% for a KGF-2 polypeptide means that the amino acid sequence of the polypeptide is the same as the reference sequence, but the polypeptide sequence is a reference amino acid of KGF-2 polypeptide Lt; RTI ID = 0.0 > 5 < / RTI > amino acid changes per 100 amino acids in each. In other words, in order to obtain a polypeptide having 95% or more of the same amino acid sequence as the reference amino acid sequence, 5% or less of the amino acid residue in the reference sequence is deleted or substituted with another amino acid, or 5% Means that the number of amino acids below can be inserted into the reference sequence. These changes in the reference sequence can occur either at the amino or carboxy terminus position of the reference amino acid sequence, or between those terminal positions that are interspersed either among the residues within the reference sequence or interspersed within one or more contiguous groups within the reference sequence Can occur at any location.

As a practical matter, it is contemplated that any particular polypeptide may be, for example, at least 90%, at least 95%, at least 96%, at least 97% identical to the amino acid sequence encoded by the amino acid sequence of SEQ ID NO: , 98% or more, or 99% or more is determined using a known computer program such as Bestfit program [Wisconsin Sequencing Package for Unix (Version 8) of Genetics Computer Group, 575 University Park, Madison Science Drive, Wisconsin, As shown in FIG. If Bestfit or other sequence alignment program is used to determine whether a particular sequence is 95% identical to a reference sequence according to the invention, the percent identity is calculated over the entire length of the reference amino acid sequence by setting parameters, A homology difference of no more than 5% of the total number of amino acid residues in the reference sequence is allowed.

Proteins of the present invention can be naturally occurring, recombinantly produced, or chemically synthesized using methods known per se (Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., NY and Hunkapiller, M. et al., Nayure 310: 105-111 (1984)]. For example, a peptide corresponding to a fragment of the KGF-2 polypeptide of the present invention can be synthesized using a peptide synthesizer. Further, if necessary, the atypical amino acid or the chemical amino acid analog can be introduced into the KGF-2 polypeptide sequence by substitution or addition. Unconventional amino acids include the D-isomers of common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2- aminobutyric acid,? -Abu,? -Ahx, Norbornene, hydroxyproline, sacocin, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenyl But are not limited to, glycine, cyclohexyl alanine,? -Alanine, fluoro-amino acids, designer amino acids such as? -Methyl amino acid, Ca-methyl amino acid, Na-methyl amino acid and common amino acid analogs . In addition, the amino acid may be D (preferential) or L (left).

Variants that are not naturally occurring can be produced using known mutagenesis techniques, including oligonucleotide-mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis For example, Carter et al., Nucl. Acids Res. 13: 4331 (1986); And Zoller et al., Nucl. 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 present invention also relates to the use of the compounds of the invention in the preparation of a medicament for use in the treatment of a disease or disorder, for example during glycation, acetylation, phosphorylation, amidation, derivatization by known protecting groups / Lt; RTI ID = 0.0 > KGF-2 < / RTI > Any modification of a number of chemical transformation may be performed using known techniques, these examples of known techniques are cyanogen bromide, trypsin, chymotrypsin, specific chemical degradation by papain, V8 protease, NaBH _4, acetylation But are not limited to, hydrogenation, formylation, oxidation, reduction, metabolic synthesis in the presence of tocinamicin, and the like.

Additional post-translational modifications such as treatment of N-linked or O-linked carbohydrate chains, N-terminus or C-terminus, attachment of chemical moieties to amino acid backbones, N-linked or O-linked carbohydrate chains As well as the addition or deletion of N-terminal methionine as a result of prokaryotic host cell expression. In addition, the polypeptides of the invention may be modified with detectable labels, such as enzymatic labels, fluorescent labels, radioisotope labels, or affinity labels to enable detection and isolation of the protein.

Increased solubility, increased stability, and increased circulation time of the polypeptide or reduced immunogenicity (see, U.S. Patent No. 4,179,337). The chemical moiety for the derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like.

The polypeptide may be modified at random positions within the molecule, or at predetermined locations within the molecule, and may include one, two, or three or more attached chemical moieties.

The polymer may be of any molecular weight and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight for handling and preparation is from about 1 kDa to about 100 kDa, wherein " about " means that in a polytetrahydroglycol formulation some molecules have a molecular weight somewhat less than the above molecular weight ). Depending on the desired therapeutic profile (e. G., The duration of the desired sustained release, the effect on biological activity if present, ease of handling, degree or lack of antigenicity and other known effects of polyethylene glycol on the therapeutic protein or analog) Other molecular weights can be used. For example, the average molecular weight of polyethylene glycol is 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.

As described above, the 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), all incorporated herein by reference).

The polyethylene glycol molecule (or other chemical moiety) must be attached to the protein in view of its effect on the action or antigenic domains of the protein. There are a number of attachment methods available to those of skill in the art and are described, for example, in EP 0 401 384 (herein incorporated by reference) (coupling of PEG to G-CSF) . Hematol. 20: 1028-1035 (1992)), a report of pegylation of GM-CSF using tresilyl chloride. For example, polyethylene glycol can be covalently bonded through a reactive group, for example, an amino acid residue by a free amino group or a carboxyl group. The reactive groups are those to which the activated polyethylene glycol molecules can bind. Examples of amino acid residues having a free amino group include lysine residues and N-terminal amino acid residues; Examples of the amino acid residue having a free carboxyl group include an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid residue. The sulfhydryl group can also be used as a reactive group for attaching the polyethylene glycol molecule. For therapeutic purposes, it is preferably attached to amino groups or lysine groups such as those attached at the N-terminus.

As described above, polyethylene glycol can be attached to a protein by binding to any amino acid residue in a plurality of amino acid residues. For example, polyethylene glycol can be coupled to proteins by covalent linkage to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. (Eg, lysine, histidine, aspartic acid, glutamic acid, or cysteine) or an amino acid residue in one or more forms of the protein (eg, lysine, histidine, aspartic acid , Glutamic acid, cysteine, and combinations thereof).

Proteins in which the N-terminus is chemically modified may be particularly preferred. When polyethylene glycol is used as an example of the composition of the present invention, various polyethylene glycol molecules (molecular weight, branching etc.), the ratio of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mixture, the type of pegylation reaction performed, - a method of obtaining a terminal pegylated protein. The method of obtaining an N-terminal pegylated preparation (i. E. Isolating this moiety from other monopegylated moieties as necessary) is carried out by purifying the N-terminal pegylated material from a population of pegylated protein molecules . Selective proteins that are chemically modified in the N-terminal modification are obtained by reductive alkylation which utilizes the differential reactivity of different forms of primary amino groups (lysine to N-terminal) available for derivatization in a particular protein. Under suitable reaction conditions, a substantially selective derivatization of the protein at the N-terminus using the carbonyl group-containing polymer is obtained.

As already described, pegylation of the proteins of the present invention can be accomplished by any number of means. For example, polyethylene glycol can be attached to the protein either directly or through an intervening linker. No linker systems for attaching polyethylene glycols 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; All of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to the amino acid residues of a protein without the use of an intervening linker uses trisylated MPEG, which is a monomethoxypolyethylene glycol using tremyl chloride (ClSO ₂ CH ₂ CF ₃ ) MPEG). In the reaction of proteins with tremelylated MPEG, polyethylene glycol is attached directly to the amine group of the protein. Accordingly, the present invention includes a protein-polyethylene glycol conjugate produced by the reaction of a protein of the present invention with a poly (ethylene glycol) molecule having 2,2,2-trifluoroethanesulfonyl group.

In addition, polyethylene glycol can be attached to proteins using a number of different intervening linkers. For example, U.S. Patent No. 5,612,460, incorporated herein by reference, discloses a urethane linker for linking polyethylene glycol to a protein. A protein-polyethylene glycol conjugate wherein the polyethylene glycol is attached to the protein by the linker is selected from the group consisting of MPEG-succinimidyl succinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4, 5-trichlorophenyl carbonate, MPEG-p-nitrophenol carbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycols to proteins are described in WO 98/32466, incorporated herein by reference. Pegylated protein products produced using the reaction chemistries disclosed herein are included within the scope of the present invention.

In addition, the number of polyethylene glycol moieties attached to each of the proteins of the present invention (i.e., degree of substitution) may vary. For example, the pegylated protein of the present invention can be linked to an average of 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 may be, for example, 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 polyethylene glycol moieties. Methods for determining the degree of substitution are described, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992).

KGF-2 deletion mutant

Natural KGF-2 is relatively unstable in the aqueous phase and undergoes chemical and physical degradation, resulting in loss of biological activity during processing and storage. In addition, natural KGF-2 tends to aggregate in aqueous solution at elevated temperatures and is inactivated under acidic conditions.

A particularly preferred KGF-2 polypeptide is a deletion mutant described below (counted from the first amino acid (Met) of the 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)

Ala (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)

Phe (57) -Ser (208) Met (1), Thr (36) or Cys (37)

Ser (198) Met (1), Thr (36) or Cys (37) -Ser (198)

Ser (62) -Ser (208) Met (1), Thr (36) or Cys (37)

Ala (63) -Ser (208) Met (1), Thr (36) or Cys (37)

Gly (64) -Ser (208) Met (1), Thr (36) or Cys (37)

Arg (65) -Ser (208) Met (1), Thr (36) or Cys (37)

Val (67) -Ser (208) Met (1), Thr (36) or Cys (37)

Ser (69) -Ser (208) Met (1), Thr (36) or Cys (37)

Val (77) -Ser (208) Met (1), Thr (36) or Cys (37)

Arg (80) -Ser (208) Met (1), Thr (36) or Cys (37)

Met (1), Thr (36) or Cys (37) -Arg (187)

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 deletion Ala (63) -Ser (208) (KGF-2Δ28) and Ser (69) -Ser (208) (KGF-2Δ33). Other preferred N-terminal and C-terminal deletion mutants are 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).

The invention also includes deletion mutants that retain amino acids deleted from both the N-terminus and the C-terminus. Such mutants include all combinations of the aforementioned N-terminal deletion mutants and C-terminal deletion mutants such as Ala (39) -His (200), Met (44) -Arg (63) -Lys (153), Ser (69) -Lys (153), and the like. These combinations can be prepared using recombinant techniques known in the art.

Thus, preferred KGF polypeptides for use in the pharmaceutical compositions of the present invention include deletions (i.e., at least Met (1) -) of the first 38 to the first 147 amino acid residues of the Figure 1 (SEQ ID NO: 2) Terminal deletion mutant comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) except for the deletion of Gln (38). Alternatively, the agent may be a deletion (i.e., deletion of at least Met (1) -Gin (38)) that comprises the first 38 to the first 137 amino acid residues of the N-terminal amino acid residue of Figure 1 (SEQ ID NO: Lt; / RTI > Alternatively, the agent comprises a mutant having from the first 46 to the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the preparation comprises a mutant having a deletion comprising the first 62 to the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the preparation comprises a mutant having a deletion comprising the first 68 to the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the agent comprises a mutant having a deletion comprising the first 76 or more first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the agent comprises a mutant having a deletion comprising the first 92 to the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the agent comprises a mutant having a deletion comprising an initial 103 to first 137 N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2).

In addition to formulations comprising a KGF-2 mutant having the above range of N-terminal deletion mutants, the present invention also relates to formulations having all combinations of the above-mentioned ranges, Deletion of the first 62 to the first 68 amino acids of the N-terminal amino acid residue of SEQ ID NO: 2); Deletion of the first 62 or more and the first 76 or fewer N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); Deletion of the first 62 or more and the first 92 or fewer N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2); Deletion of the first 62 or more to the first 103 or fewer N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2); Deletion of the first 68 or more and the first 76 or fewer N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); Deletion of the first 68 or more and first 92 or less N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); Deletion of the first 68 or more and the first 103 or fewer N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2); Deletion of the first 46 to the first 62 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); Deletion of the first 46 to the first 68 amino acids of the N-terminal amino acid residue of Figure 1 (SEQ ID NO: 2); Deletion of the first 46 or more and the first 76 or fewer N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2).

In another embodiment, a formulation comprising a C-terminal deletion mutant is provided by the present invention. Preferably, the N-terminal amino acid residue of the C-terminal deletion mutant is 1 (Met), 36 (Thr) or 37 (Cys) of Figure 1 (SEQ ID NO: 2). Such a preparation may comprise at least a deletion of the last C-terminal amino acid residue (Ser (208)), but not more than the last 55 C-terminal amino acid residues (i.e., the amino acid residue Glu (154) -Ser (SEQ ID NO: 2) except for the deletion of the amino acid sequence shown in SEQ ID NO: 208). Alternatively, the preparation comprises a mutant having a deletion comprising at least the last C-terminal amino acid residue to the last 65 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the preparation comprises a mutant having a deletion comprising the final 10 to last 55 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the preparation comprises a mutant having a deletion comprising the final 20 to more than 55 final C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the preparation comprises a mutant having a deletion comprising a final 30 to more than 55 final C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the preparation comprises a mutant having a deletion comprising the final 40 to more than 55 final C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2). Alternatively, the agent comprises a mutant having a deletion comprising the last 50 or more to the last 55 or fewer C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2).

In addition to a formulation comprising a KGF-2 mutation having a range of C-terminal deletion mutations as described above, the present invention encompasses the aforementioned range, such as at least the last C-terminal amino acid residue in the figure 1 (SEQ ID NO: 2) Terminal amino acid residue deletion; At least the last C-terminal amino acid residue to the last 20 C-terminal amino acid residue deletions in Figure 1 (SEQ ID NO: 2); At least the last C-terminal amino acid residue to the last 30 C-terminal amino acid residue deletions of Figure 1 (SEQ ID NO: 2); At least the last C-terminal amino acid residue to the last 40 C-terminal amino acid residue deletions in Figure 1 (SEQ ID NO: 2); Deletion of the last 10 or more C-terminal amino acid residues to the last 20 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 2); Terminal < / RTI > amino acid residue to the last 30 C-terminal amino acid residue deletions in Figure 1 (SEQ ID NO: 2); Terminal < / RTI > amino acid residue to the last 40 or fewer C-terminal amino acid residue deletions in Figure 1 (SEQ ID NO: 2); Terminal 20 amino acid residues to the last 30 C-terminal amino acid residue deletions in Figure 1 (SEQ ID NO: 2); And the like.

In another embodiment, the KGF-2 polypeptide can be a deletion mutant having an amino acid deleted from both the N-terminal and C-terminal residues. Such mutants include all combinations of the aforementioned N-terminal deletion mutants and C-terminal deletion mutants. Such mutants include deletions of the first 46 or more N-terminal amino acid residues to the first 137 or fewer N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2); Includes those comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) except for deletion of at least the last C-terminal amino acid residue to the last 55 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2). Alternatively, deletions can be made with at least the first 62, 68, 76, 92, 103 or 122 N-terminal amino acids to the first 137 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 2) 20, 30, 40 or 50 C-terminal amino acid residues to not more than 55 final C-terminal amino acid residues of the last 10, 20, 30, 40 or 50 C-terminal amino acid residues. It also includes all combinations of the ranges disclosed above.

KGF-2 replacement mutant

Useful KGF-2 polypeptides include those having amino acid substitutions. Natural mature KGF-2 contains 44 charged residues, 32 of which possess positive charge. Depending on the position of these residues in the three dimensional structure of the protein, substituting one or more of these residues with an amino acid having a negative charge or a neutral charge alters the electrostatic interactions of adjacent residues, resulting in increased protein stability and decreased cohesion Lt; / RTI > Protein aggregation can not cause loss of activity, but can cause problems in the manufacture of pharmaceutical preparations because these proteins can be immunogenic (see 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 be considered to minimize charge repulsion in the tertiary structure of the protein molecule. Therefore, it is of particular interest to replace the charged amino acid with another charge and a neutral or negatively charged amino acid. The latter improves the properties of KGF-2 by resulting in a positive charge reduced protein. Wherein the improvement includes increased stability and reduced aggregation of the analogue as compared to native KGF-2 protein.

Substitution of amino acids can alter the binding specificity for cell surface receptors. Ostade et al., Nature 361: 266-268 (1993) disclose specific TNF alpha mutations that result in selective binding of TNF alpha to only one of two existing TNF receptors.

The KGF-2 molecule may comprise one or more amino acid substitutions, deletions or additions by natural mutagenesis or human manipulation. Some of the preferred mutations are as follows: Ala (49) Gln, Asn (51) Ala, Ser (54) Val, Ala (63) Pro, Gly (64) Glu, Val Val, Arg (80) Lys, Lys (87) Arg, Tyr (88) Trp, Phe (89) Tyr, Lys Glu (104) Met, Asn (105) Lys, Pro (107) Asn, Ser (109) Asn, Leu (111) Met, Thr 123) Ile, Ala (125) Gly, Ile (126) Val, Asn (127) Glu, Asn (127) Gln, Tyr (130) Phe, Met (134) Thr, Lys Glu, Glu (142) Ala, Ser (143) Lys, Phe (146) Ser, Asn (148) Glu, Lys (151) Asn, Leu (152) Phe, Glu Arg (155) Leu, Glu (157) Leu, Gly (160) His, Phe (167) Ala, Asn (168) Lys, Gln 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.

According to the designation, for example, Ala (49) Gln means that Ala is replaced with Gln at position 49 of Figure 1 (SEQ ID NO: 2).

Changes in unimportant properties such as conservative amino acid substitutions that do not significantly affect protein folding or activity are desirable. Examples of conservative amino acid substitutions known to those skilled in the art are as follows:

Aromatic: phenylalanine

Tryptophan

Tyrosine

Hydrophobic: Leucine

Isoleucine

Balin

Polarity: Glutamine

Asparagine

Basic: arginine

Lysine

Histidine

Acid: Aspartic acid

Glutamic acid

Miniature alanine

Serine

Threonine

Methionine

Glycine

Of course, the number of amino acid substitutions that can be made by those skilled in the art will depend on a variety of factors, including those described above. Generally speaking, the number of substitutions for any given KGF-2 polypeptide will not be 50, 40, 30, 20, 10, 5 or 3 or more, depending on the subject. For example, a number of substitutions can be made at the C-terminus of KGF-2 to improve stability.

The amino acid of KGF-2 that is essential for function can be identified by methods known in the art such as site-directed mutagenesis or alanine injection mutagenesis (Cunningham and Wells, Science 244: 1081-1085 (1989)). The procedure of alanine injection mutagenesis induces a single alanine mutation in all residues in the molecule. Next, mutant molecules generated for biological activity such as receptor binding or in vitro or in vivo proliferative activity are tested. (See Example 1, for example). Important sites for ligand-receptor binding can be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (see, 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 wherein the cysteine residue is replaced with a serine residue at amino acid positions 37, 106 and 150. An odd number of cysteine means that at least one cysteine residue is available for intermolecular crosslinking or linkage that can cause the protein to adopt an undesirable tertiary structure. A novel KGF-2 protein bearing serine or at least one cysteine substituted for example with alanine is generally a soluble, correctly folded protein that is purified at high yield. While not wishing to be bound by theory, it is believed that the cysteine residue at position 106 is important for function. This cysteine residue is highly conserved in all other FGF family members.

Also, KGF-2 polypeptides are described in PCT / US95 / 01790 filed February 14, 1995, 08 / 461,195 filed June 5, 1995, 08 / 696,135 filed August 13, 1996 60 / 033,852 filed on August 13, 1996, 60 / 039,045 filed on February 28, 1997, 08 / 862,432 filed May 23, 1997, and August 13, 1997 09 / 023,082 filed on February 13, 1998, 09 / 345,373 filed on July 1, 1999, 1999/00 / 055,561 filed on August 13, 1997, 60 / 142,343, filed July 2, 1999, 60 / 143,648 filed July 14, 1999, 60 / 144,024 filed July 15, 1999, filed on August 12, 1999, 60 / 148,628, 60 / 149,935 filed September 24, 1999, 60 / 163,375 filed November 3, 1999, 60 / 171,677 filed December 22, 1999, 60 / 198,322, filed April 19, the respective disclosures of which are incorporated herein by reference.

Therapeutic Uses of KGF-2 Polypeptide Compositions

The polypeptide of the present invention can stimulate keratinocyte growth and proliferation. Thus, the compositions of the present invention can be used to stimulate epithelial cell proliferation and basal keratinocytes for wound healing purposes, and stimulate hair follicle production and healing of skin wounds. These wounds can be trauma or deep wounds and include damage to the dermis and epithelium of the skin.

KGF-2 is useful for the treatment of many diseases and conditions. For example, KGF-2 is in vitro and in vivo active in various wound healing models. See, for example, U.S. Serial No. 08 / 910,875 (filed on August 13, 1997) and 09 / 023,082 (filed February 13, 1998).

The individual to which KGF-2 is administered may have wound healing at normal speed, or there may be a healing disorder. When administered to individuals without healing impairment, KGF-2 is administered to accelerate the normal healing process. When administered to a subject with a healing disorder, administration of KGF-2 facilitates healing of wounds that would otherwise slowly heal or not heal at all. Many diseases and symptoms can lead to healing disorders. These diseases and conditions include diabetes (e. G., Type II diabetes mellitus), treatment of steroid and non-steroidal drugs, and ischemic blockade or impairment.

A number of growth factors have been identified that promote wound healing in healing disorder individuals. These growth factors include growth hormone releasing factor, platelet derived growth factor, and basal fibroblast growth factor. Thus, the present invention also includes administration of the KGF-2 composition in combination with one or more additional growth factors or other agents that promote wound healing.

The compositions of the present invention promote the healing of anastomoses and other wounds induced by surgical procedures in individuals and in healing disorders that heal wounds at normal rates.

The composition of the present invention can be used for stimulating cell differentiation such as muscle cells, cells constituting nerve tissue, prostate cells and lung cells.

The composition of the present invention is useful for the treatment of symptoms that lead to abnormal wound healing such as uremia, malnutrition, vitamin deficiency, obesity, infection, immunosuppression and complications associated with steroids, radiation therapy and anti- And a normal subject may be treated with a surgical wound, a wound wound, a deep wound including damages of the dermis and epithelium, an eye tissue wound, a tooth tissue wound, an oral wound, a diabetic ulcer, a dermal ulcer, a venous ulcer, Or to stimulate wound healing of wounds such as burns resulting from exposure to cryogenic temperatures or exposure to chemicals. The composition may be used for the promotion of wound healing associated with chronic venous leg ulcers caused by ischemic and ischemic damage, such as venous circulatory system restoration and / or lack of damage; Promotion of dermal restoration following dermis loss, increase in epithelial tensile strength and epithelium thickness; And to increase the adhesion of the skin graft to the wound layer and to re-epithelialization stimulation from the wound layer.

Other useful therapeutic uses of KGF-2 polypeptides include, but are not limited to, stimulating epithelial cell proliferation and basal keratinocytes for the purpose of treating skin defects and burns such as psoriasis and epidermal blisters. KGF-2 can be used to increase the attachment of skin grafts to the wound bed and to stimulate re-epithelization from the wound bed. KGF-2 can be used to reduce the side effects of gastro-toxicity resulting from radiation, chemotherapy or viral infection. KGF-2 can be used to treat diseases and symptoms of the liver, lungs, kidneys, chest, pancreas, stomach, small intestine and colon. KGF-2 can be used to treat inflammatory bowel disease, diabetes, thrombocytopenia, hypofibrinogenemia, hypoalbuminemia, hypoglobulinemia, hemorrhagic cystitis, dry mouth, and keratoconjunctivitis sicca. KGF-2 can be used to stimulate epithelial cells of salivary glands and lacrimal gland to stimulate the proliferation and re-epithelialization of the nasal mucosa.

A number of other indications that can be treated with the compositions of the present invention are disclosed in U.S. Serial Nos. 08 / 910,875 and 09 / 023,082, which are incorporated herein by reference.

The present invention relates to novel liquid and lyophilized preparations of KGF-2 and its deletion mutants. The present invention also relates to KGF-2 preparations for use in therapy. This formulation is more stable than the active KGF-2 polypeptide and, in some cases, enhances and rapidly increases wound healing activity of the polypeptide.

As used herein, an "animal" refers to an animal, preferably a mammal (eg, a monkey, cow, horse, pig, boar, sheep, rodent, goat, dog, cat, chicken, monkey, rabbit, Whales and dolphins), more preferably humans.

The KGF-2 [Delta] 33 polypeptide used in the formulation of the present invention may or may not have N-terminal methionine, and it is preferred that the polypeptide lacks N-terminal methionine. The stability of the KGF-2 polypeptide preparation of the present invention is determined by proliferation assay as described below.

Other therapeutic uses of KGF-2 are described in U.S. Serial No. 60 / 074,585 (filed February 13, 1998), 60 / 114,484 (filed December 30, 1998), and 09 / 248,998 , All of which are incorporated herein by reference.

Keratinocyte proliferation assay

The keratinocytes are cells in the epithelium of the skin. The growth and spread of keratinocytes in the skin is an important process for wound healing. Thus, proliferation assay of keratinocytes is a valuable indicator of protein activity that stimulates keratinocyte proliferation and, as a result, heals wounds.

However, keratinocytes are difficult to grow in vitro. There are few keratinocyte lines present. These cell lines have different cell and genetic defects. Primary dermal keratinocytes for this assay are chosen to avoid complicating analysis by cell defects such as loss of major growth factor receptor or dependence of major growth factors for growth. These primary keratinocytes are available from Cronetics, Inc. (San Diego, Calif., USA).

The viability of the KGF-2 polypeptide can be determined by cell proliferation assay using murine Baf3 2b cells transfected with the fibroblast growth factor 2iiib receptor (FGFR2iiib). Proliferation of the cells, as will be described later after the cells are exposed to protein-measured by ^[methyl-3 H] thymidine insertion. Analysis is performed on 96-well tissue culture cluster plates with approximately 22,000 Baf3 2b cells in each well. 3 times to expose the cells to different concentrations of KGF-2 polypeptide and incubate at 37 [deg.] C in a CO ₂ incubator for about 48 hours. An approximate amount of cell culture medium containing labeled thymidine is added to each well and incubated for another 5 hours. Cells are harvested from a 96 well type glass fiber filter mat using a cell sorter. The filter mat is dried and the radioactivity introduced into each sample is counted using a flat layer liquid scintillation counter. Under these analytical conditions, cells exposed to KGF-2 exhibit an increased uptake of radioactivity compared to control cells treated with a suitable dilution of placebo buffer or simply phosphate buffered saline.

Another useful keratinocyte proliferation assay is the use of alamarbium. Allamac Blue is a viable blue dye that is metabolized by mitochondria when added to the culture medium. This dye turns red from the tissue culture supernatant. The amount of red dye can be quantified directly by reading the optical density difference between 570 nm and 600 nm. This reading reflects cell activity and cell number.

Normal primary dermal keratinocytes (CC-0255, NHEK-Neo pulverized) are purchased from Clonetics, Inc. These cells are subcultured twice. The keratinocytes are grown in complete keratinocyte growth medium (CC-3001, KGM; Cronetics Inc.) until 80% full grown. Cells are trypsinized according to manufacturer's specifications. Briefly, cells are washed twice with Hank's balanced salt solution. Add 2-3 ml of trypsin to the cells at room temperature for 3-5 minutes. Trypsin neutralization solution is added and cells are collected. Cells were spun at 600 x g for 5 minutes at room temperature and plated in fresh flasks at 3,000 cells / cm ² using prewarmed media.

For proliferation analysis, 1,000-2,000 keratinocytes are plated on a Corning flat bottom 96-well flat plate in complete medium except for the outermost column. The outer wells are filled with 200 [mu] l of sterile water. This helps to keep the well temperature and moisture fluctuations to a minimum. Cells were grown overnight at 37 ° C in 5% CO ₂ . Cells are washed twice with keratinocyte basal medium (CC-3101, KBM, Cronetics, Inc.) and 100 μl of KBM is added to each well. Incubate for 24 hours. Dilute the growth factor in KBM with a series of dilutions and add 100 μl 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. Incubate for 2 to 3 days. At the end of incubation, the cells are washed once with KBM and 100 μl of pre-mixed 10% v / v Allama Blue and KBM are added to the medium. Incubate for 6 to 16 hours until the color of the medium turns red in the KGM positive control. Measure the OD 570 nm - OD 600 nm by placing the plate directly on a flatbed reader.

Composition of KGF-2 deletion mutants

Useful deletion mutants for use in the compositions of the present invention can be constructed with the following protocols.

Using the optimized KGF-2 construct as a template, a deletion mutant was constructed from the 5 'and 3' ends of the KGF-2 gene. The deletion was selected based on the region of the gene that has a negative effect on expression in E. coli. In the case of 5 'deletion, the primer described below was used as the 5' primer. These primers include ATG encoding a restriction site and initiation factor methionine. KGF-2 (FGF-12) 208 amino acid 3'HindIII primer was used for the 3 'primer. PCR amplification was performed for 25 rounds using standard conditions. The product for deletion mutation KGF-2 36aa / 208aa was restricted to BspHI at the 5 'site and HindIII at the 3' site and cloned into pQE60 digested with BspHI and HindIII. All other products were restricted to NcoI for the 5 'restriction enzyme and to HindIII for the 3' region and cloned into pQE60 digested with NcoI and HindIII. In the case of KGF-2 (FGF-12) 36aa / 153aa, FGF-12 36aa / 208aa was used as a 5 'primer using 128aa 3' HindIII as a 3 'primer. In the case of FGF-12 62aa / 153aa, 128aa 3 'HindIII was used as a 3' primer and FGF-12 62aa / 208aa was used as a 5 'primer. The naming of the resulting clone is to specify the first and last amino acids of the polypeptide resulting from deletion. For example, KGF-2 36aa / 153aa is the first amino acid of the deletion mutant 36 and the final amino acid is the amino acid 153 of KGF-2. The construction of a KGF-2 deletion mutant is disclosed in U.S. Serial Nos. 08 / 910,875 and 09 / 023,082, the disclosures of which are incorporated herein by reference. Also, as shown below, each mutant retains the N-terminal methionine added thereto. However, the KGF-2 deletion polypeptide used in the preparation according to the present invention may or may not have N-terminal methionine, and the polypeptide is preferably deficient in N-terminal methionine.

Sequence of deletion primer

FGF12 3 'HindIII : (used for all deletion clones above)

FGF12 36aa / 153aa:

5 ' BsphI (as described above)

FGF12 63aa / 153aa:

5'NcoI and 3'HindIII, as described above.

Composition of the N-terminal deletion mutant KGF-2? 33

Configuration of KGF-2? 33 in pQE6

For the polymerase chain reaction-induced amplification and subcloning of KGF2Δ33 into the E. coli protein expression vector pQE6, two oligonucleotide primers (5952 and 19138) complementary to a given region of KGF2 were synthesized by the following base sequence Respectively.

For the N-terminal primer (5952), the HindIII restriction site was introduced in the case of the C-terminal primer (19138) while the AflIII restriction site was introduced. Primer 5952 contains an ATG sequence in the frame adjacent to the KGF2 coding region to translate the cloned fragment in E. coli while a primer 19138 is located within the frame adjacent to the KGF-2 coding region that is correctly translated in E. coli Containing two end codons (which are used primarily in E. coli).

Polymerase chain reaction was performed using standard conditions known to those skilled in the art and nucleotide sequences for mature KGF-2 (aa36-208) as template. The resulting amplicon was restricted to AflIII and HindIII and subcloned into the NcoI / HindIII digested pQE6 protein expression vector.

Composition of KGF-2Δ33 in pHE1

For polymerase chain reaction-induced amplification and KGF2Δ33 subcloning into E. coli protein expression vector pHE1, two oligonucleotide primers (6153 and 6150) corresponding to a predetermined region of KGF2 were synthesized by the following base sequence Respectively.

For the N-terminal primer (6133), the NdeI restriction site was introduced, while for the C-terminal primer (6150) the Asp718 restriction site was introduced. Primer 6153 includes an ATG sequence within the frame adjacent to the KGF2 encoding region to translate the cloned fragment in E. coli while the primer 6150 is adjacent to the KGF-2 encoding region that is correctly translated in E. coli, And two end codons (which are used primarily in E. coli).

Polymerase chain reaction was performed using standard conditions known to those skilled in the art and nucleotide sequences for mature KGF-2 (aa36-208) as template. The resulting amplicon was restricted to NdeI and Asp718 and subcloned into the NdeI / Asp718 digested pHE1 protein expression vector.

The nucleotide sequence of KGF-2? 33

The amino acid sequence of KGF-2? 33

B. Composition of the optimized KGF-2 [Delta] 33 polynucleotide sequence

To increase the expression level of KGF-2 Δ33 in E. coli, the codons of the complete gene were optimized to match the most highly used ones in E. coli. Since the template used to generate KGF2? 33 has been optimized within the N-terminal region, the C-terminal amino acid (84-208) has to be optimized.

First, amino acids 172-208 were codon optimized to form KGF2? 33 (s172-208). This was accomplished using a nested PCR strategy. The oligonucleotides PM07 and PM08 (corresponding to amino acids 172-208) were combined and heated to 70 DEG C and cooled to 37 DEG C and annealed together. The annealed oligonucleotides were then used as templates for standard PCR reactions induced by primers PM09 and PM10. In a separate PCR reaction using standard conditions known to those skilled in the art and using KGF2? 33 as template, the oligonucleotide PM05 (overlapping with the Pst1 site in the coding region of KGF2) and PM11 were used to amplify the region of KGF2 corresponding to amino acids 84-172 Lt; / RTI > In a third PCR reaction, the first PCR reaction product (corresponding to the codon optimized amino acid 172-208) and the second PCR reaction product (corresponding to the non-codon optimized amino acids 84-172) were combined and oligonucleotides PM05 and PM10 ≪ / RTI > was used as the template for the standard PCR reaction induced. The resulting amplicon was digested with Pst1 / HindIII and subcloned into pQE6KGF2Δ33 digested with Pst1 / HindIII to substitute the corresponding non-codon optimized region to form pQE6KGF2Δ33 (s172-208).

To complete the codon optimization of KGF2, overlapping oligonucleotides were used to form optimized synthetic gene codons for the region of KGF2 corresponding to amino acids 84-172. Four oligonucleotides (PM31, PM32, PM33 and PM34) were combined and subjected to 7 cycles of the following PCR: 94 占 폚, 30 seconds; 46.5 DEG C, 30 seconds; And 72 ° C for 30 seconds.

The second PCR reaction induced with primers PM35 and PM36 was carried out using 1 l of the first PCR reaction as a template according to the following standard procedure. The resulting codon-optimized gene fragment was digested with Pst1 / Sal1 and subcloned with pstE / Sal1 digested pQE6KGF2Δ33 (s172-208) to form a fully optimized KGF2 encoding gene pQE6KGF2Δ33s.

In order to form an alternative E. coli expression vector, KGF2? 33s was PCR amplified using primers PM102 and PM130 on pQE6KGF2? 33s. The resulting amplicon was digested with NdeI and EcoRV and subcloned into pHE1 expression vector digested with NdeI and Asp718 (blunt ends) to form pHE1 [Delta] 33s.

Lt; RTI ID = 0.0 > of KGF-2? 33s < / RTI >

Nucleotide sequence of KGF-2? 33 (s172-208)

Amino acid sequence of KGF-2? 33 (s172-208):

Summary of the Invention

The present invention relates to a liquid preparation and a lyophilized preparation of KGF-2 and its deletion mutants or point mutants or substitution mutants (hereinafter referred to as KGF-2 polypeptides). The present invention also relates to the use of such KGF-2 polypeptide preparations to promote or accelerate soft tissue growth or regeneration, for example, in wound healing, or in the treatment of mucocytis or inflammatory bowel disease. A preferred formulation of the present invention utilizes a novel mutant of KGF-2, in one embodiment, a deletion mutant referred to herein as KGF-2? 33. The adjunct ingredients used in the formulation can provide (1) storage stability to the KGF-2 polypeptide, (2) also enhance and / or enhance the soft tissue healing activity of the therapeutic composition, (3) Thereby providing an active form of KGF-2 polypeptide while allowing for one application and administration.

A first aspect of the invention relates to a formulation comprising a KGF-2 polypeptide and a buffer having a buffering capacity between about pH 5.0 and about pH 8.0. Useful buffering agents include phosphate, acetate, aconitate, succinate, maleate, carbonate and citrate buffers, with citrate being preferred.

A second aspect of the invention relates to a formulation comprising a KGF-2 polypeptide, a lyophilized bulk agent and a buffer having a buffering capacity between about pH 5.0 and about pH 8.0. Useful buffering agents include phosphate, aconitate, succinate, maleate, carbonate and citrate buffers, with citrate being preferred.

A third aspect of the invention relates to a formulation comprising a KGF-2 polypeptide and a thiol-containing compound, preferably a monothiol glycerol, capable of stabilizing a KGF-2 polypeptide. Preferably the formulation comprises a buffer having a buffering capacity between about pH 5.0 and about pH 8.0. The formulation may also include one or more antioxidants or one or more metal chelating agents.

A fourth aspect of the invention is directed to a formulation comprising a KGF-2 polypeptide, a buffer, and a high molecular weight compound that gels the formulation at a predetermined temperature. Preferred high molecular weight compounds are pluronic or poloxamer polyoxyethylene-polyoxypropylene block copolymers. Thiol-containing compounds, such as monothioglycerol, may be included in the formulation to further stabilize the polypeptide.

A fifth aspect of the invention relates to a formulation comprising a KGF-2 polypeptide, a buffering agent and a thickening agent. A thickener is used to increase the viscosity of the formulation. Preferred thickeners are carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC), natozole, and carbomer.

The formulations of the present invention may also be formulated with metal chelating agents, antioxidants or thiol-containing compounds such as ascorbic acid esters, monothiol glycerol, cysteine, tocopherol, butylated hydroxyanisole, sodium sulfate, sodium bisulfite and sodium meta- Sulphite) and preservatives (e.g., m-cresol, phenol, chlorobutane, chlorobutanol, benzyl alcohol, methylparaben and propylparaben). Antimicrobial preservatives may reduce the stability of the KGF-2 formulation. Surprisingly, it has been found that the combined use of methylparaben and propylparaben is suitable for use in KGF-2 preparations. The formulations of the invention may also have a nitrogen blanket overlay in the headspace of the vial. The formulation of the present invention may also be cleaned using helium, argon or nitrogen.

Example 1

KGF-2 liquid formulation

The following ingredients were mixed to form a liquid KGF-2 [Delta] 33 formulation, which was stored at -20 [deg.] C.

2 mg / ml KGF-2 [Delta] 33 polypeptide,

20 mM sodium acetate,

125 mM sodium chloride,

1 mM EDTA,

Water, pH 6.2

This formulation retained in vitro viability for up to 10 months at storage conditions of 2-8 ° C or less. The bioactivity at the 10th month is shown in Fig. This formulation retained all physiological-chemical properties for up to 11 months in storage conditions.

The viability was measured using the cell proliferation assay as follows. BaF3 cells were cultured in RPMI 1640 containing 10% NBCS, 10% WEHI cell conditioned medium, 2 mM glutamine, 600 / / ml GENETICIN, 1 쨉 β-mercaptoethanol / 500 ㎖ growth medium, 50 units penicillin and 50 / / 0.0 > RPMI 1640 < / RTI > medium (Ornitz, DM et al., J. Biol. Chem. 271: 15292-15297 (1996)). For cell proliferation analysis, BaF3 cells were harvested by centrifugation and washed with a basal medium (which had the same composition as the growth medium, but without WEHI-conditioned medium, supplemented with 1 ug / ml heparin). After this operation, cells were resuspended in basal medium and 22,000 cells / 180 [mu] l were plated per well in a 96 well cell culture cluster dish. A suitable dilution of KGF2 (10X higher than the required final concentration) was made in PBS in another 96 well plate and added to the cells in a final volume of 200 [mu] l. Cell plates were incubated in a 37 ° C, 5% CO ₂ incubator for 36-40 hours and 0.5 μCi methyl- ³ H thymidine in 50 μL basal medium was added to each well. The plate was incubated for another 5 hours in a thermostat, and the cells were collected by filtration through a glass fiber filter using TomTek water 96. Thymidine introduced from the monoclonal beta scintillation scintillation counter was counted.

Example 2

KGF-2 Lyophilized formulation

The following ingredients were mixed to form a lyophilized KGF-2 [Delta] 33 formulation.

10 mg / ml KGF-2? 33,

10 mM sodium citrate,

20 mM sodium chloride,

1 mM EDTA,

7% w / v sucrose,

Water (removed upon lyophilization)

pH 6.2

This formulation retained in vitro viability for up to 9 months at 45 ° C or below storage conditions. The life style for the ninth month is shown in Fig. The bioactivity was measured using the cell proliferation assay detailed in Example 1. Reversed phase HPLC demonstrated that the formulation had physiological-chemical properties for up to 8 months at 45 ° C and 75% relative humidity.

Example 3

The thickened KGF-2 formulation

The following ingredients were mixed to form a thickened KGF-2 [Delta] 33 formulation.

2 mg / ml KGF-2? 33,

10 mM sodium citrate,

20 mM sodium chloride,

1 mM EDTA,

7% w / v sucrose,

1.25% carboxymethylcellulose,

water,

pH 6.2

This preparation is prepared by adding KGF-2 [Delta] 33 polypeptide to a carboxymethylcellulose solution. The viscosity of the resulting formulation was about 250 cps as measured by a rotary method using a spindle viscometer. In the presence of carboxymethylcellulose, the KGF-2 polypeptide retained its viability. The viability of the formulation was analyzed using the cell proliferation assay detailed in Example 1.

Example 4

Gel-type KGF-2 preparation

The following ingredients were mixed to form a gel-like KGF-2 [Delta] 33 formulation.

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

Add KGF-2 [Delta] 33 to the pluronic solution at about 2-8 [deg.] C. The viscosity of the resulting formulation was about 50 cps at 20 캜 and was a solid at about 37 캜. KGF-2 retained viability in the presence of pluronic F127 as measured by the cell proliferation assay detailed in Example 1.

Example 5

Activation of KGF 2 by monothioglycerol

KGF-2Δ33 protein raw material preparation (0.1 to 2.0 mg / ml) was prepared with and without monothioglycerol (MTG).

Protein preparations were diluted in 1 x phosphate buffered saline (PBS) at pH 7.2 to obtain the required concentrations for use in cell proliferation assays.

Cell culture

BaF32b cells were cultured in RPMI 1640 containing 10% NBCS, 10% WEHI cell conditioned medium, 2 mM glutamine, 600 / / ml GENETICIN, 1 쨉 β mercaptoethanol / 500 ㎖ growth medium, 50 units penicillin and 50 / / Were grown and maintained in RPMI 1640 medium (Ornitz, DM Et al., J. Biol. Chem. 271: 15292-15297 (1996)).

Cell proliferation assay

For cell proliferation analysis, BaF32b cells were harvested by centrifugation and washed with a basal medium, which had the same composition as the growth medium, but no WEHI-conditioned medium, supplemented with 1 g / ml heparin. After this operation, cells were resuspended in basal medium and 22,000 cells / 180 [mu] l were plated per well in a 96 well cell culture cluster dish. A suitable dilution of KGF2 (10X higher than the required final concentration) was made in PBS in another 96 well plate and added to the cells in a final volume of 200 [mu] l. Cell plates were incubated in a 37 ° C, 5% CO ₂ incubator for 36-40 hours and 0.5 μCi methyl- ³ H thymidine in 50 μL basal medium was added to each well. The plate was incubated for another 5 hours in a thermostat, and the cells were collected by filtration through a glass fiber filter using TomTek water 96. Thymidine introduced from the monoclonal beta scintillation scintillation counter was counted.

result

A. Effect of MTG Concentration on KGF-2 Activity

Cell proliferation assays were performed with KGF-2 exposed to different concentrations of monothioglycerol (MTG). Control samples did not contain excipients. For MTG, stimulation of KGF-2 activity was observed at various concentrations of MTG, as shown in FIG. The increase in activity was 10 to 150% of the control group, depending on the concentration of MTG used. Increases in cell proliferative activity were not observed using other members of the proliferative factor family. From these observations, we concluded that the stimulation of KGF-2 activity by MTG is very specific.

conclusion

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

Example 6

KGF-2 gel formulation with citrate

The following ingredients were mixed to form a KGF-2 formulation, which is liquid at room temperature and becomes a gel upon application to the skin.

20 mM sodium citrate,

125 mM sodium chloride,

1 mM EDTA disodium salt,

17% pluronic 127, pH 6.0,

water.

Example 7

KGF-2 gel formulation having acetic acid salt

The following ingredients were mixed to form a KGF-2 formulation that could be gelled upon application to the skin.

20 mM sodium citrate,

125 mM sodium chloride,

1 mM EDTA disodium salt,

17% pluronic 127, pH 6.0,

water.

Example 8

Liquid KGF-2 preparation

Suitability of sodium citrate as a buffer to maintain KGF-2? 33 was evaluated in four separate preparations and three separate pHs (pH 5.0, pH 5.5 and pH 6.0).

Formulation:

A. KGF-2? 33 1 or 2 mg / ml

20 mM sodium citrate,

125 mM sodium chloride,

1 mM EDTA disodium salt,

water.

B. Same as "A" above, but additionally containing 1% glycerol.

C. Same as "A" above, but additionally contains 0.05% methionine.

D. Same as "A" above, but additionally contains 1% monothioglycerol.

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

2. Freeze-dried preparations

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

Formulation:

A. 10 mM sodium citrate, 20 mM sodium chloride, 1 mM EDTA 2 sodium salt and 4% mannitol, pH 6.0

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

C. 10 mM sodium citrate, 20 mM sodium chloride, 1 mM EDTA 2 sodium salt and 8% trehalose, pH 6.0

The concentration of KGF-2 polypeptide was 3 mg / ml and 8 mg / ml. The evaluation parameters were RP-HPLC, SDS-PAGE, and appearance before and after reconstitution with water.

3. 10 mg / ml KGF-2 lyophilized

Whether the formulation allows lyophilization of the protein at 10 mg / ml was assessed with the subsequent stability of the protein after reconstitution.

Formulation:

10 mM sodium citrate, 20 mM sodium chloride, 1 mM EDTA 2 sodium salt and 4% mannitol, pH 6.0

The lyophilized product was reconstituted with water or water containing 1% monothioglycerol.

Example 9

Thickener stability

The following formulations are prepared according to the methods of the above examples. Constitutions 1 and 2 include only lyophilized KGF-2, Constituents 3 and 4 include a thickening agent as part of a lyo cake, and Configurations 5 through 8 include a thickening agent as part of a liquid diluent.

Configuration number The lyophilized vial Liquid bottle One 7.0 mM sucrose, 10 mM sodium citrate, 20 mM NaCl, 1 mM EDTA, pH 6.2, 0.05 mg / ml KGF-2 Used water 2 7.0 mM sucrose, 10 mM sodium citrate, 20 mM NaCl, 1 mM EDTA, pH 6.2, 1.0 mg / ml KGF-2 Used water 3 0.46 HEC, 7.0% sucrose, 10 mM sodium citrate, 20 mM NaCl, 1 mM EDTA, pH 6.2, 0.05 mg / ml KGF-2 Used water 4 1 mM EDTA, pH 6.2, 1.0 mg / ml KGF-2 < RTI ID = 0.0 > Used water 5 7.0 mM sucrose, 10 mM sodium citrate, 20 mM NaCl, 1 mM EDTA, pH 6.2, 0.05 mg / ml KGF-2 0.46% HEC 10 mM sodium citrate pH 6.2 6 7.0 mM sucrose, 10 mM sodium citrate, 20 mM NaCl, 1 mM EDTA, pH 6.2, 1.0 mg / ml KGF-2 0.46% HEC 10 mM sodium citrate pH 6.2 7 7.0 mM sucrose, 10 mM sodium citrate, 20 mM NaCl, 1 mM EDTA, pH 6.2, 0.05 mg / ml KGF-2 0.92% HPMC 10 mM sodium citrate pH 6.2 8 7.0 mM sucrose, 10 mM sodium citrate, 20 mM NaCl, 1 mM EDTA, pH 6.2, 1.0 mg / ml KGF-2 0.46% HPMC 10 mM sodium citrate pH 6.2

Example 10

The use of KGF-2 in the treatment of chronic wounds is expected to include multiple administrations of the drug, topical administration. Since the lyophilized composition does not contain any preservative therein, the drug product to be reconstituted upon administration should be placed in a separate bottle. This results in a labor-intensive product formulation that must be carried out at each administration, as well as a significant amount of waste medicine. Ideally, the commercial configuration of KGF-2 includes a single bottle of KGF-2, which is used for several applications. To investigate the success of this construction, the compatibility of KGF-2 with the preservative was investigated.

Preservatives were selected from a list of compounds approved by the FDA. The five selected candidate preservatives were based on the dissemination of previously published studies of biopharmaceuticals as well as the Physicians Desk Reference (PDR). Since formulations and dosage forms were not complete, they did not initially focus on stability at pH 6.2 or compatibility with elastomeric closure. Concentrations were selected based on the values provided in the FDA / USP and BP guidelines and literature. The following table shows the five candidate preservatives tested in this study.

Preservative density Commentary Benzyl alcohol 0.9% 1. Not to be used for newborn babies 2. Optimal activity at pH <5 Chlorobutanol 0.5% 1. Rubber closure and incompatibility 2. Poor stability at neutral pH phenol 0.5% 1. It should not be lyophilized. 2. May be corrosive if used topically 3. Excellent effect under acidic conditions Cresol 0.3% 1, not freeze dried 2. May be corrosive if used topically 3. Excellent effect at pH 4-8 Parabens ¹ 0.2% 1. Excellent effect at pH 4 ~ 8 2 weeks use may be irritant 1. " Parabens " are defined in this Example as 0.18% methylparaben and 0.02%

To use a preservative, two criteria must be met. First, compatibility and stability with drug products should be established. Second, the microbial effect must be established (according to USP <51>). The aim of this study was to examine the short-term compatibility of these preservatives with KGF-2.

Materials and methods

Formulation

All chemicals were purchased in the spectrum and were USP / NF grades or equivalents. KGF-2 was dyed with basic formulation buffer containing one of the following formulation buffers: 0.9% benzyl alcohol, 0.5% chlorobutanol, 0.5% phenol, 0.3% m-cresol or 0.18% methylparaben + 0.02% Ryu "). Diafiltration was performed in an Amicon agitation cell with a Biomax 10 kD membrane. All procedures were carried out at 2-8 ° C. A total of five buffer changes were performed for each filtration. The recovery rate in the filtration process was> 90%. KGF-2 was diluted to 1.0 mg / ml, filled in a 2 ml Schott Purform bottle, and sealed with a Diakyo D-7771 serum stopper and aluminum wrinkle seal. The bottles were stored at -80 ° C, 2-8 ° C and 25 ° C / 60% relative humidity. The formulation containing the precipitate was filtered through 0.2 μm and then further analyzed.

Exterior

Under the normal magnification, a visual inspection was performed using forward lighting fluorescent light against a black and white background.

SDS-PAGE

SDS-PAGE was performed under non-reducing conditions using 16% tris-glycine Novex gel. A total of 2 ug KGF-2 per lane was loaded. The gel was manipulated at a constant pressure (125V) for about 2 hours. Dyeing was carried out using a Daiichi silver dyeing kit according to the manufacturer's instructions.

Life castle

The viability of KGF-2 is determined using the murine lymphocyte cell line Baf3 2b stably transfected with the fibroblast growth factor 2iiib receptor. Activity after exposure to the KGF-2 - will be based on the cell proliferation measured by the introduction of the thymidine [methyl- ³ H].

Differential scanning calorimetry (DSC)

DSC thermograms of each formulation were obtained from Calorimetry Scientific Nano DSC (Model 5100). The injection was carried out at a temperature of 5 to 80 캜 at a rate of 1 캜 / min in both heating and cooling directions. The melting point (T _m ) was measured at the peak of the thermal transition. No signal was observed in the cooling direction in any sample, indicating that unfolding is irreversible

RP-HPLC (concentration, purity,% oxidation)

RP-HPLC was performed on a Waters 2690 Alliance system equipped with a Waters 996 photodiode array detector. A Waters Delta-pack C18 column (2.1 x 150 mm, 5 [mu] m, 300 ANGSTROM) was used for separation using a linear gradient from solvent A (0.1% TFA in water) to solvent B (0.07% TFA in acetonitrile). The concentration is measured by the correlation between the standard curve using the internal reference standard of known concentration and the unknown total peak area. Typical chromatograms of standards are given below. Two major peaks were isolated and they were already identified as the complete KGF-2 peak and the oxidized form of KGF-2. The oxidation rate (%) is reported as the area ratio of the total area. Purity (%) is the areal percentage (%) due to the total form and the sum of the oxidized forms.

result

Chlorobutanol and parabens showed compatibility with KGF-2 at 2-8 ° C. Benzyl alcohol, m-cresol and phenol caused rapid aggregation of KGF-2 upon storage at 2-8 ° C. When stored at 25 ° C, all preparations showed poor appearance one week after storage. When freezing / thawing conditions were applied to the solution in the presence of m-cresol and phenol, KGF-2 precipitated from solution.

All preservatives tested showed some de-stabilizing effect on KGF-2 thermal stability. The melting point as measured by DSC showed a drop of about 5 ° C in the presence of all preservatives. Of the preservatives tested, parabens have minimal effect.

The specific activity of KGF-2 was not affected in the presence of any preservatives tested. The total activity of the formulation stored at 25 ° C decreased after 6 weeks of storage due to the precipitation of KGF-2, but the soluble protein in the chloro-butanol, paraben and benzyl alcohol formulations retained the specific activity.

When stored at 2-8 [deg.] C, all preparations are within 10% of the starting concentration as determined by RP-HPLC. When stored at 25 ° C, the formulation containing m-cresol or phenol showed the highest sedimentation rate. Among the formulations tested, preparations containing paraben or chlorobutanol retained maximal KGF-2 in solution during the short-term stability test.

Benzyl alcohol causes the rapid oxidation of KGF-2 and then proceeds with the degradation of the oxidized form. This is manifested by an initial high level of oxidation (formed during the diafiltration process) and a slow transition to additional oxidized forms. When stored at 2-8 ° C, the remaining formulation shows a similar oxidation rate of approximately 4% per month. When stored at 25 ° C, the oxidation rate increases to 30-40% per month. Of the formulations tested, those containing parabens exhibit the lowest average oxidation rate.

Purity is determined as a total of the major oxidized forms of KGF-2 as a ratio of the total chromatographic area. Preparations containing benzyl alcohol or phenol have low initial purity, gradually increase after increasing purity. This corresponds to a sharp increase in chromatographic peak at zero time, which appears to correspond to soluble aggregates. As the precipitate continues to form, this peak disappears and there is no subsequent increase at any other peak area. When stored at 25 ° C, preparations containing parabens retain their maximum purity.

SDS-PAGE shows a slight increase in dimer band in the formulation containing benzyl alcohol at time 0 as well as after 6 weeks of storage. A slight increase in lane streaking and dimer levels is observed in formulations stored at 25 ° C.

conclusion

KGF-2 is incompatible with benzyl alcohol, m-cresol and phenol under the conditions tested. In these formulations, flocculation and subsequent precipitation were the primary pathways of degradation. Of the preservatives tested, methyl / propyl paraben had minimal effect on the short-term stability of KGF-2.

Example 11

Among the lyophilized preparations, KGF-2

The following ingredients were mixed to form a KGF-2? 33 premix formulation.

3.3 mg / ml KGF-2? 33,

10 mM sodium citrate,

20 mM sodium chloride,

1 mM EDTA,

2% w / v glycine,

0.5% sucrose,

Water (removed upon lyophilization),

pH 6.2

This preparation was lyophilized according to the second lyophilization cycle described above.

This formulation retained in vitro viability for up to 12 months at storage conditions below 25 ° C.

Example 12

KGF-2 topical preparation

The following ingredients were mixed to form a KGF-2 formulation for topical application.

10 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 is obvious that the present invention can be carried out by methods other than those specifically disclosed in the foregoing description and examples.

Many modifications and variations of the present invention are possible in light of the above teaching, and are therefore within the scope of the appended claims.

All publications (eg, patents, patent applications, publications, laboratory manuals, books, or other documents) cited herein are incorporated by reference.

Certificate of Microorganism Deposit (PCT Rule 13bis)

A. The following certificate relates to the microorganism mentioned on line 4 on page 21 of the specification B. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: December 16, 1994 Accession number: ATCC 75977 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmid, 366885A D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For office International Office This paper has been repaired with the international application This paper has been repaired by the international collection office. Authorized Authorized

Certificate of Microorganism Deposit (PCT Rule 13bis)

A. The following certificate is about the microorganism mentioned on line 10 on page 4 of the specification B. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: 1998. 1. 9 Accession number: ATCC 209575 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmid, pHEKGF-2? 33 D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For office International Office This paper has been repaired with the international application This paper has been repaired by the international collection office. Authorized Authorized

Claims (78)

(a) a KGF-2 polypeptide having a concentration range of from about 0.02 to about 40 mg / ml (w / v); (b) a buffer having a buffering capacity of from about pH 5.0 to about pH 8.0 in a concentration range of from about 5 mM to about 50 mM; (c) a pharmaceutically acceptable diluent for making the composition into a specified volume; and (d) preservatives selected from the group consisting of m-cresol, chloro butanol, and mixtures of methyl paraben and propyl paraben; or Its reaction product &Lt; / RTI &gt; The method according to claim 1, (a) a chelating agent wherein the concentration range is from about 0 mM to about 10 mM; And (b) an isotonic agent having a concentration range of from about 0 mM to about 150 mM &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof. 3. The pharmaceutical composition of claim 2, wherein said isotonic agent is selected from the group consisting of NaCl, glycine, sucrose, mannitol, and mixtures thereof. The method according to claim 1, (1) from about 0.5% to about 2% w / v glycerol, (2) from about 0.1% to about 1% w / v methionine or (3) from about 0.1% to about 2% w / v monothioglycerol &Lt; / RTI &gt; 2. The pharmaceutical composition of claim 1, wherein 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 of claim 5, wherein 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 of claim 6, wherein the KGF-2 polypeptide is present in a concentration range of about 0.2 to 4 mg / ml. 2. The pharmaceutical composition according to claim 1, wherein the KGF-2 polypeptide is KGF-2? 33. 2. The pharmaceutical composition according to claim 1, wherein the diluent is water. 3. The pharmaceutical composition of claim 2, wherein the chelating agent is at a concentration of about 1 mM EDTA and the isotonic agent is present at a concentration of about 125 mM. The pharmaceutical composition of claim 1, wherein the pH is from about pH 5.5 to about pH 6.5. 12. The pharmaceutical composition according to claim 11, wherein the pH is about pH 6.0. The pharmaceutical composition according to claim 1, wherein the buffer is selected from the group consisting of phosphonic acid, acetic acid, aconitic acid, citric acid, glutaric acid, malic acid, succinic acid, carboxylic acid and alkali or alkaline earth salts thereof. 14. The pharmaceutical composition of claim 13, wherein the buffer is a phosphate, acetate, or citrate salt. 14. The pharmaceutical composition of claim 13, wherein the buffer is a citrate salt. The pharmaceutical composition of claim 1, wherein the buffer is present in a concentration range of from about 5 mM to about 30 mM. 17. The pharmaceutical composition of claim 16, wherein the buffer is a citrate salt present in a concentration from about 10 mM to about 20 mM. The pharmaceutical composition of claim 1, further comprising a stabilizing amount of at least one of (a) an antioxidant or (b) a thiol-compound. The pharmaceutical composition of claim 1, wherein the composition is maintained at a temperature of -20 占 폚 or lower. The method of claim 1, wherein the KGF-2 33 polypeptide is selected from the group consisting of a KGF-2 33 polypeptide having an N-terminal methionine, a KGF-2 33 polypeptide lacking N-terminal methionine, and mixtures thereof &Lt; / RTI &gt; The pharmaceutical composition of claim 1, further comprising a bulk agent. 22. The pharmaceutical composition according to claim 21, wherein the bulk agent is selected from the group consisting of sucrose, glycine, mannitol, trehalose and mixtures thereof. 23. The pharmaceutical composition according to claim 22, wherein the bulk agent is sucrose or a mixture of sucrose and glycine. The pharmaceutical composition of claim 2, further comprising a bulk agent. 23. The pharmaceutical composition of claim 22, wherein the bulk agent is present in a concentration from about 2% to about 10% w / v. 23. The pharmaceutical composition according to claim 22, wherein the bulk agent is 5% mannitol, 7% sucrose, 8% trehalose, or 2% glycine + 0.5% sucrose. 22. The pharmaceutical composition of claim 21, wherein the pH is about pH 6.2. 22. The pharmaceutical composition according to claim 21, wherein the diluent is water. 22. The pharmaceutical composition according to claim 21, wherein the buffer is selected from the group consisting of phosphonic acid, acetic acid, aconitic acid, citric acid, glutaric acid, malic acid, succinic acid, carboxylic acid and alkali or alkaline earth salts thereof. 30. The pharmaceutical composition of claim 29, wherein said buffer is a phosphate or citrate salt. 31. The pharmaceutical composition of claim 30, wherein the buffer is a citrate salt. 29. The pharmaceutical composition of claim 28, wherein at least 90% of the water is removed by lyophilization. 33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is reconstituted in an amount effective to maintain an isotonic condition of about 290 mOsm. 22. The pharmaceutical composition according to claim 21, wherein the KGF-2 polypeptide is KGF-2? 33. 35. The method of claim 34, wherein said KGF-2 33 polypeptide is selected from the group consisting of KGF-2 33 polypeptide bearing N-terminal methionine, KGF-2 33 polypeptide lacking N-terminal methionine, and mixtures thereof &Lt; / RTI &gt; 22. The pharmaceutical composition of claim 21, wherein the buffer is added at a concentration of about 5 mM to about 50 mM. 37. The pharmaceutical composition of claim 36, wherein the buffer is a citrate at a concentration of about 10 mM. 22. The pharmaceutical composition of claim 21, further comprising a stabilizing amount of at least one of (a) an antioxidant or (b) a thiol-compound. (B) about 0.01% to about 2% w / v ascorbic acid; (c) about 0.01% to about 2% w / v ascorbic acid; 2% w / v methionine, or (d) a combination thereof, in a sterile water containing a stabilizing amount of an antioxidant. The pharmaceutical composition of claim 1, further comprising an amount of a thickener effective to increase the viscosity to from about 50 to about 10,000 cps. 41. The pharmaceutical composition of claim 40, wherein the thickener is present in an amount effective to increase the viscosity to from about 50 to about 1,000 cps. 42. The pharmaceutical composition of claim 41, wherein the thickener is present in an amount effective to increase the viscosity from about 200 to about 300 cps. 41. The pharmaceutical composition of claim 40, wherein the thickener is present at a concentration of 0 to 5% (w / w). 41. The pharmaceutical composition according to claim 40, wherein the thickener is an acrylic acid high molecular weight polymer crosslinked with water soluble etherified cellulose, or allyl sucrose or allyl ether of pentaerythritol. 45. The pharmaceutical composition of claim 44, wherein the etherified cellulose is alkylcellulose, hydroxyalkylcellulose, carboxyalkylcellulose, or alkylhydroxyalkylcellulose. 41. The pharmaceutical composition according to claim 40, wherein the etherified cellulose is methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose or carboxymethyl cellulose. 47. The pharmaceutical composition of claim 46, wherein the etherified cellulose derivative has a molecular weight of from about 50,000 to about 700,000 and a concentration of from about 0 to about 20% by weight. 50. The pharmaceutical composition of claim 47, wherein the etherified cellulose derivative has a molecular weight of from about 80,000 to about 240,000 and a concentration of from about 2 to about 8 weight percent. 43. The pharmaceutical composition of claim 42, wherein the buffer is a citrate having a concentration of from about 10 mM to about 50 mM. 50. The pharmaceutical composition of claim 49, wherein the buffer is a citrate having a concentration of from about 10 mM to about 20 mM. 50. The pharmaceutical composition of claim 49, wherein the bulk agent is sucrose at a concentration of from about 0.01% to about 5%. 52. The pharmaceutical composition of claim 51, wherein the thickener is added directly to the liquid formulation and then lyophilized. 52. The pharmaceutical composition of claim 51, wherein the thickener is added to the lyophilized formulation by reconstituting the lyophilized formulation by adding a suitable diluent wherein the thickener is dissolved. 22. The pharmaceutical composition of claim 21, further comprising an amount of a thickener effective to increase the viscosity to from about 50 to about 10,000 cps. The pharmaceutical composition of claim 1, further comprising an amount of a gelling agent effective to increase the viscosity at room temperature from about 0.1 to about 10,000 cps. 22. The pharmaceutical composition of claim 21, further comprising an effective gelling agent effective to increase the viscosity at room temperature from about 0.1 to about 10,000 cps. 56. The pharmaceutical composition according to claim 55, wherein the gel-forming agent is a water-soluble polymer capable of forming a viscous aqueous solution, or a water-insoluble water-swellable polymer capable of forming a viscous solution. 58. The composition of claim 57 wherein said gel formers are high molecular weight polymers selected from the group consisting of vinyl polymers, polyoxyethylene-polyoxypropylene copolymers, polysaccharides, proteins, poly (ethylene oxide), acrylamide polymers, A pharmaceutical composition. 59. The composition of claim 58, wherein the gel-forming agent is selected from the group consisting of (1) a vinyl polymer selected from the group consisting of polyacrylic acid, polymethacrylic acid, polyvinylpyrrolidone, polyvinyl alcohol, and salts and esters thereof; Or (2) a polysaccharide selected from the group consisting of cellulose derivatives, glycosaminoglycan, agar, pectin, alginic acid, dextran, alpha-amylose, amylopectin, chitosan and salts and esters thereof. 59. The pharmaceutical composition according to claim 58, wherein the gel-forming agent is a glycosaminoglycan selected from the group consisting of hyaluronic acid, chondroitin, chondroitin-4-sulfate, heparan sulfate, heparin and salts and esters thereof. 61. The pharmaceutical composition according to claim 60, wherein the glycosaminoglycan is present together with collagen, gelatin or fibronectin. 59. The pharmaceutical composition according to claim 58, wherein the gel-forming agent is an acrylamide polymer selected from the group consisting of polyacrylamide or polymethacrylamide. 59. The pharmaceutical composition according to claim 58, wherein the gel-forming agent is a polyoxyethylene-polyoxypropylene block copolymer. 66. The pharmaceutical composition of claim 63 comprising from about 10% to about 60% by weight of a polyoxyethylene-polyoxypropylene block copolymer having an average molecular weight of from about 500 to about 50,000. 65. The pharmaceutical composition of claim 64 comprising from about 14 to about 18 weight percent of a polyoxyethylene-polyoxypropylene block copolymer having a molecular weight range of from 1,000 to 15,000. The pharmaceutical composition of claim 1, wherein the KGF-2 polypeptide is present in a concentration from about 0.01 mg / ml to about 10 mg / ml. The method of claim 1, wherein the KGF-2 polypeptide is selected from the group consisting of Ala (63) -Ser (208) (KGF-2Δ28) and Ser (69) Terminal deletion selected from the group consisting of: &lt; RTI ID = 0.0 &gt; 69. The pharmaceutical composition of claim 67, wherein said KGF-2 polypeptide possesses an N-terminal methionine, is deficient in N-terminal methionine, or a mixture thereof. 2. The method of claim 1, wherein the KGF-2 polypeptide is 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). &Lt; / RTI &gt; 70. The pharmaceutical composition of claim 69, wherein the KGF-2 polypeptide has N-terminal methionine, N-terminal methionine is deficient, or a mixture thereof. The method according to claim 1, (a) lysine; (b) hydroxypropyl -? - cyclodextrin; And (c) sulfated-beta-cyclodextrin; &Lt; / RTI &gt; or a combination thereof. The pharmaceutical composition according to claim 1, wherein the preservative is a mixture of methylparaben and propylparaben. 73. The pharmaceutical composition of claim 72, wherein said composition comprises 0.18% methyl paraben and 0.02% propyl paraben. (a) about 1.0 mg / ml KGF-2; (b) 20 mM citrate (pH 5-5.5); And (c) 0.01% polysorbate 80 &Lt; / RTI &gt; 74. The pharmaceutical composition of claim 74, further comprising 1 mM EDTA. (a) about 3.3 mg / ml of 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) about pH 6.2; Or a reaction product thereof &Lt; / RTI &gt; 78. The pharmaceutical composition of claim 77, wherein at least 90% of the water is removed by lyophilization. (a) about 1.0 mg / ml of KGF-2; (b) 0.46% hydroxyethylcellulose; (c) 7% sucrose; (d) 20 mM sodium citrate; (e) 20 mM sodium chloride; (f) 1 mM EDTA; And (g) about pH 6.2; Or a reaction product thereof &Lt; / RTI &gt;