CA2586213A1 - Formulations, methods of production and uses of fgf-20 - Google Patents

Abstract

The present invention provides improved formulations comprising FGF-20, its fragments, derivatives, variants, homologs, analogs, or a combination thereof, improved methods for production, and methods of use of the compositions of the invention.

Description

DEMANDE OU BREVET VOLUMINEUX

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PLUS D'UN TOME.

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FORMULATIONS, METHODS OF PRODUCTION AND USES OF FGF-20 1. FIELD OF THE INVENTION
The present invention relates to improved formulations comprising FGF-20, its fragments, derivatives, variants, homologs, analogs, or a combination thereof, improved methods for production, and methods of use thereof.

2. BACKGROUND OF THE INVENTION
The fibroblast growth factor ("FGF") family consists of more than 20 members, each containing a conserved amino acid core (see, e.g., Powers et al., Endocr.
Relat. Cancer, 7(3):65-197 (2000)). FGFs regulate diverse cellular functions such as growth, survival, apoptosis, motility, and differentiation (see, e.g., Szebenyi et aL, lnt. Rev. Cytol., 185:45-106 (1999)). Members of the FGF family are also involved in various physiological and pathological processes during embryogenesis and adult life, including morphogenesis, limb development, tissue repair, inflammation, angiogenesis, and tumor growth and invasion (see, e.g., Powers et al., Endocr. Relat.
Cancer, 7(3):165-197 (2000); and Szebenyi et aL, Int. Rev. Cytol. 185:45-106 (1999)).

Through a homology-based genomic mining process, a novel human FGF, FGF-20, was discovered. See U.S. Patent Application Nos. 09/494,585, filed January 13, 2000, and 09/609,543, filed July 3, 2000, the disclosure of each references is incorporated herein by reference. The amino acid sequence of FGF-20 shows close homology with human FGF-9 (70% identity) and FGF16 (64% identity).

Recombinant full length FGF-20 has been shown to induce a proliferative response in mesenchymal and epithelial cells, but not in human smooth muscle, erythroid, or endothelial cells (see, e.g., Jeffers et al., Cancer Res. 61(7):3131-3138 (2001)). FGF-20 and its variants or derivatives have also been shown to be effective in preventing and/or treating certain diseases, such as oral mucositis (see International Patent Application Publication No. WO
2003/099201, filed May 9, 2003), inflammatory bowel disease ("IBD") (see International Patent Application Publication No.
WO 2002/058716, filed November 6, 2001), and certain diseases related to central nerve system, such as Parkinson's Disease, and certain diseases related to cardiovascular system, such as stroke (see International Patent Application Publication No. 2004/100892, filed May 10, 2004). FGF-20 and its variants or derivatives have also been shown to be effective in preventing and/or treating symptoms associated with radiation exposure (see International Patent Application Publication No.
WO 2005/025489, filed May 10, 2004). The disclosure of each reference is incorporated herein by reference in its entirety.

Therefore, there is a great need for pharmaceutical formulations comprising FGF-20 and/or its variants or derivatives that are suitable for clinical uses, of which formulations are stable and can be produced at a commercial scale.

Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.

3. SUMMARY OF THE INVENTION
The present invention provides improved formulations comprising a fibroblast growth factor, preferably FGF-20, or its fragments, derivatives, variants, homologs, analogs, or a combination thereof. The present invention also provides improved production methods for isolating one or more CG53135 proteins. The present invention further provides methods of use of CG53135 proteins and the improved formulations comprising one or more CG53135 proteins.

In one embodiment, the present invention provides a formulation comprising about 0.1-1 M
arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, about 0.01-0.1 M
sodium phosphate monobasic (NaH2PO4=H2O), about 0.01 %-0.1 % weight/volume ("w/v") polysorbate 80 or polysorbate 20, and an isolated fibroblast growth factor ("FGF"). In a specific embodiment, the concentration of the FGF in the formulations of the invention is about 0.005 mg/mI
to about 50 mg/mI. The FGF protein is preferably a CG53135 protein. In a specific embodiment, the formulations of the present invention comprise one or more isolated proteins selected from the group consisting of: (a) a protein comprising an amino acid sequence of SEQ ID
NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; (b) a protein with one or more amino acid substitutions to the protein of (a), wherein said substitutions are no more than 15% of the amino acid sequence of SEQ
ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and wherein said protein with one or more amino acid substitutions retains cell proliferation stimulatory activity;
and (c) a fragment of the protein of (a) or (b), which fragment retains cell proliferation stimulatory activity. In some embodiments, the formulations of the invention comprise one or more isolated proteins, where the concentration of the proteins is of 0.5-30 mg/mI. In a specific embodiment, the concentration of the proteins is 10 mg/ml. In some embodiments, the formulations of the invention are lyophilized or spray dried.

In a specific embodiment, the formulations of the invention comprise an arginine in a salt form, which is selected from the group consisting of arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride. In one embodiment, the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose in the formulations of the invention has a concentration of 0.01-0.7 M, preferably 0.5 M.

In another embodiment, the sodium phosphate monobasic in the formulations of the invention has a concentration of 0.05 M. In another embodiment, polysorbate 80 or polysorbate 20 of the formulations of the invention is 0.01 % (w/v). In one embodiment, the formulations of the invention comprise polysorbate 80. In another embodiment, the formulations of the invention comprise polysorbate 20.

In a specific embodiment, a formulation of the invention comprises about 10 mg/mI of an isolated protein comprising an amino acid sequence of SEQ ID NO:24, 0.5 M
arginine sulfate, 0.05 M sodium phosphate monobasic, and 0.01 % (w/v) polysorbate 80. In another specific embodiment, a formulation of the invention comprises about 10 mg/ml of an isolated protein comprising an amino acid sequence of SEQ ID NO:2, 0.5 M arginine sulfate, 0.05 M sodium phosphate monobasic, and 0.01 %(w/v) polysorbate 80. In another embodiment, a formulation of the invention comprises 0.5 M
arginine sulfate, 0.05 M sodium phosphate monobasic, 0.01 %(w/v) polysorbate 80, and about 10 mg/mi of a mixture of isolated proteins, wherein said proteins comprise a first protein comprising an amino acid sequence of SEQ ID NO:24, and a second protein comprising an amino acid sequence of SEQ ID NO:2. In a specific embodiment, a formulation of the invention further comprises one or more isolated proteins, wherein said proteins comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:26, 28, 30 and 32. In some embodiments, the formulations of the invention comprise one or more isolated proteins that are carbamylated.

In another embodiment, the present invention provides methods of increasing solubility of a fibroblast growth factor ("FGF") in an aqueous solution by adding arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, or a combination thereof to said solution to a final concentration of 0.01 - 1 M. In some embodiment, the fibroblast growth factor is an isolated CG53135 protein. In a specific embodiment, the fibroblast growth factor is an isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID
NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

In some embodiments, an arginine in a salt form is selected from the group consisting of arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride. In one embodiment, the final concentration of arginine in a salt form is 0.01 - 0.7 M, preferably 0.5 M. In some embodiments, the methods of the invention further comprise adding acetate, succinate, tartrate, or a combination thereof to the solution to increase the solubility of the protein.
Preferably, the acetate, succinate, tartrate, or a combination thereof has a final concentration of 0.01-0.2 M in the solution.

In another embodiment, the present invention provides a method of producing an isolated protein comprising the steps of: (1) fermenting an E. coli cell containing a vector comprising SEQ ID
NO:8; (2) chilling the fermented culture to 10-15 C; (3) diluting the chilled culture with a lysis buffer comprising 50-100 mM sodium phosphate, 60 mM ethylene diamine tetraacetic acid, 7.5 mM DTT, and 3.5-5 M urea; (4) lysing the cells in the diluted culture; (5) loading the resultant cell lysate onto a pre-equilibrated cation exchange column, and flushing the column with a buffer comprising 50-100 mM sodium phosphate, 40 mM EDTA, 10 mM sodium sulfate, and 3-5 M urea; (6) washing the flushed column with a buffer comprising 50-100 mM sodium phosphate, 5 mM EDTA, 10-25 mM
sodium sulfate, and 2.22 mM dextrose; (7) washing the column again with an elution buffer comprising 50-100 mM sodium phosphate, 5 mM EDTA, 150-250 mM sodium sulfate, and 0.5-1 M
L-arginine; (8) loading the resultant eluate onto a hydrophobic interaction chromatography column pre-equilibrated with 50-100 mM sodium phosphate, 150-250 mM sodium sulfate, 5 mM EDTA, and 1 M arginine; (9) washing the resulting column with a solution comprising 100-250 mM sodium phosphate, 5 mM EDTA, and 0.8-1 M arginine; and (10) washing the column again with a solution comprising 50-100 mM sodium phosphate, 5 mM EDTA, and 0.1-0.3 M arginine to elute the protein.

In a specific embodiment, the method further comprises the steps of: (11) concentrating the resultant eluate; (12) filtering the retentate obtained together with a solution comprising 50 mM
sodium phosphate, 0.5 M arginine; (13) concentrating the filtered retentate;
and (14) filtering the concentrated retentate.

In some embodiments, the cells are fermented by a method comprising the steps of: (a) culturing E. coli cells containing a vector comprising SEQ ID N0:8 to exponential growth phase with 2.5 to 4.5 OD600 units in a chemically defined seed medium; (b) inoculating cells of step (a) to a seed medium and culturing the cells to an exponential growth phase with 3.0 to 5.0 OD600 units; (c) transferring the cells of step (b) to a chemically defined batch medium; (d) culturing the cells of step (c) to 25-35 units OD600, and adding additional chemically defined medium with a feeding rate of 0.7 g/kg broth/minute; (e) culturing the cells of step (d) to 135 to 165 units OD600; and (f) culturing the cells of step (e) for about four hours.

The present invention also provides isolated proteins produced by the methods of the invention.

Uses of the compositions and formulations of the invention for preventing and/or treating a disease, e.g., alimentary mucositis, arthritis, a disorder or symptom associated with radiation exposure, a disorder of central nerve system or cardiovascular system, are also provided.

3.1 TERMINOLOGY
As used herein, the term "about" in the context of a given numerate value or range refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range.

As used herein, the term "CG53135", refers to a class of proteins (including peptides and polypeptides) or nucleic acids encoding such proteins or their complementary strands, where the proteins comprise an amino acid sequence of SEQ ID N0:2 (211 amino acids), or its fragments, derivatives, variants, homologs, or analogs. In a preferred embodiment, a CG53135 protein retains at least some biological activity of FGF-20. As used herein, the term "biological activity" means that a CG53135 protein possesses some but not necessarily all the same properties of (and not necessarily to the same degree as) FGF-20.

A member (e.g., a protein and/or a nucleic acid encoding the protein) of the CG53135 family may further be given an identification name. For example, CG53135-01 (SEQ ID
NOs:1 and 2) represents the first identified FGF-20 (see U.S. Patent Application No.
09/494,585); CG53135-05 (SEQ ID NOs:8 and 2) represents a codon-optimized, full length FGF-20 (i.e., the nucleic acid sequence encoding FGF-20 has been codon optimized, but the amino acid sequence has not been changed from the originally identified FGF-20); CG53135-12 (SEQ ID NOs:21 and 22) represent a single nucleotide polymorphism ("SNP") of FGF-20 where one amino acid in CG53135-12 is different from SEQ ID N0:2 (the aspartic acid at position 206 is changed to asparagine, c206D->N").
Some members of the CG53135 family may differ in their nucleic acid sequences but encode the same CG53135 protein, e.g., CG53135-01, CG53135-03, and CG53135-05 all encode the same CG53135 protein. An identification name may also be an in-frame clone ("IFC") number, for example, IFC 250059629 (SEQ ID NOs:33 and 34) represents amino acids 63-196 of the full length FGF-20 (cloned in frame in a vector). Table 1A shows a summary of some of the CG53135 family members. In one embodiment, the invention includes a variant of FGF-20 protein, in which some amino acids residues, e.g., no more than 1%, 2%, 3%, 5%, 10% or 15% of the amino acid sequence of FGF-20 (SEQ ID NO:2), are changed. In another embodiment, the invention includes nucleic acid molecules that can hybridize to FGF-20 under stringent hybridization conditions.

Table 1A. Summary of some of the CG53135 family members Name SEQ ID NO Brief Description (DNA/Protein) CG53135-01 1 and 2 FGF-20 wild type, stop codon removed CG53135-02 3 and 4 Codon optimized, amino acids 2-54 (as numbered in SEQ ID
NO:2) were removed CG53135-03 5 and 2 FGF-20 wild type CG53135-04 6 and 7 Amino acids 20-51 (as numbered in SEQ ID NO:2 were removed, also valine at position 85 is changed to alanine ("8 V->A") CG53135-05 8 and 2 Codon optimized, full length FGF-20 CG53135-06 9 and 10 Amino acids 20-51 (as numbered in SEQ ID NO:2) were removed CG53135-07 11 and 12 Protein consisting of amino acids 1-18 (as numbered in SEQ ID
NO:2) CG53135-08 13 and 14 Protein consisting of amino acids 32-52 (as numbered in SEQ ID
NO:2) CG53135-09 15 and 16 Protein consisting of amino acids 173-183 (as numbered in SEQ
ID NO:2) CG53135-10 17 and 18 Protein consisting of amino acids 192-211 (as numbered in SEQ
ID NO:2) CG53135-11 19 and 20 Protein consisting of amino acids 121-137 (as numbered in SEQ
ID NO:2) CG53135-12 21 and 22 FGF-20 SNP, aspartic acid at position 206 is changed to asparagines (i206D--).N") as compared to CG53135-01 CG53135-13 23 and 24 CG53135-05 minus first 2 amino acids at the N-terminus CG53135-14 25 and 26 CG53135-05 minus first 8 amino acids at the N-terminus CG53135-15 27 and 28 CG53135-05 minus first 11 amino acids at the N-terminus CG53135-16 29 and 30 CG53135-05 minus first 14 amino acids at the N-terminus CG53135-17 31 and 32 CG53135-05 minus first 23 amino acids at the N-terminus IFC 250059629 33 and 34 In frame clone, open reading frame comprising a nucleotide sequence encoding amino acids 63-196 of FGF-20 (SEQ ID NO:2) IFC 250059669 35 and 36 In frame clone, open reading frame comprising a nucleotide sequence encoding amino acids 63-211 of FGF-20 (SEQ ID NO:2) IFC 317459553 37 and 38 In frame clone, open reading frame comprising a nucleotide sequence encoding amino acids 63-194 of FGF-20 (SEQ ID NO:2) with 1G,E
IFC 317459571 39 and 40 In frame clone, open reading frame comprising a nucleotide sequence encoding amino acids 63-194 of FGF-20 (SEQ ID NO:2) IFC 250059596 41 and 10 In frame clone, open reading frame comprising a nucleotide sequence encoding amino acids 1-19 and 52-211 of FGF-20 (SEQ
ID NO:2) IFC 316351224 41 and 10 In frame clone, open reading frame comprising a nucleotide sequence encoding amino acids 1-19 and 52-211 of FGF-20 (SEQ
ID NO:2).

As used herein, the term "effective amount" refers to the amount of a therapy (e.g., a formulation comprising a CG53135 protein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof, prevent the advancement of a disease, cause regression of a disease, prevent the recurrence, development, or onset of one or more symptoms associated with a disease, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

As used herein, the term "FGF-20" refers to a protein comprising an amino acid sequence of SEQ ID NO:2, or a nucleic acid sequence encoding such a protein or the complementary strand thereof.

As used herein, the term "hybridizes under stringent conditions" describes conditions for hybridization and washing under which nucleotide sequences at least 30%
(preferably, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. In one, non limiting example, stringent hybridization conditions comprise a salt concentration from about 0.1 M to about 1.0 M sodium ion, a pH from about 7.0 to about 8.3, a temperature is at least about 60 C, and at least one wash in 0.2 X sodium chloride/sodium citrate (SSC), 0.01% bovine serum albumin (BSA). In another non-limiting example, stringent hybridization conditions are hybridization at 6X SSC at about 45 C, followed by one or more washes in 0.1X SSC, 0.2% sodium dodecyl sulfate (SDS) at about 68 C. In yet another non-limiting example, stringent hybridization conditions are hybridization in 6XSSC at about 45 C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 C (i.e., one or more washes at 50 C, 55 C, 60 C or 65 C). It is understood that the nucleic acids of the invention do not include nucleic acid molecules that hybridize under these conditions solely to a nucleotide sequence consisting of only A or T
nucleotides.

As used herein, the term "isolated" in the context of a protein agent refers to a protein agent that is substantially free of cellular material or contaminating proteins from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material"
includes preparations of a protein agent in which the protein agent is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a protein agent that is substantially free of cellular material includes preparations of a protein agent having less than about 30%, 20%, 10%, or 5% (by dry weight) of host cell proteins (also referred to as a "contaminating proteins"). When the protein agent is recombinantly produced, it is also preferably substantially free of culture medium, f.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein agent preparation. When the protein agent is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein agent.
Accordingly, such preparations of a protein agent have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the protein agent of interest. In a specific embodiment, protein agents disclosed herein are isolated.

As used herein, the term "isolated" in the context of nucleic acid molecules refers to a nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, nucleic acid molecules are isolated.

As used herein, the terms "prevent," "preventing," and "prevention" refer to the prevention of the recurrence, onset, or development of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof in a subject resulting from the administration of a therapy (e.g., a composition comprising a CG53135 protein), or the administration of a combination of therapies.

As used herein, the term "prophylactically effective amount" refers to the amount of a therapy (e.g., a composition comprising a CG53135 protein) which is sufficient to result in the prevention of the development, recurrence, or onset of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof, or to enhance or improve the prophylactic effect(s) of another therapy.

As used herein, the term "stability" in the context of a protein formulation, refers to the ability of a particular protein formulation to maintain the native, active structure of a protein as the protein is exposed to thermo-mechanical stresses over time. In some embodiments, stability of a protein formulation generally refers to the tendency of a protein formulation to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses, as well as the tendency of a protein formulation to form biologically inactive and/or insoluble aggregates of the protein as a result of interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. A related parameter to the "stability"
of a protein formulation is its solubility in that higher molecular weight aggregates and denatured forms of a protein, including partially denatured forms of a protein, which are generally less soluble than their non-aggregated, lower molecular weight counterparts and native forms of the protein.

Another related parameter to the "stability" of a protein formulation is the protein concentration in that physically stable formulations may become less physically stable as the concentration of the protein is increased or decreased.

As used herein, the terms "subject" and "subjects" refer to an animal, preferably a mammal, including a non-primate (e.g., a cow, pig, horse, cat, or dog), a primate (e.g., a monkey, chimpanzee, or human), and more preferably a human. The term "subject" is used interchangeably with "patient" in the present invention.

As used herein, the terms "treat," "treatment," and "treating" refer to the reduction of the progression, severity, and/or duration of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or amelioration of one or more symptoms thereof, wherein such reduction and/or amelioration result from the administration of one or more therapies (e.g., a composition comprising a CG53135 protein).

As used herein, the term "therapeutically effective amount" refers to the amount of a therapy (e.g., a composition comprising a CG53135 protein), which is sufficient to reduce the severity or duration of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases), prevent the advancement of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases), cause regression of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases), ameliorate one or more symptoms associated with a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases), or enhance or improve the therapeutic effect(s) of another therapy.

4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an example of manufacturing a drug product comprising one or more CG53135 proteins.

FIG. 2 (A) shows SDS-PAGE analysis (gel code blue) of CG53135 produced by Process I
and Process 2 (as described in Section 6), respectively. Lane 1: molecular weight markers (kDa);
lane 2-4: purified CG53135 (10 pg), reduced. Lane 5-8: Process 1 reference standard DEV10 (720, 380, 45, and 28 ng) reduced. (B) shows SDS-PAGE analysis (silver stain) of CG53135. Lane 1:
molecular weight markers (kDa) are shown on the left; lane 3: purified CG53135 by Process 2 (5 pg); lane 5: purified CG53135 reference standard Process 1(5 pg); lane 7:
purified CG53135 Process 2 (10 pg); lane 9: purified CG53135 reference standard Process 1 (10 pg).

FIG. 3 shows RP-HPLC analysis of CG53135 purified by Process 1 and Process 2, respectively (Process 1 is represented by the solid line).

FIG. 4 shows SEC-HPLC analysis of CG53135 purified by Process I and Process 2, respectively.

FIG. 5 shows host cell protein analysis of CG53135 E. coli purified product (by Process 1 and Process 2, respectively).

FIG. 6 shows Western Blot analysis of CG53135 purified by Process I and Process 2, respectively. Western blot was probed with anti-CG53135-05 antibody. Lane 1:
molecular weight marker (kDa); lane 3: CG53135 purified by Process 2 (10 pg); lane 5: CG53135 purified by Process 1 (10 pg).

FIG. 7 shows RP-HPLC identification analysis of CG53135 purified by Process 1 and Process 2, respectively. Process 2 is represented by the dashed line.

FIG. 8 shows tryptic map of CG53135 purifled by Process 1 and Process 2, respectively.
FIG. 9 shows circular dichroism spectroscopy analysis of CG53135 produced by Process 1 and Process 2, respectively. The lower (grey) trace represents Process 1, and the upper (black) trace represents Process 2.

FIG. 10 shows near UV circular dichroism spectroscopy analysis of CG53135 purified by Process 1 and Process 2, respectively. The upper (grey) trace is the near UV
CD spectrum of Process I and the black trace (lower) is the near UV CD spectrum of Process 2.

FIG. 11 shows second derivative absorbance spectra for Process 1 (grey trace) and Process 2 (black trace).

FIG. 12 shows temperature melting curves for Process 1 and Process 2, respectively, by differential scanning calorimetry.

FIG. 13 displays the biological activity of a truncated form of recombinant (CG53135-17, denoted by (d1-23)FGF20 in the Figure) as represented by its effects on DNA
synthesis, compared to that of full length FGF-20 (denoted FGF20 in the Figure). NIH 3T3 mouse fibroblasts were serum-starved, incubated with the indicated factor for 18 hours, and analyzed by a BrdU incorporation assay.

FIG. 14 (A) shows dose response of CG53135-induced DNA synthesis in NIH 3T3 Fibroblasts. Serum starved NIH 3T3 cells were treated with purified CG53135-01 (CG53135 in figure), 10% serum or vehicle only (control). DNA synthesis was measured in triplicate for each sample, using a BrdU incorporation assay. Data points represent average BrdU
incorporation and bars represent standard error (SE). (B) CG53135 stimulates Growth of NIH 3T3 Fibroblasts.
Duplicate wells of serum starved NIH 3T3 cells were treated for 1 day with purified CG53135-01 (I
iag) or vehicle control. Cell counts for each well were determined in duplicate. Y- axis identifies cell number, which is the average of 4 cell counts (treatment duplicates x duplicate counts) and standard error (SE). (C) CG53135 induces DNA synthesis in 786-0 Kidney Epithelial cells. Serum starved 786-0 cells were left untreated or treated with partially purified CG531 35-01 (from 5 ng/pL stock), or with vehicle control (mock). DNA synthesis was measured in triplicate for each sample, using a BrdU incorporation assay. Data points represent average BrdU incorporation and bars represent standard error (SE).

FIG. 15 shows effect of Mucositis on the duration of mucositis induced by chemotherapy.
The number of days with mucositis scores > 3 was evaluated. To examine the levels of clinically significant mucositis as defined by presentation with open ulcers (score > 3), the total number of days in which an animal exhibited an elevated score was summed and expressed as a percentage of the total number of days scored for each group. Statistical significance of observed differences was calculated using Chi-square analysis. Vehicle control=disease control.

FIG. 16 shows the cell positions in the crypt.

FIG. 17 (A) shows the crypt survival curve comparing prophylactic administration of CG53135-05 E. coli purified product treatment to PBS control group following different radiation dosages. (B) Shows the effect of prophylactic administration of CG53135-05 E.
coli purified product on mice intestinal crypt survival after radiation insult.

FIG. 18 (A) and (B) show the mean daily mucositis scores following treatment with CG53135-05 E. coli purified product. Mean group mucositis scores were obtained. Error bars represent the standard error of the means (SEM). A comparison of the untreated control group and the groups that received CG53135-05 12 mg/kg IP on days 1 and 2, with the groups that received CG53135-05 on day -1 only was performed. (A) shows groups that received CG53135-05 at 6 mg/kg or 12 mg/kg; and (B) shows groups that received CG53135-05 at 24 mg/kg or 48 mg/kg.

FIG. 19 shows mean daily mucositis scores following treatment with CG53135-05 E. coli purified product once, twice, thrice or four times. Mean group mucositis scores were obtained.
Error bars represent the standard error of the means (SEM). A comparison of the untreated and vehicle control groups with the groups that received CG53135-05 E. coli purified product 12 mg/kg IP was performed. (A) Groups that received CG53135-05 E. coli purified product for one or two days; (B) Groups that received CG53135-05 purified product for three or four days.

FIG. 20 shows percent weight gain in animals with mucositis treated with purified product. Animals were weighed daily, the percent weight change from day -4 was calculated, and group means and standard errors of the mean (SEM) calculated for each day. A
comparison of the untreated control group and the groups receiving CG53135-05 E. coli purified product 12 mg/kg IP on days 1 and 2, with the groups receiving CG53135-05 E.
coli purified product on day -1 only was performed. (A) Groups that received CG53135-05 E. coli purified product at 6 mg/kg or 12 mg/kg; (B) Groups that received CG53135-05 E. coli purified product at 24 mg/kg or 48 mg/kg.

FIG. 21 shows Weight change represented as Area Under the Curve (AUC) gain in animals with mucositis treated with CG53135-05 E. coli purified product. The area under the curve (AUC) was calculated for the percent weight change exhibited by each animal in the study. This calculation was made using the trapezoidal rule transformation. Group means were calculated and are shown with error bars representing SEM for each group. A One Way ANOVA was performed to compare groups.

FIG. 22 shows mean daily mucositis scores following treatment with CG53135-05 E. coli purified product once, twice, thrice or four times. Mean group mucositis scores were obtained.
Error bars represent the standard error of the means (SEM). A comparison of the untreated and vehicle control groups with the groups that received CG53135-05 E. coli purified product 12 mg/kg IP was performed. (A) Groups that received CG53135-05 E. coli purified product for one or two days; (B) Groups that received CG53135-05 purified product for three or four days.

FIG. 23 shows weight change represented as Area Under the Curve (AUC) for animals treated with single dose of CG53135-05 E. coli purified product for one, two, three or four days. The area under the curve (AUC) was calculated for the percent weight change exhibited by each animal in the study. This calculation was made using the trapezoidal rule transformation. Group means were calculated and are shown with error bars representing SEM for each group.
A One Way ANOVA was performed to compare groups.

FIG. 24 (A) and (B) show effects of CG53135 on body weight in animals with gastrointestinal injury induced by whole body irradiation as analyzed by one-way ANOVA and Dunnett's Multiple Comparison Test, respectively.

FIG. 25 (A) and (B) show effects of CG53135 on diarrhea score in mice with gastrointestinal injury induced by whole body irradiation as analyzed by one-way ANOVA and Tukey's Multiple Comparison Test, respectively.

FIG. 26 shows analysis of diarrhea score for each day of observation.

FIG. 27 shows the effect of Phosphate Buffered Saline (PBS) control on mice survival after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy, or 641 cGy.

FIG. 28 (A) shows the effect of prophylactic administration of CG53135 (day-1) on survival of mice after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy, or 641 cGy. (B) shows Kaplan-Meier plots for survival at 570 cGy and 606 cGy, with statistically significant differences between CG53135-treated and PBS-treated control animals. (C) Probit analysis for survival over the range of radiation doses.

FIG. 29 shows the effect of prophylactic administration of CG53135 (day-2 and -1) on survival of mice after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy, or 641 cGy.

FIG. 30 shows the effect of CG53135 multiple-dose administration prior to irradiation on crypt survival curves. Animals (n = 6/group) were administered PBS or CG53135-05 E. coli purified product (12 mg/kg) by intraperitoneal (IP) injection once daily for 4 consecutive days prior to a single 10, 11, 12, 13, or 14 Gy dose of X-ray whole-body irradiation on Day 0. The plot represents the radiation dose-response for crypt survival. Data points represent crypt survival in individual animals analyzed using a multi-target (Puck) analysis model, DRFIT.

FIG. 31 shows effect of CG53135 multiple-dose administration on crypt survival curves.
Animals (n = 6/group) were administered PBS or CG53135-05 E. coli (4 mg/kg) by intraperitoneal (IP) injection once daily for either for 1, 2, 3, 4 or 5 consecutive days prior to, or post a single dose (13 Gy) whole body irradiation on Day 0. The plot represents the level of protection of crypt cells in response to treatment schedule. Protection factor value indicates the number of surviving crypts per circumference in the CG53135-05-treated animals compared to PBS, expressed as a ratio.

FIG. 32 (A)and (B) show CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in NIH 3T3 cells. (C) shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in CCD1070sk cells. (D) shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in CCD18Co cells. (E) shows activation of ERK and AKT kinases by CG53135. (F) shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in human umbilical vein endothelial cells (HUVEC).

FIG. 33 (A) shows the effect of CG53135 on the survival of IEC 18 cells irradiated with different X-ray doses. (B) shows the effect of CG53135 on the survival of NIH
3T3 cells irradiated with different X-ray doses.

FIG. 34 shows the effect of CG53135 on the survival of HUVEC irradiated with different X-ray doses.

FIG. 35 shows survival curves for irradiated cells. Cells of various types (hematopoietic -32D; mesenchymal - CCD18-Co and NIH3T3; epithelial - IEC18, IEC6 and bone -U2OS and Saos-2) were irradiated at the indicated doses then plated in complete growth media either with or without (untreated) 100 ng/ml CG53135-05 E. coli purified product and allowed to form colonies for 10-14 days until the colonies grew to an average diameter of 2 mm. The colonies were stained with crystal violet and counted. The natural log (Ln) of the surviving fraction is represented on the Y axis, and bars represent standard error.

FIG. 36 (A) shows the effect of CG53135 on the release of cytokine in NIH 3T3 cells. (B) shows IL-6 and IL-11 expression in response to CG53135.

FIG. 37 (A) shows dose response of CM-H2DCFDA fluorescence from IEC18 cells treated with CG53135 after 4Gy irradiation. (B) shows response of CM-H2DCFDA
fluorescence from IEC18 cells treated with CG53135 after 2Gy and 4Gy irradiation. (C) shows dose response of CM-H2DCFDA fluorescence from CCD-1 8Co cells treated with CG53135 after 4Gy irradiation.

FIG. 38 (A) shows dose response of Red CC-1 fluorescence from IEC18 cells treated with CG53135 after 4Gy irradiation. (B) shows response of Red CC-1 fluorescence from IEC18 cells treated with CG53135 after 4Gy and 6Gy irradiation. (C) shows response of Red CC-1 fluorescence from CCD-18Co cells treated with CG53135 before and after 10Gy irradiation.

FIG. 39 shows in vitro radioprotection of the myeloid cell line 32D by CG53135.

FIG. 40 shows effect of CG53135 on repopulation of thymus following bone marrow ablation and subsequent bone marrow transplant.

FIG. 41 shows the relative loss in body weight per group for study 439.

FIG. 42 (A) shows the average diarrhea score over 3 days. (B) shows the mean diarrhea severity.

FIG. 43 shows effect of CG53135 in in vitro wound repair in CaCo2, HT-29, IEC-6 human cell lines.

FIG. 44 shows effect of CG53135 on COX-2 gene expression in HT-29 cells. RT-PCR
analysis was carried out to detect the expression of COX-2 gene in HT-29 cell line, in the presence of various concentration of CG53135 (0.1, 1.0, 10, 100ng/ml). COX-2 expression was also analyzed at various time points (1, 3, 6, 24 hrs) after the addition of 100ng/ml of CG53135.

FIG. 45 shows effect of CG53135 on COX-2 gene expression in Caco2 cells. RT-PCR
analysis was carried out to detect the expression of COX-2 gene in Caco2 cell line, in the presence of various concentration of CG53135 (0.1, 1.0, 10, 100ng/mi). COX-2 expression was also analyzed at various time points (1, 3, 6, 24hrs) after the addition of 100ng/mI of CG53135.

FIG. 46 shows effect of CG53135 on COX-2 gene expression in IEC-6 cells. RT-PCR
analysis was carried out to detect the expression of COX-2 gene in I EC-6 cell line, in the presence of various concentration of CG53135 (0.1, 1.0, 10, 100ng/ml). COX-2 expression was also analyzed at various time points (1, 3, 6, 24hrs) after the addition of 100ng/mI of CG53135.

FIG. 47 shows effect of CG53135 on ITF gene expression in HT-29 and Caco2 cells. ITF
gene was detected by mRNA expression in HT-29 and Caco2 cells, in the presence of various concentration of CG53135 (0.1, 1.0, 10, 100ng/mI). ITF gene expression was also analyzed at various time points (1, 3, 6, 24hrs) after the addition of 100ng/ml of CG53135.

FIG. 48 shows effect of CG53135 in inducing COX-2, TGF-R, ITF, PPAR-y in HT-29 mRNA
expression analysis revealed that, upon induction with 100ng/ml of CG53135 for 48hours, mammalian cells expressed COX-2, TGF-E3, ITF, PPAR-y genes.

FIG. 49 shows mechanism of Epithelial Restitution by CG53135. To assess whether TGF-(3 mediates epithelial restitution by FGF-20, wound repair test was performed.
Caco2 cells were incubated with CG531 35-01 E. coli purified product (100ng/ml) and anti TGF-(3 (20 pg/mI) and percent closure was measured.

FIG. 50 (A) shows Effect of CG53135 in stimulation of kinases in Caco2 cells.
Expression of signal transducing kinases was analyzed after incubation of Caco2 cells with CG53135 E. coli purified product (100ng/ml) for different time points (10, 30, 60 minutes).
(B) Effect of kinase inhibitors in the expression of COX-2 gene in Caco2 cells. Caco2 cells were incubated with CG53135-01 E. coli purified product (100ng/mI) in the presence of 40 pM of PD098059 and 20 pM
of SB203580 and COX-2 expression was analyzed. (C) Effect of CG53135 in stimulation of kinases in THP-1 cells. Macrophage cell line, THP-1 was cultured with CG53135-01 E.
coli purified product (100ng/ml) for various time periods. Expression of signal transducing kinases was analyzed at different time points (10, 30, 60 mins). (D) Effect of CG53135 on expression of kinases in intestinal epithelial cells. Caco2 cells were incubated with FGF-20 (100ng/ml) for 10, 30, 60 minutes and expression of p-Elk-1, p-ATF-2 and p-PKC was analyzed. Similarly, HT-29 cells were incubated with FGF-20 (100ng/mi) for 10, 30, 60 minutes and expression of C-Fos and C-Jun was analyzed.
(E) Effect of CG53135 in activating ITF Transcription in HT-29 cells. ITF
promoter activity in HT-29 cells was measured by reporter assay in the presence of FGF-20 at a concentration of 100ng/ml.
FIG. 17 also shows ITF expression in HT-29 cells in the presence of CG53135-01 E. coli purified product.

FIG. 51 (A) shows presents the change in mean body weight from day 0 upon treating mice with varying doses of AB020258 (CG53135). (B) presents the percent change in mean body weight from day 0 upon treating mice with varying doses of AB020258. (C) presents mean colon blood content score upon treating mice with varying doses of AB020258.

FIG. 52 (A) presents mean distal colon inflammation score upon treating mice with varying doses of AB020258. (B) presents mean distal colon gland loss score upon treating mice with varying doses of AB020258. (C) presents mean distal colon erosion score upon treating mice with varying doses of AB020258. (D) presents mean sums of histopathology scores upon treating mice with varying doses of AB020258.

FIG. 53 presents mean spienic lymphoid atrophy score upon treating mice with varying doses of AB020258.

FIG. 54 presents mean spienic extramedullary hematopoiesis score upon treating mice with varying doses of AB020258.

FIG. 55 (A) presents the effect of CG53135 Treatment on Small Intestine Weight in Indomethacin-treated rats. (B) presents effect of CG53135 Treatment on absolute neutrophil and lymphocyte counts in indomethacin-treated rats. Blood was collected on Day 5 at necropsy and the cell counts were determined.

FIG. 56 presents effect of CG53135 Treatment on Histopathology Scores in Indomethacin-treated rats. Five sections of affected intestine were evaluated and scored for necrosis and inflammation as described in the methods.

FIG. 57 presents images showing the protective effect of CG53135 on intestinal architecture. Panel A: Small intestine from normal control animal treated iv with vehicle (BSA).
Panel. B: Small intestine from indomethacin- treated rat, further treated with vehicle (BSA) iv. Panel C: Small intestine from indomethacin-treated rat further treated with CG53135, 0.2 mg/kg iv.
Sections were stained with H&E and visualized at a magnification of 25). FIG.
60 shows the protective Effect of CG53135 on Intestinal Architecture in indomethacin treated rats. Panel A, normal control; Panel B, disease control (indomethacin treated); Panel C, disease model animal treated with 0.2 mg/kg iv CG53135. Photomicrographs were obtained on sections stained with hemotoxylin and eosin, at 25X magnification.

FIG. 58 shows the effect of CG53135 treatment on BrdU Labeling in the Intestine. BrdU
incorporation was detected by Immunoperoxidase staining. Panel A: Small intestine from normal control animal (100X). Panel B: Small intestine from indomethacin + vehicle (BSA) treated animal (50X). Panel C: Small intestine from indomethacin + CG53135 0.2 mg/kg iv treated rat (50X).

FIG. 59 shows effect of therapeutically-administered CG53135 on survival in the DSS model of colitis. Female Balb/c mice were exposed to 4% DSS in drinking water for 7 days (day 0 to day 6) and then switched to normal drinking water for 4 additional days (day 7 to day 10). CG53135 is identified as FGF-20 in FIG. 64. Disease control animals (n = 9) received daily SC injections of vehicle solution on day 4 to day 9. CG53135 groups (n = 9) received daily SC
injections of the indicated concentrations of CG53135 on day 4 to day 9. Normal control animals (n = 3) were not exposed to DSS, but did receive daily SC injections of vehicle solution on day 4 to day 9. Animal survival was recorded on a daily basis and the experiment was concluded on day 10. Note that the disease control and the 0.2 mg/kg CG53135 groups overlap.

FIG. 60 (A) shows weight change and histopathology in prophylactic group (IL-10KO mice).
IL-10 KO mice were treated with various concentrations of CG53135 E. coli purified product (0.2, 1, 5mg/kg) and weight change and histopathology was assessed. (B) shows Total Cecal Histologic Score in prophylactic group (IL-10 KO mice). IL-10 KO mice were treated with various concentrations of FGF-20 (0.2, 1, 5mg/kg) and total cecal histology was scored as described as described in the Example.

FIG. 61 (A) shows IL-12 production in prophylactic group. IL-12 production was assayed by ELISA as described in Example 40, in MLN and colonic strip culture established. (B) shows IFN-y production in prophylactic group. IFN-y production was assayed by ELISA, in MLN and colonic strip culture established. (C) shows PGE2 production in prophylactic group. PGE2 production was assayed by ELISA in MLN prepared.

FIG. 62 shows FACS analysis (prophylactic group). FACS analysis was performed to get the total MLN number as well as number of CD4+, CD8+ and CD4+CD69+ cells.

FIG. 63 shows weight change in treatment group. Weight change in the treatment group was assessed.

FIG. 64 shows Histology of Cecum (treatment) was analyzed in vehicle control as well as CG53135 treated animals.

FIG. 65 shows Histology of Rectum (treatment) was analyzed in vehicle control as well as CG53135 treated animals.

FIG. 66 shows Total Cecal Histologic Score in treatment group (IL-10 KO mice).

mice were treated with CG53135 E. coli purified product (5mg/kg) and total cecal histology was scored.

FIG. 67 shows PGE2 and TNF-a production in treatment group. PGE2 and TNF- a production was assayed by ELISA, gut culture and unseparated spienocytes of CG53135 treated IL-KO mice.

FIG. 68 (A) shows the results of Forelimb Placing Test. The mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 pg/injection CG53135-05 (square), and 2.5 pg/injection CG53135-05 (triangles) are represented overtime. Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA. (B) shows the results of Hindlimb Placing Test. The mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 pg/injection CG53135 (square), and 2.5 pg/injection CG53135 (triangles) are represented over time. Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA.
(C) shows the results of Body Swing Test. The mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 pg/injection CG53135 (square), and 2.5 pg/injection CG53135 (triangles) are represented over time. A score range of -50% swings to the right indicates no impairment, whereas 0% swings to the right swing indicates maximal impairment.
Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA. (D) shows the results of Cylinder Test. The mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 pg/injection CG53135 (square), and 2.5 pg/injection CG53135 (triangles) are represented over time. (E) shows the results of Body Weight. The mean and standard errors of the weights for groups receiving vehicle (diamonds), 1.0 pg/injection CG53135 (square), and 2.5 pg/injection CG53135 (triangles) is represented over time.

FIG. 69 (A) shows the effect of CG53135 on Pro-MMP production in SW1353 cells in the presence of IL-1 beta. (B) shows the effect of CG53135 on Pro-MMP production in SW1353 cells in the presence of TNF-alpha. (C) shows the effect of CG53135 on TIMP production in SW1353 cells.

FIG. 70 (A) shows the effect of intra-articular injection of CG53135 in the Meniscal Tear Model of Rat Osteoarthritis (Prophylactic Dosing): Mean Tibial Cartilage Degeneration. (B) shows results of intra-articular injection of CG53135 in the Meniscal Tear Model of Rat Osteoarthritis:
(Prophylactic Dosing): Total Cartilage Degeneration Width. (C) shows results of intra-articular injection of CG53135 in the Meniscal Tear Model of Rat Osteoarthritis:
(Prophylactic Dosing):
Significant Tibial Cartilage Degeneration Width.

FIG. 71 (A) shows results of intra-articular injection CG53135 in the Meniscal Tear Model of Rat Osteoarthritis (Therapeutic Dosing): Mean Tibial Degeneration. (B) shows results of intra-articular injection of CG53135 in the Meniscal Tear Model of Rat Osteoarthritis (Therapeutic Dosing): Total Cartilage Degeneration Width. (C) shows results of intra-articular injection of CG53135 in Meniscal Tear Model of Rat Osteoarthritis (therapeutic Dosing):
Significant Tibial Cartilage Degeneration Width.

FIG. 72 (A) shows trophic action of EGF, NGF and CG53135; (B) shows the time course of CG53135-inhibited serum withdrawal-induced apoptosis.

FIG. 73 shows CG53135 inhibits serum withdrawal-induced caspase activation.
FIG. 74 shows neuritogenic action of CG53135 as compared to NGF.

FIG. 75 shows activation of MAPK by NGF and CG53135, and the inhibition of activity by PD98059, a MAPKK inhibitor.

5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides improved formulations comprising one or more proteins, which are more stable and soluble, and can be easily lyophilized by commercial equipments. The improved formulations comprise 0.01-1 M of a stabilizer, such as arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, 0.01-0.1 M sodium phosphate monobasic (NaH2PO4=H2O), 0.01 %-0.1 % weight/volume ("w/v") polysorbate 80 or polysorbate 20, and one or more isolated CG53135 proteins. In a specific embodiment, the concentration of CG53135 protein(s) in the improved formulations of the invention is less than 50 mg/ml, less than 30 mg/mi, less than 10 mg/mI, less than 5 mg/mI, or less than 1 mg/mI. In another embodiment, the concentration of CG53135 protein(s) in the improved formulations of the invention is between 0.005-50 mg/mI. In a preferred embodiment, the formulation is lyophilized.

The present invention also provides methods for increasing solubility of a FGF
protein in a solution (e.g., an aqueous solution). In one embodiment, the present invention provides a method for increasing solubility of a FGF protein in a solution by adding arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose to the solution. In another embodiment, the present invention provides a method for increasing solubility or stability of a FGF
protein in a solution by adding buffering salts such as acetate, succinate, tartrate, phosphate, or a combination thereof to the solution. In yet another embodiment, buffering salts such as acetate, succinate, tartrate, phosphate, or a combination thereof is added in combination with arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose to the solution to increase the solubility of a FGF protein.
The arginine in a salt form can be, but is not limited to, arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride. In a preferred embodiment, arginine sulfate is used. In some embodiments, the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose is between 0.01 M to 1 M. In one embodiment, the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose is 0.5 M. In some embodiment, the final concentration of the buffering salts such as acetate, succinate, tartrate, phosphate, or a combination thereof is 0.05 M. In a preferred embodiment, the FGF protein is a FGF-20 protein, a fragment, a derivative, a variant, a homolog, or an analog of FGF-20, or a combination thereof.

The present invention further provides improved production methods for CG53135 proteins and/or formulations comprising one or more CG53135 proteins. The improved production methods allow for commercial scale production of CG53135 proteins and/or formulations comprising one or more CG53135 proteins. The improved production methods also allow for purifying CG53135 proteins to a high degree of purity. In some embodiments, the purity of the CG53135 purified by the improved production methods is at least 97%, at least 98%, at least 99%. In a preferred embodiment, the purity of CG53135 purified by the improved production methods is from 99% up to 100% (including 100%).

The present invention also provides methods of use of the improved formulations and compositions of the invention for preventing or treating a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof, and dosage regiments.

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections:

(i) CG53135 (ii) Methods of Preparing CG53135 (iii) Characterization of CG53135 (iv) Prophylactic and Therapeutic Uses (v) Dosage Regimens (vi) Pharmaceutical Compositions and Formulations 5.1 CG53135 The present invention provides for improved formulations comprising one or more CG53135 proteins and improved production methods. As used herein, the term "CG53135"
refers to a class of proteins (including peptides and polypeptides) or nucleic acids encoding such proteins or their complementary strands, where the proteins comprise an amino acid sequence of SEQ ID NO:2 (211 amino acids, "FGF-20"), or its fragments, derivatives, variants, homologs, or analogs.

In one embodiment, a CG53135 protein is a variant of FGF-20. It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the FGF-20 protein may exist within a population (e.g., the human population). Such genetic polymorphism in the FGF-20 gene may exist among individuals within a population due to natural allelic variation. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the FGF-20 gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in the FGF-20 protein, which are the result of natural allelic variation of the FGF-20 protein, are intended to be within the scope of the invention. In one embodiment, a CG53135 is CG53135-12 (SEQ ID NOs:21 and 22), which is a single nucleotide polymorphism ("SNP") of FGF-20 (i.e., 206D->N). (For more detailed description of CG53135-12, see e.g., U.S.
Patent Application No. 10/702,126, filed November 4, 2003, the disclosure of which is incorporated herein by reference in its entirety.) Other examples of SNPs of FGF-20 are also described in U.S.
Patent Application No. 10/435,087, the content of which is incorporated herein by reference.

In another embodiment, CG53135 refers to a nucleic acid molecule encoding a protein from other species or the protein encoded thereby, and thus has a nucleotide or amino acid sequence that differs from the human sequence of FGF-20. Nucleic acid molecules corresponding to natural allelic variants and homologues of the FGF-20 cDNAs of the invention can be isolated based on their homology to the human FGF-20 nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

In another embodiment, CG53135 refers to a fragment of an FGF-20 protein, including fragments of variant FGF-20 proteins, mature FGF-20 proteins, and variants of mature FGF-20 proteins, as well as FGF-20 proteins encoded by allelic variants and single nucleotide polymorphisms of FGF-20 nucleic acids. An example of an FGF-20 protein fragment includes, but is not limited to, residues 2-211, 3-211, 9-211, 12-211, 15-211, 24-211, 54-211, or 55-211 of FGF-20 (SEQ ID NO:2). In one embodiment, CG53135 refers to a nucleic acid encodes a protein fragment that includes residues 2-211, 3-211, 9-211, 12-211, 15-211, 24-211, 54-211, or 55-211 of SEQ ID
NO:2.

The invention also encompasses derivatives and analogs of FGF-20. The production and use of derivatives and analogs related to FGF-20 are within the scope of the present invention.

In a specific embodiment, the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type FGF-20.
Derivatives or analogs of FGF-20 can be tested for the desired activity by procedures known in the art, including but not limited to, using appropriate cell lines, animal models, and clinical trials.

In particular, FGF-20 derivatives can be made via altering FGF-20 sequences by substitutions, insertions or deletions that provide for functionally equivalent molecules. In one embodiment, such alteration of an FGF-20 sequence is done in a region that is not conserved in the FGF protein family. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as FGF-20 may be used in the practice of the present invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of FGF-20 which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. In a preferred embodiment, a wild-type FGF-20 nucleic acid sequence is codon optimized to the nucleic acid sequence of SEQ ID NO:8 (CG53135-05). Likewise, the FGF-20 derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of FGF-20 including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.

Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. FGF-20 derivatives of the invention also include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of FGF-20 including altered sequences in which amino acid residues are substituted for residues with similar chemical properties. In a specific embodiment, 1, 2, 3, 4, or 5 amino acids are substituted.

Derivatives or analogs of FGF-20 include, but are not limited to, those proteins which are substantially homologous to FGF-20 or fragments thereof, or whose encoding nucleic acid is capable of hybridizing to the FGF-20 nucleic acid sequence.

The FGF-20 derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned FGF-20 gene sequence can be modified by any of numerous strategies known in the art (e.g., Maniatis, T., 1989, Molecular Cloning, A
Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of FGF-20, care should be taken to ensure that the modified gene remains within the same translational reading frame as FGF-20, uninterrupted by translational stop signals, in the gene region where the desired FGF-20 activity is encoded.

Additionally, the FGF-20-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C. et aL, 1978, J. Biol.
Chem 253:6551), use of TAB® linkers (Pharmacia), etc.

Manipulations of the FGF-20 sequence may also be made at the protein level.
Included within the scope of the invention are FGF-20 fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, reagents useful for protection or modification of free NH2- groups, free COOH- groups, OH- groups, side groups of Trp-, Tyr-, Phe-, His-, Arg-, or Lys-; specific chemical cleavage by cyanogen bromide, hydroxylamine, BNPS-Skatole, acid, or alkali hydrolysis; enzymatic cleavage by trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

In addition, analogs and derivatives of FGF-20 can be chemically synthesized.
For example, a protein corresponding to a portion of FGF-20 which comprises the desired domain, or which mediates the desired aggregation activity in vitro, or binding to a receptor, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the FGF-20 sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, a-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, R-alanine, designer amino acids such as (3-methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids.

In a specific embodiment, the FGF-20 derivative is a chimeric or fusion protein comprising FGF-20 or a fragment thereof fused via a peptide bond at its amino- and/or carboxy-terminus to a non-FGF-20 amino acid sequence. In one embodiment, the non-FGF-20 amino acid sequence is fused at the amino-terminus of an FGF-20 or a fragment thereof. In another embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising an FGF-20-coding sequence joined in-frame to a non-FGF-20 coding sequence). Such a chimeric product can be custom made by a variety of companies (e.g., Retrogen, Operon, etc.) or made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. In a specific embodiment, a chimeric nucleic acid encoding FGF-20 with a heterologous signal sequence is expressed such that the chimeric protein is expressed and processed by the cell to the mature FGF-20 protein. The primary sequence of FGF-20 and non-FGF-20 gene may also be used to predict tertiary structure of the molecules using computer simulation (Hopp and Woods, 1981, Proc. Nati.
Acad. Sci. U.S.A. 78:3824-3828); the chimeric recombinant genes could be designed in light of correlations between tertiary structure and biological function. Likewise, chimeric genes comprising an essential portion of FGF-20 molecule fused to a heterologous (non-FGF-20) protein-encoding sequence may be constructed. In a specific embodiment, such chimeric construction can be used to enhance one or more desired properties of an FGF-20, including but not limited to, FGF-20 stability, solubility, or resistance to proteases. In another embodiment, chimeric construction can be used to target FGF-20 to a specific site. In yet another embodiment, chimeric construction can be used to identify or purify an FGF-20 of the invention, such as a His-tag, a FLAG tag, a green fluorescence protein (GFP), 0-galactosidase, a maltose binding protein (MaIE), a cellulose binding protein (CenA) or a mannose protein, etc. In one embodiment, a CG53135 protein is carbamylated.

In some embodiment, a CG53135 protein can be modified so that it has improved solubility and/or an extended half-life in vivo using any methods known in the art. For example, Fc fragment of human IgG, or inert polymer molecules such as high molecular weight polyethyleneglycol (PEG) can be attached to a CG53135 protein with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the protein or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation can be closely monitored by SDS-PAGE
and mass spectrometry to ensure proper conjugation of PEG molecules to the CG53135 protein.
Unreacted PEG can be separated from CG53135-PEG conjugates by size-exclusion or by ion-exchange chromatography. PEG-derivatized conjugates can be tested for in vivo efficacy using methods known to those of skill in the art.

A CG53135 protein can also be conjugated to albumin in order to make the protein more stable in vivo or have a longer half life in vivo. The techniques are well known in the art, see e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413, 622, all of which are incorporated herein by reference.

In some embodiments, CG53135 refers to CG53135-01 (SEQ ID NOs:1 and 2), 02 (SEQ ID NOs:3 and 4), CG53135-03 (SEQ ID NOs:5 and 2), CG53135-04 (SEQ ID
NOs:6 and 7), CG53135-05 (SEQ ID NOs:8 and 2), CG53135-06 (SEQ ID NOs:9 and 10), CG53135-07 (SEQ
ID NOs:11 and 12), CG53135-08 (SEQ ID NOs:13 and 14), CG53135-09 (SEQ ID
NOs:15 and 16), CG53135-10 (SEQ ID NOs:17 and 18), CG53135-11 (SEQ ID NOs:19 and 20), CG53135-12 (SEQ
ID NOs:21 and 22), CG53135-13 (SEQ ID NOs:23 and 24), CG53135-14 (SEQ ID
NOs:25 and 26), CG53135-15 (SEQ ID NOs:27 and 28), CG53135-16 (SEQ ID NOs:29 and 30), CG53135-17 (SEQ
ID NOs:31 and 32), IFC 250059629 (SEQ ID NOs:33 and 34), IFC 20059669 (SEQ ID
NOs:35 and 36), IFC 317459553 (SEQ ID NOs:37 and 38), IFC 317459571 (SEQ ID NOs:39 and 40), IFC
250059596 (SEQ ID NOs:41 and 10), IFC316351224 (SEQ ID NOs:41 and 10), or a combination thereof. In a specific embodiment, a CG53135 is carbamylated, for example, a carbamylated CG53135-13 protein or a carbamylated CG53135-05 protein.

Examples of prophylactic and/or therapeutic uses of CG53135 have been described in previously filed patent applications (see e.g., U.S. Patent Application Nos.
09/992,840, 10/011,364, 10/321,962, 10/435,087, 10/842,206, 10/842,179, and U.S. Patent No.
6,797,695). The disclosure of each reference is incorporated by reference herein in its entirety.

5.2 METHODS OF PREPARING CG53135 The present invention provides for formulations comprising one or more isolated CG53135 proteins and improved methods of production. In accordance with the methods described herein, CG53135 proteins employed in a formulation of the invention or produced by the production methods of the invention can have a purity in the range of 80 to 100 percent, or at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In one embodiment, one or more CG53135 proteins employed in a formulation of the invention or produced by the production methods of the invention have a purity of at least 99%. In another embodiment, CG53135 is purified to apparent homogeneity, as assayed, e.g., by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Any techniques known in the art can be used in purifying a CG53135 protein, including but are not limited to, separation by precipitation, separation by adsorption (e.g., column chromatography, membrane adsorbents, radial flow columns, batch adsorption, high-performance liquid chromatography, ion exchange chromatography, inorganic adsorbents, hydrophobic adsorbents, immobilized metal affinity chromatography, affinity chromatography), or separation in solution (e.g., gel filtration, electrophoresis, liquid phase partitioning, detergent partitioning, organic solvent extraction, and ultrafiltration). See e.g., Scopes, PROTEIN PURIFICATION, PRINCIPLES AND
PRACTICE, 3rd ed., Springer (1994). During the purification, the biological activity of CG53135 may be monitored by one or more in vitro or in vivo assays as described in Section 5.3, infra. The purity of CG53135 can be assayed by any methods known in the art, such as but not limited to, gel electrophoresis. See Scopes, supra.

Methods known in the art can be utilized to recombinantly produce CG53135 proteins. A
nucleic acid sequence encoding a CG53135 protein can be inserted into an expression vector for propagation and expression in host cells.

An expression construct, as used herein, refers to a nucleic acid sequence encoding a CG53135 protein operably associated with one or more regulatory regions which enable expression of a CG53135 protein in an appropriate host cell. "Operably-associated" refers to an association in which the regulatory regions and the CG53135 sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation.

The regulatory regions necessary for transcription of CG53135 can be provided by the expression vector. A translation initiation codon (ATG) may also be provided if a CG53135 gene sequence lacking its cognate initiation codon is to be expressed. In a compatible host-construct system, cellular transcriptional factors, such as RNA polymerase, will bind to the regulatory regions on the expression construct to effect transcription of the modified CG53135 sequence in the host organism. The precise nature of the regulatory regions needed for gene expression may vary from host cell to host cell. Generally, a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence. Such regulatory regions may include those 5' non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. The non-coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.

In order to attach DNA sequences with regulatory functions, such as promoters, to a CG53135 gene sequence or to insert a CG53135 gene sequence into the cloning site of a vector, linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of the cDNAs by techniques well known in the art (see e.g., Wu et al., 1987, Methods in Enzymol, 152:343-349). Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation. Alternatively, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA using PCR with primers containing the desired restriction enzyme site.

An expression construct comprising a CG53135 sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of a CG53135 protein without further cloning. See, e.g., U.S.
Patent No. 5,580,859. The expression constructs can also contain DNA sequences that facilitate integration of a CG53135 sequence into the genome of the host cell, e.g., via homologous recombination.
In this instance, it is not necessary to employ an expression vector comprising a replication origin suitable for appropriate host cells in order to propagate and express CG53135 in the host cells.

A variety of expression vectors may be used, including but are not limited to, plasmids, cosmids, phage, phagemids or modified viruses. Such host-expression systems represent vehicles by which the coding sequences of a CG53135 gene may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express CG53135 in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing CG53135 coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing CG53135 coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing CG53135 coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing CG53135 coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli and eukaryotic cells are used for the expression of a recombinant CG531 35 molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO) can be used with a vector bearing promoter element from major intermediate early gene of cytomegalovirus for effective expression of a CG53135 sequence (Foecking et al., 1986, Gene 45:101; and Cockett et a/., 1990, Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the CG53135 molecule being expressed. For example, when a large quantity of a CG531 35 is to be produced, for the generation of pharmaceutical compositions of a CG53135 molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E.coli expression vector pCR2.1 TOPO (Invitrogen); pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509) and the like.
Series of vectors like pFLAG (Sigma), pMAL (NEB), and pET (Novagen) may also be used to express the foreign proteins as fusion proteins with FLAG peptide, malE-, or CBD- protein. These recombinant proteins may be directed into periplasmic space for correct folding and maturation. The fused part can be used for affinity purification of the expressed protein.
Presence of cleavage sites for specific protease like enterokinase allows to cleave off the CG53135 protein. The pGEX vectors may also be used to express foreign proteins as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, many vectors to express foreign genes can be used, e.g., Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes.
The virus grows in cells like Spodoptera frugiperda cells. A CG53135 coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a CG53135 coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing CG53135 in infected hosts (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad.
Sci. USA 8 1:355-359). Specific initiation signals may also be required for efficient translation of inserted CG53135 coding sequences. These signals include the ATG initiation codon and adjacent sequences.
Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products.
Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript and post-translational modification of the gene product, e.g., glycosylation and phosphorylation of the gene product, may be used.
Such mammalian host cells include, but are not limited to, PC12, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.
Expression in a bacterial or yeast system can be used if post-translational modifications turn to be non-essential for a desired activity of CG53135. In a preferred embodiment, E.
coli is used to express a CG53135 sequence.

For long term, high yield production of properly processed CG53135, stable expression in cells is preferred. Cell lines that stably express CG53135 may be engineered by using a vector that contains a selectable marker. By way of example but not limitation, following the introduction of the expression constructs, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the expression construct confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines. Such cells can be cultured for a long period of time while CG53135 is expressed continuously.

A number of selection systems may be used, including but not limited to, antibiotic resistance (markers like Neo, which confers resistance to geneticine, or G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; May, 1993, TIB TECH 11(5):155-2 15); Zeo, for resistance to Zeocin; Bsd, for resistance to blasticidin, etc.); antimetabolite resistance (markers like Dhfr, which confers resistance to methotrexate, Wigler et al., 1980, Nati. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl.
Acad. Sci. USA 78:1527);
gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Nati. Acad. Sci.
USA 78:2072); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). In addition, mutant cell lines including, but not limited to, tk-, hgprt- or aprt- cells, can be used in combination with vectors bearing the corresponding genes for thymidine kinase, hypoxanthine, guanine- or adenine phosphoribosyltransferase. Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegier, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1.

The recombinant cells may be cultured under standard conditions of temperature, incubation time, optical density and media composition. However, conditions for growth of recombinant cells may be different from those for expression of CG53135.
Modified culture conditions and media may also be used to enhance production of CG53135. Any techniques known in the art may be applied to establish the optimal conditions for producing CG53135.

An alternative to producing CG53135 or a fragment thereof by recombinant techniques is peptide synthesis. For example, an entire CG53135, or a protein corresponding to a portion of CG53135, can be synthesized by use of a peptide synthesizer. Conventional peptide synthesis or other synthetic protocols well known in the art may be used.

Proteins having the amino acid sequence of CG53135 or a portion thereof may be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc., 85:2149. During synthesis, N-a-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support, i.e., polystyrene beads. The proteins are synthesized by linking an amino group of an N-a-deprotected amino acid to an a-carboxyl group of an N-a-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide.
The attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
The most commonly used N-a-protecting groups include Boc which is acid labile and Fmoc which is base labile. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are not discussed in detail herein (See, Atherton et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag).

Purification of the resulting CG53135 is accomplished using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography. The choice of appropriate matrices and buffers are well known in the art and so are not described in detail herein.

5.2.1 Improved Production Methods The present invention provides improved manufacturing processes for producing compositions comprising one or more CG53135 proteins. The improved manufacturing processes provide benefits such as more stable and more pure drug product, and are also suitable for commercial scale production of a composition comprising one or more CG53135 proteins.

The present invention provides methods of isolating a protein, where the methods comprises the steps of: (1) fermenting a host cell, such as E. coli, that containing a vector, where the vector comprises a nucleotide sequence encoding a CG53135 protein. In a preferred embodiment, the vector comprises a codon-optimized, full length CG53135-05 (SEQ ID NO:8);
(2) lysing the cultured cells. Cells may be lysed by any methods known in the art. In one embodiment, cells are lysed by homogenization. In another embodiment, the fermented cultured cells are chilled, and diluted with cell lysis buffer comprising 50-100 mM sodium phosphate, 60 mM
EDTA, 7.5 mM DTT, 3.5-5 M urea, pH 7.2, and then lysed by, e.g., homogenization. In a preferred embodiment, polyethyleneimine ("PEI") is added to the fermentation broth before homogenization; (3) purification by a cation exchange column. In a preferred embodiment, a pre-equilibrated expanded bed cation exchanger, such as STREAMLINE SPT"' is used. In one embodiment, after the cation exchange column is loaded with the protein to be isolated, the column is flushed with additional equilibration buffer comprising 50-100 mM sodium phosphate, 40 mM EDTA, 10 mM sodium sulfate, 3-5 M urea, pH 7Ø The column may be further washed with a buffer comprising 50-100 mM
sodium phosphate, mM EDTA, 10-25 mM sodium sulfate, 2.22 M dextrose, pH 7Ø The elution buffer to elute the protein from the cation exchange column comprises, e.g., 50-100 mM sodium phosphate, 5 mM

EDTA, 150-250 mM sodium sulfate, 0.5-1 M L-arginine, pH 7.0; and (4) further purification by using a hydrophobic interaction chromatography column (e.g., PPG 650M). In one embodiment, the hydrophobic interaction chromatography column, e.g., PPG 650M, is equilibrated and washed with 50-100 mM sodium phosphate, 150-250 mM sodium sulfate, 5 mM EDTA, 1 M
arginine, pH 7Ø In another embodiment, the column is further washed with 100-250 mM sodium phosphate, 5 mM
EDTA, 0.8-1 M arginine, pH 7Ø In another embodiment, the protein is eluted with 50-100 mM
sodium phosphate, 5 mM EDTA, and 0.1-0.3 M arginine, pH 7Ø

In a preferred embodiment, the eluted protein from step (4) described above may be further purified by either one or both of the following steps: (5) further purification by filtering the eluted protein. In a preferred embodiment, a charged endotoxin binding filter (e.g., CUNOTM 30 ZA depth filter) is used. In one embodiment, the filter is first flushed with water for injection, and then with 50-100 mM sodium phosphate, 5 mM EDTA, 0.1-03 M arginine, pH 7.0; and (6) further purification by using a hydrophobic interaction chromatography column (e.g., Phenyl Sepharose HP
Chromatography). In one embodiment, the column is equilibrated and washed with 50-100 mM
sodium phosphate, 10-100 mM ammonium sulfate, 800-1000 mM sodium chloride, 0.5-1 M arginine, pH 7Ø In another embodiment, the protein is eluted with 50-100 mM sodium phosphate, 0.5-1 M
arginine, pH 7Ø

A protein isolated by the methods of the present invention may be further concentrated and filtered to produce a drug product. Pharmaceutical carriers may be added to produce a desired formulation, such as formulations provided by the present invention.

5.3 CHARACTERIZATION OF CG53135 The characteristics of the protein(s) purified by the production methods of the instant invention or the immediate products of the production methods of the instant invention (e.g., purity, various characters including the biological activity of CG53135) may be determined by methods known in the art. Compositions for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to in rats, mice, chicken, cows, monkeys, rabbits, etc (examples of such tests can be found in U.S. Patent Application Nos.
09/992,840, 10/011,364, 10/321,962, 10/435,087, 10/842,206, and 10/842,179, the disclosure of each is incorporated herein by reference).

For examples, methods known in the art, such as but not limited to, sodium dodecyl sulphate polyacrylamide gel electrophoresis ("SDS-PAGE"), reversed phase high-performance liquid chromatography ("RP-HPLC"), size exclusion high-performance liquid chromatography ("SEC-HPLC"), Western Blot (e.g., host cell protein Western Blot), can be used to analyze the purity of the product of the manufacturing processes of the instant invention. In a preferred embodiment, a product of the manufacturing processes of the instant invention is at least 97%, at least 98%, or at least 99% pure by densitometry. In another preferred embodiment, a product of the manufacturing processes of the instant invention is more than 97%, more than 98%, or more than 99% pure by densitometry.

Methods known in the art, such as but not limited to, Western Blot, sequencing (e.g., N-terminal Edman sequencing), liquid chromatography (e.g., HPLC, RP-HPLC with both UV and electrospray mass spectrometric detection), mass spectrometry, total amino acid analysis, peptide mapping, SDS-PAGE, can be used to determine the identity of the product of the manufacturing processes of the instant invention. The secondary, tertiary and/or quaternary structure of a product of the manufacturing processes of the instant invention can analyzed by any method know in the art, for example, far UV circular dichroism spectrum can be used to analyze the secondary structure, near UV circular dichroism spectroscopy and second derivative UV absorbance spectroscopy can be used to analyze the tertiary structure, and light scattering SEC-HPLC can be used to analyze quaternary structure.

Potency of a product of the manufacturing processes of the instant invention can be measured by methods known in the art or any bioassays that measuring one or more biological activities of a CG53135 protein. In one embodiment, potency of a product of the manufacturing processes of the instant invention is measured by the ability of the product to stimulate cell growth of NIH 3T3 cells (for an example of such assay, see Section 6.5).

Other characters of a product of the manufacturing processes of the instant invention, such as safety (e.g., residual DNA, endotoxin, bioburden), pH, osmolarity, suifyhydryl content, can also be analyzed by any method known in the art (for examples of some of these methods, see Section 6).
Such methods are well known in the art and are not described in detail herein.

In one embodiment, a CG53135 protein reference standard, which is representative of the bulk drug substance from the improved manufacturing process, is prepared and characterized.
Preferably, this reference standard is characterized for its purity, identity (e.g., molecular weight, amino acid sequence), potency, structure (e.g., secondary, tertiary and quaternary structures), safety (e.g., endotoxin, bioburden), and other characters, such as but not limited to, pH, osmolality, and suifyhydryl content. Upon visual inspection, a CG53245 protein reference standard should be clear and colorless. Once a CG53135 protein reference is established, it can be used for, e.g., quality control of the manufacturing process, and as a positive control for other assays related to CG53135.

5.4 PROPHYLACTIC AND THERAPEUTIC USES
The present invention provides methods of preventing and/or treating a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof comprising administering to a subject in need thereof an effective amount of a composition comprising one or more isolated CG53135 proteins.

5.4.1. Alimentary Mucositis In one embodiment, the present invention provides methods of preventing and/or treating alimentary mucositis. Alimentary mucositis that can be prevented and/or treated by the methods of the invention includes, but is not limited to, oral mucositis, esophagitis, stomatitis, enteritis, and proctitis. In some embodiments, the methods of the invention comprise administering an effective amount of a composition comprising one or more isolated CG53135 proteins to a subject with mucositis at more than one area in the alimentary canal (e.g., a subject with both oral mucositis and enteritis). In some embodiments, the methods of the invention comprise administering an effective amount of a composition comprising one or more isolated CG53135 proteins to a subject with mucositis at only one area in the alimentary canal (e.g., a subject with only oral mucositis, or a subject with only enteritis). In a preferred embodiment, the alimentary mucositis that can be prevented and/or treated by the methods of the invention is oral mucositis. In some embodiments, the alimentary mucositis that can be prevented and/or treated by the methods of the invention is not an oral mucositis. Alimentary mucositis may be induced by, e.g., chemical insult, radiation insult, biological insult (e.g., bacteria), or a combination thereof.

In some embodiments, the present invention provides methods of preventing and/or treating alimentary mucositis in patient populations with alimentary mucositis and populations at risk to develop alimentary mucositis. In one embodiment, the present invention provides methods of preventing and/or treating alimentary mucositis in a subject who has been treated with radiation therapy and/or chemotherapy. In another embodiment, the present invention provides methods of preventing alimentary mucositis by administering a composition comprising one or more CG53135 proteins to a subject who is going to be treated with radiation therapy and/or chemotherapy. In a specific embodiment, the present invention provides methods of preventing and/or treating alimentary mucositis in a subject who has been treated with conditioning myeloablative radiation therapy and /or chemotherapy in preparation for autologous or allogenic hematopoietic stem cell transplant. In another specific embodiment, the present invention provides methods of preventing and/or treating alimentary mucositis in a subject who has received or is receiving mucosatoxic chemotherapy with mucositis-inducing agents (e.g., leukemia patients treated with cytarabine). In yet another specific embodiment, the present invention provides methods of preventing and/or treating alimentary mucositis in a subject who has head and/or neck cancer treated with radiation therapy with or without adjuvant chemotherapy.

In one embodiment, the present invention provides a method of preventing alimentary mucositis comprising administering a composition comprising one or more CG53135 proteins prior to an insult (e.g., a chemical insult, a radiation insult, a biological insult, or a combination thereof) that may induce alimentary mucositis occurs to a subject. In another embodiment, the present invention provides a method of preventing alimentary mucositis comprising administering a composition comprising one or more CG53135 proteins after an insult (e.g., a chemical insult, a radiation insult, a biological insult, or a combination thereof) that may induce alimentary mucositis occurs to a subject, but prior to the development of alimentary mucositis in the subject. In yet another embodiment, the present invention provides a method of treating alimentary mucositis comprising administering a composition comprising one or more CG53135 proteins after alimentary mucositis developed in a subject.

In some embodiments, the present invention provides a method of preventing and/or treating alimentary mucositis comprising cyclically administering a composition comprising one or more CG53135 proteins. In one embodiment, cycling therapy involves the administration of a first therapy for a period of time, followed by the administration of a second therapy for a period of time and repeating this sequential administration, f.e., the cycle, in order to, e.g., to avoid or reduce the side effects of one of the therapies and/or to improve the efficacy of the therapies. In another embodiment, cycling therapy involves the administration of a therapy for a period of time, stop the therapy for a period of time, and repeat the administration of the therapy. In accordance to the present invention, a composition comprising one or more CG53135 proteins can be administered to a subject prior to, during, or after the administration of a radiation therapy and/or chemotherapy, where such radiation therapy and/or chemotherapy is a cycling therapy.

In accordance to the instant invention, a composition comprising one or more isolated CG53135 proteins can also be used in combination with other therapies to prevent and/or treat alimentary mucositis. In one embodiment, a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other agents that have prophylactic and/or therapeutic effect(s) on alimentary mucositis and/or have amelioration effect(s) on one or more symptoms associated with alimentary mucositis to a subject to prevent and/or treat alimentary mucositis. Non-limiting examples of such agents are: mucosal protective agents (e.g., sucralfate, colloidal bismuth), antibiotics, antifungal agents (e.g., fluconazole, amphotericin B), antiviral agents (e.g., acyclovir), antiemetic agents (e.g., phenothiazines, butyrophenones, benzodiazepines, corticosteroids, cannabinoids, 5-HT3 serotonin receptor blockers), antidiarrhea agents (e.g., diphenoxylate, loperamide, kaolin, pectin, methylacellulose, activated attapulgite, magnesium aluminum silicate, non-steroidal anti-inflammatory agents (NSAIDs)), transforming growth factor (TGF), interieukin-11 (IL-11), granulocyte-macrophage colony stimulating factor (GM-CSF), keratinocyte growth factor (KGF), L-glutamine, Amifostene, and Granulocyte colony stimulating factor (G-CSF). In another embodiment, a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other therapies that have palliative effect on alimentary mucositis. Non-limiting examples of such therapies are:
application of topical analgesics such as lidocaine and/or systemic administration of narcotics and antibiotics, topical fluoride application with or without calcium phosphate, mechanical plaque removal, tooth sponges, sucking ice chips resulting in oral cooling, oral rinses with various anti-infective agents, oral mouthwashes with local anesthetics.

5.4.2. Radiation Protection and Stimulatory Effects on Stem Cells In one embodiment, the present invention provides methods of preventing and/or treating one or more disorders associated with (e.g., caused by) radiation exposure, chemotherapy, chemical/biological warfare agents, and/or any other insults affecting rapidly proliferating tissues in the body, or one or more symptoms thereof by administering to a subject a prophylactically or therapeutically effective amount of a composition comprising one or more isolated CG53135 proteins.

In some embodiments, the present invention provides methods of preventing and/or treating a pathology of epithelial cells and/or mesenchymal cells comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins. In another embodiment, the present invention provides methods of stimulating proliferation, differentiation or migration of epithelial cells and/or mesenchymal cells comprising administering to a subject in need thereof an effective amount of a composition comprising one or more CG53135 proteins.

Epithelial membranes are continuous sheets of cells with contiguous cell borders that have characteristic specialized sites of close contact called cell junction. Such membrane, which can be one or more cells thick, contain no capillaries. Epithelia are attached to the underlying connective tissue by a component known as a basement membrane, which is a layer of intercellular material of complex composition that is distributed as a thin layer between the epithelium and the connective tissue.

Stratified squamous nonkeratinizing epithelium is common on wet surfaces that are subject to considerable wear and tear at sites where absorptive function is not required. The secretions necessary to keep such surfaces wet have to come from appropriately situated glands. Sites lined by this type of epithelium include the esophagus and the floor and sides of the oral cavity.

Simple columnar epithelium is made up of a single layer of tall cells that again fit together in a hexagonal pattern. In simple secretory columnar epithelium, the columnar cells are all specialized to secret mucus in addition to being protective. Sites of this type of epithelium is present include the lining of the stomach.

A simple columnar epithelium that is made up of absorptive cells as well as secretory cells lines the intestine. To facilitate absorption, this membrane is only one cell thick. Interspersed with cells that are specialized for absorption, there are many goblet cells that secrete protective mucus.

Mesenchymal cells are stem cells that can differentiate into, e.g., osteoblasts, chondrocytes, myocytes, and adipocytes. Mesenchymal-epithelial interactions play an important role in the physiology and pathology of epithelial tissues. Mesenchymal cells may associate with epithelium basement membrane (e.g., pericytes and perivascular monocyte-derived cells (MDCs)), or reside within epithelium (MDCs and T cells). The nature of the interactions between mesenchymal cells and tissue-specific cells may depend on the tissue type (e.g., brain versus epidermis), or on the prevention or allowance/stimulation of differentiation of cells into the suicidal state (apoptosis) by mesenchymal cells in a given epithelium. Specialized mesenchymal cells, such as pericytes, MDCs, and T lymphocytes, may significantly influence the differentiation and aging of epithelial cells.

The stromal compartment of the cavities of bone is composed of a net-like structure of interconnected mesenchymal cells. Stromal cells are closely associated with bone cortex, bone trabecule and to the hemopoietic cells. The bone marrow-stromal micro-environment, is a complex of cells, extracellular matrix (ECM) with growth factors and cytokines that regulate osteogenesis and hemopoiesis locally throughout the life of the individual. The role of the marrow stroma in creating the microenvironment for bone physiology and hemopoiesis lies in a specific subpopulation of the stroma cells. They differentiate from a common stem cell to the specific lineage each of which has a different role. Their combined function results in orchestration of a 3-D-architecture that maintains the active bone marrow within the bone.

In adults, blood cells are produced by the bone marrow, the spongy material filling the body's bones. The bone marrow produces two blood cell groups, myeloid and lymphoid. The myeloid cell line includes, e.g., the following: (1) Immature cells called erythrocytes that later develop into red blood cells; (2) Blood clotting agents ( platelets); (3) Some white blood cells, including macrophages (which act as scavengers for foreign particles), eosinophils (which trigger allergies and also defend against parasites), and neutrophils (the main defenders against bacterial infections). The lymphoid cell line includes, e.g., the lymphocytes, which are the body's primary infection fighters. Among other vital functions, certain lymphocytes are responsible for producing antibodies, factors that can target and attack specific foreign agents (antigens). Lymphocytes develop in the thymus gland or bone marrow and are therefore categorized as either B-cells (bone marrow-derived cells) or T-cells (thymus gland-derived cells).

According to the present invention, CG53135 can regulate proliferation, differentiation, and/or migration of epithelial cells and/or mesenchymal cells, and thus have prophylactic and/or therapeutic effects on a disorder associated with a pathology of epithelial cells and/or mesenchymal cells.

In some embodiments, a composition used in accordance to the methods of the invention comprises a FGF-20 protein, a fragment, a derivative, a variant, a homolog, or an analog of FGF-20, or a combination thereof. In some embodiments, a composition used in accordance to the methods of the invention comprises CG53135-01 (SEQ ID NO:2), CG53135-02 (SEQ ID NO:
4), CG53135-03 (SEQ ID NO:2), CG53135-04 (SEQ ID N0:7), CG53135-05 (SEQ ID NO: 2), CG53135-06 (SEQ ID
NO: 10), CG53135-07 (SEQ ID NO:12), CG53135-08 (SEQ ID NO:14), CG53135-09 (SEQ
ID
NO:16), CG53135-10 (SEQ ID NO:18), CG53135-11 (SEQ ID NO:20), CG53135-12 (SEQ
ID
NO:22), CG53135-13 (SEQ ID NO:24), CG53135-14 (SEQ ID NO:26), CG53135-15 (SEQ
ID
NO:28), CG53135-16 (SEQ ID NO:30), CG53135-17 (SEQ ID NO:32), IFC 250059629 (SEQ ID
NO:34), IFC 20059669 (SEQ ID NO:36), IFC 317459553 (SEQ ID NO:38), IFC
317459571 (SEQ ID
NO:40), IFC 250059596 (SEQ ID N0:10), or IFC316351224 (SEQ ID NO:10), or any two or more combinations of CG53135 proteins. In one embodiment, the compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, and (2) a protein comprising an amino acid sequence of SEQ ID
NO:24. In another embodiment, the compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID
NO:26, (4) a protein comprising an amino acid sequence of SEQ ID NO:28, (5) a protein comprising an amino acid sequence of SEQ ID NO:30, and (6) a protein comprising an amino acid sequence of SEQ ID NO:32. In another embodiment, the compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:28, (4) a protein comprising an amino acid sequence of SEQ ID
NO:30, and (5) a protein comprising an amino acid sequence of SEQ ID NO:32. In another embodiment, the compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:32; (2) a protein comprising an amino acid sequence of SEQ ID NO:30, (3) a protein comprising an amino acid sequence of SEQ ID
NO:28; and (4) a protein comprising an amino acid sequence of SEQ ID NO:24. In yet another embodiment, the compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID
NO:28, (4) a protein comprising an amino acid sequence of SEQ ID NO:30, (5) a protein comprising an amino acid sequence of SEQ ID NO:32, (6) a carbamylated protein comprising an amino acid sequence of SEQ ID NO:24, and (7) a carbamylated protein comprising an amino acid sequence of SEQ ID NO:2.

In some embodiments, an insult affecting rapidly proliferating tissues is radiation exposure.
In a specific embodiment, the insult is ionizing radiation. In another embodiment, the insult may be one or more chemotherapies or one or more chemical/biological warfare agents (such as a vesicant agent or bacteria), or a combination thereof. Non-limiting examples of chemotherapy and chemical/biological warfare agent are alkylating agents, vesicant agents (e.g., mustard agents) and microorganisms. In some embodiments, an insult affecting rapidly proliferating tissues is one or more radiation exposures, one or more chemotherapies, one or more chemical/biological warfare agents, or a combination thereof.

Organs and body systems most sensitive to the effects of insult such as ionizing radiation include, but are not limited to, skin, hematopoietic and lymphatic systems, gonads, lungs, nerve tissues, and the GI tract. In one embodiment, the insult are particularly damaging to hematopoietic and/or gastrointestinal tissues of a subject. In a specific embodiment, the disorder to be prevented or treated is a disorder of hematopoiesis, including but not limited to, anemia, leukopenia (e.g., neutropenia), thrombocytopenia, pancytopenia, and a clotting disorder. In another embodiment, the disorder to be prevented or treated is alimentary mucositis, including but not limited to, oral mucositis, esophagitis, stomatitis, enteritis, and proctitis. In another embodiment, the disorder to be prevented or treated is a cerebrovascular syndrome. In some embodiments, the symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents) include, but are not limited to, diarrhea, skin burn, sores, fatigue, dehydration, inflammation, hair loss, ulceration of oral mucosa, xerostomia, and bleeding (e.g., from the nose, mouth or rectum).

The present invention also provides methods of upregulating oxygen scavenging pathways in a subject, where the methods comprise administering to the subject a composition comprising one or more CG53135 proteins. In one embodiment, the oxygen scavenging pathways comprise one or more superoxide dismutases ("SOD"), including but are not limited to, intracellular CuZnSOD and MnSOD, and extracellular-SOD ("EC-SOD"). In another embodiment, the oxygen scavenging pathways comprise genes selected from the group consisting of ERK, AKT, a superoxide dismutase, cyclooxygenase-2 ("COX-2"), and Nrf-2. Cells exposed to radiation must be able to deal with the detrimental effects of ionized radicals (reactive oxygen species or ROS), of which the most reactive species within the cell are generated by the ionization of H20. Administering one or more CG53135 proteins to a subject increases transcription of enzymes, like superoxide, that scavenge ROS and convert them to less reactive intermediates, like hydrogen peroxide.
Administering one or more CG53135 proteins to a subject also reduces the load of reactive oxygen species induced by an insult, such as radiation.

The present invention further provides methods of stimulating secretion of one or more endogenous cytokines and/or endogenous chemokines from cells of a subject comprising administering to the subject a composition comprising one or more CG53135 proteins. The endogenous cytokines secreted can be, but are not limited to, interleukin ("IL") - 1 b, IL-6, IL-7, IL-8, IL-11, and granulocyte-colony forming factor ("G-CSF"). The endogenous chemokines secreted can be, but are not limited to, chemokine (C-X-C motif) ligand 1("CXCL1") and monocyte chemoattractant protein ("MCP-1"). Some of these endogenous cytokines and chemokines have been shown to be involved in endogenous radioprotective responses.

The present invention provides methods of stimulating proliferation of hematopoietic stem cells and/or gastrointestinal stem cells of a subject, where the methods comprise administering to the subject a composition comprising one or more CG53135 proteins. In one embodiment, administration of one or more CG53135 proteins to a subject stimulates fibroblast cells within the bone marrow stroma to secret factors that facilitate the health and proliferative capacity of hematopoietic stem cells. In another embodiment, administration of one or more CG53135 proteins to a subject leads to a rapid proliferative burst of gastrointestinal stem cells, which is followed by a counter-regulatory inhibition in proliferation 24 hours later. This leads to a synchronization of the cell cycle at the tissue level, which is more radio-resistant.

The present invention also provides methods of optimizing engraftment of hematopoietic stem cells in a subject, where the methods comprise administering to the subject a composition comprising one or more CG53135 proteins. In one embodiment, administration of one or more CG53135 proteins improves successful engraftment or repopulation of T-cells following a bone marrow transplant after marrow radioablation. In another embodiment, administration of one or more CG53135 proteins increases the speed of T-cell reconstitution within the thymus after bone marrow transplant.

The patient population that can be targeted using the methods of the present invention include, but are not limited to, subjects who have been exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents), subjects who are suspected to have been exposed an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents), subjects who will be exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents), and subjects who are at risk to be exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents).

In one embodiment, a composition comprising one or more CG53135 proteins is administered to a subject prior to the subject's exposure to an insult affecting rapidly proliferating tissue in a body. In another embodiment, a composition comprising one or more CG53135 proteins is administered to a subject after the subject's exposure to an insult affecting rapidly proliferating tissue in a body but prior to a disorder associated with the insult or a symptom thereof developed in the subject. In another embodiment, a composition comprising one or more CG53135 proteins is administered to a subject after a disorder associated with an insult affecting rapidly proliferating tissue in a body or a symptom thereof developed in the subject. In yet another embodiment, a composition comprising one or more CG53135 proteins is administered to a subject who is at risk for exposure to an insult affecting rapidly proliferating tissues.

Compositions comprising one or more CG53135 proteins can also be administered in combination with one or more other therapies to prevent, treat, or ameliorate a disorder or one or more symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents). In a preferred embodiment, a composition comprising one or more CG53135 proteins is administered in combination with one or more other therapies known to be used in preventing, treating, or ameliorating a disorder or one or more symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents). Examples of such other therapies include, but are not limited to, Mesna (sodium 2-mercaptoethene sulfonate) and other analogues with free thiol moieties, dimesna (disodium 2,2'-dithiobis ethane sulfonate) and other disulfides, and compounds such as, for example, described in U.S. Application Publication No.
20030092681, and KGF (see e.g., U.S. Patent No. 6,743,422). Examples of other agents that can be used in combination with a composition comprising CG53135 is shown in Table 1 B.

Table 1 B.
Trade name, Company / Administration Mechanism Treatment Mode common or Supplier Method(s) chemical name Amifostine Medimmune IV (200 mg/m2) Free radical Possible prophylaxis Ethyol, WR-2721 Inc. approved scavenger for first responders phosphorylated SC (500 mg/m2) Protection from 15-30 minutes prior to aminothiol possible reactive oxygen exposure to radiation species Sodium Pharmacies 4 g initial Alkalinization of Treatment following bicarbonate followed by 2 g urine leading to in~estion of uranium every 4 h rapid secretion (2 U) of uranium carbonate complex Ca-DTPA HEYL Chemical 1 g IV or Metal chelation Treatment following Zn-DTPA Pharmaceutical nebulizer (Calcium is ingestion or inhalation calcium or zinc Factory (Berlin) substituted by of plutonium, salt of diethylene Distributed by: other metals) americium triamine penta- Oak Ridge Promotes acetate Associated increased renal Universities / clearance DOE
Potassium iodide Pharmacies 16-130 mg oral Thyroid blocker Prophylaxis prior to depending on Prevents exposure, or exposure and accumulation of treatment age radioiodines immediately following (hrs) Radiogardase HEYL Chemical 1 g orally three Complex with Treatment following Prussian Blue Pharmaceutical times per day Cesium (137Cs) ingestion or other ferric Factory (Berlin) or Thallium internal contamination hexacyanoferrate Administered leading to of the gut by: Oak Ridge enhanced Inst. for secretion Science and Industry / DOE
G-CSF Amgen Inc. SC or IV Stimulates the Off label use following filgrastim, 5 g/kg/day proliferation, radiation exposure to Neupogen filgrastim differentiation, limit life threatening pegfilgrastim, SC 6mg and function of infections Neulasta pegfilgrastim neutrophils Prophylaxis GM-CSF Berlex SC or IV Stimulates the Off label use following sargramostim, (Schering AG) 250 mg/m2/day proliferation, radiation exposure to Leukine differentiation, limit life threatening and function of infections neutrophils Prophylaxis In one embodiment, during a combination therapy, a CG53135 protein and/or another therapy are administered in a sub-optimal amount, e.g., an amount that does not manifest detectable therapeutic benefits when administered alone, as determined by methods known in the art. In such methods, co-administration of a CG53135 protein and another therapy results in an overall improvement in effectiveness of treatment.

In one embodiment, a composition comprising one or more CG53135 proteins and one or more other therapies are administered within the same patient visit. In another embodiment, a composition comprising one or more CG53135 proteins is administered prior to the administration of one or more other therapies. In yet another embodiment, a composition comprising one or more CG53135 proteins is administered subsequent to the administration of one or more other therapies.
In a specific embodiment, a composition comprising one or more CG53135 proteins and one or more other therapies are cyclically administered to a subject. Cycling therapy involves the administration of a composition comprising one or more CG53135 proteins for a period of time, followed by the administration of one or more other therapies for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment.

5.4.3. Inflammatory Bowel Disease and Irritable Bowel Syndrome In one embodiment, the present invention provides methods of preventing and/or treating inflammatory bowel disease or irritable bowel syndrome comprising administering to a subject in need thereof an effective amount of a composition comprising one or more isolated CG53135 proteins. Inflammatory bowel disease that can be prevented and/or treated by the methods of the invention includes, but is not limited to, ulcerative colitis and Crohn's disease.

The present invention provides methods of preventing and/or treating inflammatory bowel disease in patient populations with inflammatory bowel disease and populations at risk to develop inflammatory bowel disease. The present invention also provides methods of preventing and/or treating irritable bowel syndrome in patient populations with irritable bowel syndrome and populations at risk to develop irritable bowel syndrome.

In one embodiment, the present invention provides a method of preventing or treating inflammatory bowel disease or irritable bowel syndrome comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of an isolated protein selected from the group consisting of: (a) a protein comprising an amino acid sequence of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; (b) a protein with one or more amino acid substitutions to the protein of (a), wherein said substitutions are no more than 15% of the amino acid sequence of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and wherein said protein with one or more amino acid substitutions retains cell proliferation stimulatory activity; and (c) a fragment of the protein of (a) or (b), which fragment retains cell proliferation stimulatory activity.

In some embodiments, the present invention provides a method of preventing and/or treating inflammatory bowel disease or irritable bowel syndrome comprising cyclically administering a composition comprising one or more CG53135 proteins. In one embodiment, cycling therapy involves the administration of a first therapy for a period of time, followed by the administration of a second therapy for a period of time and repeating this sequential administration, i.e., the cycle, in order to, e.g., to avoid or reduce the side effects of one of the therapies and/or to improve the efficacy of the therapies. In another embodiment, cycling therapy involves the administration of a therapy for a period of time, stop the therapy for a period of time, and repeat the administration of the therapy.

In accordance to the instant invention, a composition comprising one or more isolated CG53135 proteins can also be used in combination with other therapies to prevent and/or treat inflammatory bowel disease or irritable bowel syndrome. In one embodiment, a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other therapies (e.g., therapeutic agents) that have prophylactic and/or therapeutic effect(s) on inflammatory bowel disease and/or have amelioration effect(s) on one or more symptoms associated with inflammatory bowel disease to a subject to prevent and/or treat inflammatory bowel disease. Non-limiting examples of such therapies are: 5-aminosalicylates, antibiotics, corticosteroids, immunomodulators (e.g., 6-mercaptoputine, azathioprine, methotrexate, cyclosporine), and biological response modifiers (e.g., infliximab). In another embodiment, a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other therapies (e.g., therapeutic agents) that have prophylactic and/or therapeutic effect(s) on irritable bowel syndrome and/or have amelioration effect(s) on one or more symptoms associated with irritable bowel syndrome to a subject to prevent and/or treat inflammatory bowel disease. Non-limiting examples of such therapies are: laxatives;
antidiarrheals (e.g., diphenoxylate (e.g., Lomotil, Lomocot); loperamide (e.g., Imodium, Pepto Diarrhea), cholestyramine (e.g., Questran, Cholybar)); antispasmodics (e.g., dicyclomine, hyoscyamine, and clidinium (in combination with chlordiazepoxide hydrochloride)); peppermint oil; direct smooth muscle relaxants;
antidepressants; 5-HT3 antagonists (e.g., Alosetron (Lotronex), cilansetron);
5-HT4 agonists (e.g., tegaserod (Zelnorm/Zelmac) and prucalopride); M3 receptor antagonists (e.g., zamifenacin and darifenacin).

5.4.4. Arthritis and Diseases Associated with CNS System or Cardiovascular System In one embodiment, the present invention provides methods of preventing and/or treating a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases) comprising administering to a subject in need thereof an effective amount of a composition comprising one or more isolated CG53135 proteins.

In one embodiment, the present invention provides methods of preventing and/or treating arthritis (e.g., osteoarthritis or rheumatic arthritis) comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins.

In another embodiment, the present invention provides methods of reducing cartilage degeneration comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins. In another embodiment, the present invention provides methods of stimulating cartilage repair comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins. In a specific embodiment, the present invention provides methods of stimulating cartilage healing after surgery in a subject comprising administering to a subject a composition comprising one or more CG53135 proteins.

In another embodiment, the present invention provides methods of preventing and/or treating a cardiovascular disease, such as stroke (e.g., ischemic stroke, hemorrhagic stroke), comprising administering to a subject a composition comprising one or more CG53135 proteins. In a specific embodiment, the present invention provides methods of preventing and/or treating a cardiovascular disease, such as stroke, comprising administering to a subject a composition comprising an isolated protein comprising an amino acid sequence of SEQ ID NO:
4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a combination thereof.

In another embodiment, the present invention provides methods of preventing and/or treating a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis, Huntington's disease) comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins. In a specific embodiment, the present invention provides methods of preventing and/or treating a neurodegenerative disease comprising administering to a subject in need thereof a composition comprising an isolated protein comprising an amino acid sequence of SEQ ID NO: 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a combination thereof.

In some embodiments, the present invention provides a method of preventing and/or treating a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases) comprising cyclically administering a composition comprising one or more CG53135 proteins. In one embodiment, cycling therapy involves the administration of a first therapy for a period of time, followed by the administration of a second therapy for a period of time and repeating this sequential administration, i.e., the cycle, in order to, e.g., to avoid or reduce the side effects of one of the therapies and/or to improve the efficacy of the therapies. In another embodiment, cycling therapy involves the administration of a therapy for a period of time, stop the therapy for a period of time, and repeat the administration of the therapy. In accordance to the present invention, a composition comprising one or more CG53135 proteins can be administered to a subject prior to, during, or after the administration of a radiation therapy and/or chemotherapy, where such radiation therapy and/or chemotherapy is a cycling therapy.

In accordance to the instant invention, a composition comprising one or more isolated CG53135 proteins can also be used in combination with other therapies to prevent and/or treat a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases). In one embodiment, a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other agents that have prophylactic and/or therapeutic effect(s) on a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases) and/or have amelioration effect(s) on one or more symptoms associated with the disease to a subject to prevent and/or treat the disease.
Any other agents or therapies that are known in the art that can be used to prevent and/or treat a disease, such as a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases, can be used in combination with a composition comprising one or more CG53135 proteins in accordance to the methods of the present invention.
In a specific embodiment, the present invention provides methods of stimulating cartilage healing after surgery in a subject comprising administering to a subject a composition comprising one or more CG53135 proteins.

5.5 DOSAGE REGIMENS
Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A
dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The amount of the composition of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

In one embodiment, the dosage of a composition comprising one or more G53135 proteins for administration in a human patient provided by the present invention is at least 0.001 mg/kg, at least 0.005 mg/kg, at least 0.01 mg/kg, at least 0.03 mg/kg, at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, or at least 10 mg/kg (as measured by UV assay). In another embodiment, the dosage of a composition comprising one or more CG53135 proteins for administration in a human patient provided by the present invention is between 0.001-10 mg/kg, between 0.005-5 mg/kg, between 0.005-1 mg/kg, between 0.005-0.9 mg/kg, between 0.005-0.8 mg/kg, between 0.005-0.7 mg/kg, between 0.005-0.6 mg/kg, between 0.005-0.5 mg/kg, or between 0.005-0.3 mg/kg, between 0.01-1 mg/kg, between 0.01-0.9 mg/kg, between 0.01-0.8 mg/kg, between 0.01-0.7 mg/kg, between 0.01-0.6 mg/kg, between 0.01-0.5 mg/kg, or between 0.01-0.3 mg/kg (as measured by UV assay).

Protein concentration can be measured by methods known in the art, such as Bradford assay or UV assay, and the concentration may vary depending on what assay is being used. In a non-limiting example, the protein concentration in a pharmaceutical composition of the instant invention is measured by a UV assay that uses a direct measurement of the UV
absorption at a wavelength of 280 nm, and calibration with a well characterized reference standard of CG53135 protein (instead of IgG). Test results obtained with this UV method (using CG53135 reference standard) are three times lower than test results for the same sample(s) tested with the Bradford method (using IgG as calibrator). For example, if a dosage of a composition comprising one or more CG53135 proteins for administration in a human patient provided by the present invention is between 0.001-10 mg/kg measured by UV assay, then the dosage is 0.003-30 mg/kg as measured by Bradford assay.

In one embodiment, prior to administering the first full dose, each patient preferably receives a subcutaneous injection of a small amount (e.g., 1/100 to 1/10 of the prescribed dose) of a composition of the invention to detect any acute intolerance. The injection site is examined one and two hours after the test. If no reaction is detected, then the full dose is administered.

5.6 PHARMACEUTICA COMPOSITIONS AND FORMULATIONS
The present invention encompasses pharmaceutical compositions (including formulations) comprising one or more CG53135 proteins. The pharmaceutical compositions can be administered to a subject at a prophylactically or therapeutically effective amount to prevent and/or treat one or more diseases, such as inflammatory bowel disease ("IBD"), irritable bowel syndrome, alimentary mucositis (including oral mucositis), arthritis, diseases associated with the central nerve system or cardiovascular system, and symptoms associated with radiation exposure.
Various delivery systems are known and can be used to administer a composition comprising one or more CG53135 proteins. Such delivery systems include, but are not limited to, encapsulation in liposomes, microparticies, microcapsuies, expression by recombinant cells, receptor-mediated endocytosis, construction of the nucleic acids of the invention as part of a retroviral or other vectors, etc.
Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intrathecal, intracerebroventricular, epidural, intravenous, subcutaneous, intranasal, intratumoral, transdermal, rectal, and oral routes. The compositions of the invention may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, virginal mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents.
Administration can be systemic or local. In a specific embodiment, the present invention comprises using single or double chambered syringes, preferably equipped with a needle-safety device and a sharper needle, that are pre-filled with a composition comprising one or more CG53135 proteins. In one embodiment, dual chambered syringes (e.g., Vetter Lyo-Ject dual-chambered syringe by Vetter Pharmar-Fertigung) are used. Such systems are desirable for lyophilized formulations, and are especially useful in an emergency setting.

In some embodiments, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment. This may be achieved by, for example, topical application, by injection, by infusion pump, by means of a suppository, or by means of an implant (the implant being of a reservoir with a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers).

In some embodiments, a CG53135 nucleic acid can be administered in vivo to promote expression of their encoded proteins, by constructing the nucleic acid as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector, or by direct intramuscular or intradermal injection, or by use of microparticle bombardment (e.g., a gene gun), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus, etc. Alternatively, a CG53135 nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The instant invention encompasses bulk drug compositions useful in the manufacture of pharmaceutical compositions that can be used in the preparation of unit dosage forms. In a preferred embodiment, a composition of the invention is a pharmaceutical composition. Such compositions comprise a prophylactically or therapeutically effective amount of CG53135, and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical compositions are formulated to be suitable for the route of administration to a subject.

In one embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally regarded as safe for use in humans (GRAS). The term "carrier" refers to a diluent, adjuvant, bulking agent (e.g., arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose), excipient, or vehicle with which CG53135 is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils (e.g., oils of petroleum, animal, vegetable or synthetic origins, such as peanut oil, soybean oil, mineral oil, sesame oil and the like), or solid carriers, such as one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, or encapsulating material. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include, but are not limited to, starch or its synthetically modified derivatives such as hydroxyethyl starch, stearate salts, glycerol, glucose, lactose, sucrose, trehalose, gelatin, sulfobutyl ether Beta-cyclodextrin sodium, sodium chloride, glycerol, propylene, glycol, water, ethanol, or a combination thereof. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The compositions comprising CG53135 may be formulated into any of many possible dosage forms such as, but not limited to, liquid, suspension, microemulsion, microcapsules, tablets, capsules, gel capsules, soft gels, pills, powders, enemas, sustained-release formulations and the like. The compositions comprising CG53135 may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch or its synthetically modified derivatives such as hydroxyethyl starch, stearate salts, sodium saccharine, cellulose, magnesium carbonate, etc.

A pharmaceutical composition comprising CG53135 is formulated to be compatible with its intended route of administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, intratumoral or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic or hypertonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as benzyl alcohol or lidocaine to ease pain at the site of the injection.

If a composition comprising CG53135 is to be administered topically, the composition can be formulated in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the compositions of the invention are in admixture with a topical delivery agent, such as but not limited to, lipids, liposomes, micelles, emulsions, sphingomyelins, lipid-protein or lipid peptide complexes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. The compositions comprising CG53135 may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, the compositions comprising CG53135 may be complexed to lipids, in particular to cationic lipids. For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed.
Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as Freon or hydrofluorocarbons) or in a squeeze bottle.
Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.

A composition comprising CG53135 can be formulated in an aerosol form, spray, mist or in the form of drops or powder if intranasal administration is preferred. In particular, a composition comprising CG53135 can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichiorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, other hydrofluorocarbons, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Microcapsules (composed of, e.g., polymerized surface) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as dissacharides or starch.

One or more CG53135 proteins may also be formulated into a microcapsule with one or more polymers (e.g., hydroxyethyl starch) form the surface of the microcapsule. Such formulations have benefits such as slow-release.

A composition comprising CG53135 can be formulated in the form of powders, granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets if oral administration is preferred. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);
emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).

In one embodiment, the compositions of the invention are orally administered in conjunction with one or more penetration enhancers, e.g., alcohols, surfactants and chelators. Preferred surfactants include, but are not limited to, fatty acids and esters or salts thereof, bile acids and salts thereof. In some embodiments, combinations of penetration enhancers are used, e.g., alcohols, fatty acids/salts in combination with bile acids/salts. In a specific embodiment, sodium salt of lauric acid, capric acid is used in combination with UDCA. Further penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Compositions of the invention may be delivered orally in granular form including, but is not limited to, sprayed dried particles, or complexed to form micro or nanoparticles. Complexing agents that can be used for complexing with the compositions of the invention include, but are not limited to, poly-amino acids, polyimines, polyacrylates, polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates, cationized gelatins, albumins, acrylates, polyethyleneglycols (PEG), DEAE-derivatized polyimines, pollulans, celluloses, and starches. Particularly preferred complexing agents include, but are not limited to, chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG).

A composition comprising CG53135 can be delivered to a subject by pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent.

In a preferred embodiment, a composition comprising CG53135 is formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).
Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added ,preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as benzyl alcohol or lidocaine to ease pain at the site of the injection.
Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a sealed container, such as a vial, ampoule or sachette, indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion container containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule or vial of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

A composition comprising CG53135 can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include, but are not limited to, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

In addition to the formulations described previously, a composition comprising may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.

Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A
dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The amount of the composition of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

In one embodiments, the dosage of a composition comprising one or more G53135 proteins for administration in a human patient provided by the present invention is at least 0.001 mg/kg, at least 0.01 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 60 mg/kg, at least 70 mg/kg, at least 80 mg/kg, at least 90 mg/kg, at least 100 mg/kg, at least 150 mg/kg, or at least 200 mg/kg (as measured by Bradford assay). In another embodiment, the dosage of a composition comprising one or more CG53135 proteins for administration in a human patient provided by the present invention is between 0.001-300 mg/kg, between 0.01-300 mg/kg, between 0.1-300 mg/kg, between 0.5-250 mg/kg, between 1-200 mg/kg, between 1-150 mg/kg, between 1-125 mg/kg, between 1-100 mg/kg, between 1-90 mg/kg, between 1-80 mg/kg, between 1-70 mg/kg, between 1-60 mg/kg, between 1-50 mg/kg, between 1-40 mg/kg, between 1-35 mg/kg, between 1-30 mg/kg, between 1-25 mg/kg, between 1-20 mg/kg, between 1-15 mg/kg, between 1-10 mg/kg, or between 1-5 mg/kg (as measured by Bradford assay).

Protein concentration can be measured by methods known in the art, such as Bradford assay or UV assay, and the concentration may vary depending on what assay is being used. In a non-limiting example, the protein concentration in a pharmaceutical composition of the instant invention is measured by a UV assay that uses a direct measurement of the UV
absorption at a wavelength of 280 nm, and calibration with a well characterized reference standard of CG53135 protein (instead of IgG). Test results obtained with this UV method (using CG53135 reference standard) are three times lower than test results for the same sample(s) tested with the Bradford method (using IgG as calibrator). For example, if a dosage of a composition comprising one or more CG53135 proteins for administration in a human patient provided by the present invention is between 0.1-300 mg/kg measured by Bradford assay, then the dosage is 0.033 -100 mg/kg as measured by a UV assay.

In one embodiment, prior to administering the first full dose, each patient preferably receives a bolus injection of a small amount (e.g., 1/100 to 1/10 of the prescribed dose) of a composition of the invention to detect any acute intolerance. The injection site is examined one and two hours after the test. If no reaction is detected, then the full dose is administered.

The invention also provides kits for carrying out the therapeutic regimens of the invention.
Such kits comprise in one or more containers prophylactically or therapeutically effective amounts of the composition of the invention in pharmaceutically acceptable form. The composition in a vial of a kit of the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid. Alternatively, the composition may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the composition to form a solution for injection purposes.

In another embodiment, a kit of the invention further comprises a needle or syringe (single or dual chambered), preferably packaged in sterile form, for injecting the formulation, and/or a packaged alcohol pad. In a specific embodiment, a kit of the invention comprises pre-filled needles or syringes (single or dual chambered) that are pre-filled with a composition comprising one or more CG53135 proteins. Instructions are optionally included for administration of the formulations of the invention by a clinician or by the patient.

In some embodiments, the present invention provides kits comprising a plurality of containers each comprising a pharmaceutical formulation or composition comprising a dose of the composition of the invention sufficient for a single administration.

5.6.1 Improved Formulations and Methods to Increase Solubility of a FGF
Protein The present invention provides improved formulations comprising one or more FGFs, preferably one or more CG53135 proteins, and methods for increasing solubility of FGF proteins.
The improved formulations are more stable and more favorable for commercial scale productions.

While not limited by any theory, the improved formulations are based partially on the discovery that high concentrations of arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, sucrose, acetate, succinate, or tartrate or a combination thereof can increase solubility of a growth factor, including FGF proteins. Accordingly, in one embodiment, the present invention provides a method of increasing solubility of a FGF protein in a solution (e.g., an aqueous solution) by adding arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose to the solution. In another embodiment, the present invention provides a method for increasing solubility of a FGF protein in a solution by adding acetate, succinate, tartrate, or a combination thereof to the solution. In yet another embodiment, acetate, succinate, tartrate or a combination thereof is added in combination with arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose to the solution to increase the solubility of a FGF protein. The arginine in a salt form can be, but is not limited to, arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride. In a preferred embodiment, arginine sulfate is used. In some embodiments, the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose is between 0.01 M to 1 M. In one embodiment, the final concentration of the arginine in a salt form is 0.5 M. In some embodiment, the final concentration of the acetate, succinate, tartrate or a combination thereof is 0.01 to 0.2 M.

In a preferred embodiment, the FGF protein in the formulation is a FGF-20 protein, a fragment, a derivative, a variant, a homolog, or an analog of FGF-20, or a combination thereof. In some embodiments, the FGF protein in the formulation is CG53135-01 (SEQ ID
NO:2), CG53135-02 (SEQ ID NO: 4), CG53135-03 (SEQ ID NO:2), CG53135-04 (SEQ ID NO:7), CG53135-05 (SEQ
ID NO: 2), CG53135-06 (SEQ ID NO: 10), CG53135-07 (SEQ ID NO:12), CG53135-08 (SEQ ID
NO:14), CG53135-09 (SEQ ID NO:16), CG53135-10 (SEQ ID NO:18), CG53135-11 (SEQ
ID
NO:20), CG53135-12 (SEQ ID NO:22), CG53135-13 (SEQ ID NO:24), CG53135-14 (SEQ
ID

NO:26), CG53135-15 (SEQ ID NO:28), CG53135-16 (SEQ ID NO:30), CG53135-17 (SEQ
ID
NO:32), IFC 250059629 (SEQ ID NO:34), IFC 20059669 (SEQ ID NO:36), IFC
317459553 (SEQ ID
NO:38), IFC 317459571 (SEQ ID NO:40), IFC 250059596 (SEQ ID NO:10), or IFC316351224 (SEQ
ID NO:10), or any two or more combinations of CG53135 proteins. In one embodiment, the FGF
proteins in the formulation comprise (1) a protein comprising an amino acid sequence of SEQ ID
NO:2, and (2) a protein comprising an amino acid sequence of SEQ ID NO:24. In another embodiment, the FGF proteins in the formulation comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:26, (4) a protein comprising an amino acid sequence of SEQ ID NO:28, (5) a protein comprising an amino acid sequence of SEQ ID
NO:30, and (6) a protein comprising an amino acid sequence of SEQ ID NO:32. In another embodiment, the FGF proteins in the formulation comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:28, (4) a protein comprising an amino acid sequence of SEQ ID NO:30, and (5) a protein comprising an amino acid sequence of SEQ ID
NO:32. In another embodiment, a formulation of the invention comprises (1) a protein comprising an amino acid sequence of SEQ ID NO:32; (2) a protein comprising an amino acid sequence of SEQ ID
NO:30, (3) a protein comprising an amino acid sequence of SEQ ID NO:28; and (4) a protein comprising an amino acid sequence of SEQ ID NO:24. In yet another embodiment, the FGF
proteins in the formulation comprise (1) a protein comprising an amino acid sequence of SEQ ID
NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:28, (4) a protein comprising an amino acid sequence of SEQ ID NO:30, (5) a protein comprising an amino acid sequence of SEQ ID NO:32, (6) a carbamylated protein comprising an amino acid sequence of SEQ ID NO:24, and (7) a carbamylated protein comprising an amino acid sequence of SEQ ID NO:2.

The present invention provides improved formulations comprising arginine in a salt form, sodium phosphate monobasic (NaH2PO4=H2O), a surfactant, and one or more CG53135 proteins. In one embodiment, the present invention provides improved formulations comprising 0.1-1 M arginine in a salt form, 0.01-0.1 M sodium phosphate monobasic (NaH2PO4=H2O), 0.01 %-0.1 %
weight/volume ("w/v") polysorbate 80 or polysorbate 20, and 0.005-50 mg/ml of one or more CG53135 proteins. The arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose thereof can be, but is not limited to, arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride. In a preferred embodiment, arginine sulfate is used.
In some embodiments, the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose thereof is 0.01-0.7 M. In one embodiment, the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose thereof is 0.5 M.
In some embodiments, the concentration of sodium phosphate monobasic in the formulations is between 0.02-0.09 M, 0.03-0.08 M, or 0.04-0.06M. In a specific embodiment, the sodium phosphate monobasic is 0.05M. In one embodiment, the improved formulations comprise a surfactant, which may be added, e.g., during the diafiltration and/or ultrafiltration step, to minimize the formation of aggregates. The surfactant can be, but is not limited to, polysorbate 80 and polysorbate 20.
In a specific embodiment, the concentration of polysorbate 80 or polysorbate 20 is 0.01%
(weight/volume).

The improved formulations of the present invention comprise one or more proteins: In one embodiment, the improved formulations of the invention comprise one or more proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40. In another embodiment, the improved formulations of the invention comprise one or more proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40. In a specific embodiment, the improved formulations of the invention comprise a protein comprising an amino acid sequence of SEQ ID NO:2. In another specific embodiment, the improved formulations of the invention comprise a protein comprising an amino acid sequence of SEQ ID NO:24. In yet another specific embodiment, the improved formulations of the invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, and (2) a protein comprising an amino acid sequence of SEQ ID NO:24. In a specific embodiment, the improved formulations of the invention comprise (1) a protein comprising an amino acid sequence of SEQ ID
NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:26, (4) a protein comprising an amino acid sequence of SEQ ID NO:28, (5) a protein comprising an amino acid sequence of SEQ ID NO:30, and (6) a protein comprising an amino acid sequence of SEQ ID NO:32. In one embodiment, the improved formulations of the invention comprise one or more proteins produced by any of the processes described in Section 5.2, supra. In some embodiments, the concentration of one or more CG53135 proteins in the improved formulations of the instant invention is at least 2 mg/ml, at least 10 mg/mI, at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/mI, at least 35 mg/mi, at least 40 mg/mI, at least 45 mg/ml, or at least 50 mg/ml. In some embodiments, the concentration of one or more CG53135 proteins in the improved formulations of the instant invention is no more than 50 mg/mi, no more than 30 mg/mI, no more than 10 mg/ml, no more than 5 mg/ml, no more than 1 mg/mi, or no more than 0.5 mg/ml. In some embodiments, the concentration of one or more CG53135 proteins in the improved formulations of the instant invention is 0.0005- 60 mg/mi, 0.005-50 mg/mI, 0.05-50 mg/mi, 0.5-50 mg/mI, 1-60 mg/mi, 1-50 mg/mI, 5-40 mg/mI, 5-30 mg/ml, or 5-20 mg/mi. In a specific embodiment, the concentration of one or more CG53135 proteins in the improved formulations of the instant invention is 10 mg/mI.

The improved formulations of the invention can be lyophilized or spray dried, which results more stable products with longer shelf life and the ease of handling and shipment. The process of lyophilization is very well known in the art and is not described in detail herein. Briefly, lyophilization is the process by which the moisture content of the product is reduced by freezing and subsequent sublimation under vacuum. The lyophilization process primarily consists of three stages. The first stage involves freezing the product and creating a frozen matrix suitable for drying. This step impacts the drying characteristics in the next two stages. The second stage is primary drying.

Primary drying involves the removal of the ice by sublimation by reducing the pressure (to typically around 50-500 pm Hg) of the product's environment while maintaining the product temperature at a low, desirable level. The third stage in the process is called secondary drying where the bound water is removed until the residual moisture content reaches below the target level. Any lyophilization process known in the art can be used to lyophilize the formulations of the invention.

The objective of a lyophilization process is to achieve a freeze-dried protein cake with acceptable appearance, biological potency, ease of reconstitution, and long-term storage stability. A
prudently designed lyophilization cycle is one that is robust, consumes less time and energy, and maintains product quality. Both formulation-related and cycle-related factors contribute to achieving this goal.

The addition of a lyophilization excipient in the processes described herein may be necessary. One or more excipients may be added. The lyophilization excipients contemplated for use in the present processes include, but are not limited to, sucrose, lactose, mannitol, dextran, sucrose, heparin, glycine, glucose, glutamic acid, gelatin, sorbitol, histidine, dextrose, trehalose, methocel, hydroxy ethyl cellulose, hydroxy ethyl starch, poly(ethylene glycol), poly(vinyl pyrolidone), sulfobutyl ether Beta-cyclodextrin sodium and polyvinyl alcohol, or various combinations thereof, as well as other buffers, protein stabilizers, cryoprotectants, and cryopreservatives commonly used by those skilled in the art.

Since the active ingredient of the improved formulations of the invention is a FGF protein, preferably one or more CG53135 protein, the improved formulations of the invention can be used accordingly in any situation that a FGF protein, preferably a CG53135 protein, is known to be effective. For example, the improved formulations of the invention can be used in prevention and/or treatment of disorders such as alimentary mucositis, inflammatory bowel disease, osteoarthritis, disorders of the central nerve system or cardiovascular system, and disorders associated with radiation exposure or symptoms thereof.

6. EXAMPLE
Certain embodiments of the invention are illustrated by the following non-limiting examples.
6.1 EXAMPLE 1: EXPRESSION OF CG53135 Several different expression constructs were generated to express a CG53135 protein (Table 2).

Table 2: Constructs Generated to Express CG53135 Construct Construct Description Construct Diagram 1a NIH 3T3 cells were transfected with pFGF-20, which incorporates an epitope tag (V5) and a CG53135-01 V5 His polyhistidine tag into the carboxy-terminus of the CG53135-01 protein in the pcDNA3.1 vector (Invitroge lb Human 293-EBNA embryonic kidney cells or NIH 3T3 cells were transfected with CG53135-01 using pCEP4 vector (Invitrogen) containing an IgK CG53135- V5 His IgK signal sequence, multiple cloning sites, a V5 01 epitope tag, and a polyhistidine tag 2 E.coli BL21 cells were transformed with CG53135-01 using pETMY vector (CuraGen His T7 CG53135-01 Corporation) containing a polyhistidine tag and a T7 epitope tag (this construct is also referred to as E.coli/pRSET) 3 E.coli BLR (DE3) cells (NovaGen) were transformed with CG53135-05 (full-length, CG53135-05 codon-optimized) using pET24a vector (NovaGen) 4 E.coli BLR (DE3) cells (NovaGen) were transformed with CG53135 (deletion of amino acids 2-54, codon-optimized) using pET24a CG53135-02 (deletion mutant) vector (NovaGen) In one construct, CG53135-01 (the full-length CG53135 gene) was cloned as a Bgl II-Xho I
fragment into the Bam HI-Xho I sites in mammalian expression vector, pcDNA3.lV5His (invitrogen Corporation, Carlsbad, CA). The resultant construct, pFGF-20 (construct 1 a) has a 9 amino acid V5 tag and a 6 amino acid histidine tag (His) fused in-frame to the carboxy-terminus of CG53135-01.
These tags aid in the purification and detection of CG53135-01 protein. After transfection of pFGF-20 into murine NIH 3T3 cells, CG53135-01 protein was detected in the conditioned medium using an anti-V5 antibody (Invitrogen, Carlsbad, CA).

The full-length CG53135-01 gene was also cloned as a Bgl II-Xho I fragment into the Bam HI-Xho I sites of mammalian expression vector pCEP4/Sec (CuraGen Corporation).
The resultant construct, plgK-FGF-20 (construct 1 b) has a heterologous immunoglobulin kappa (IgK) signal sequence that could aid in secretion of CG53135-01. After transfection of plgK-FGF-20 into human 293 EBNA cells (Invitrogen, Carlsbad, CA; catalog # R620-07), CG53135-01 was detected in the conditioned medium using an anti-V5 antibody.

In order to increase the yield of CG53135 protein, a Bgl II-Xho I fragment encoding the full-length CG53135-01 gene was cloned into the Bam HI-Xho I sites of E.coli expression vector, pETMY (CuraGen Corporation). The resultant construct, pETMY-FGF-20 (construct 2) has a 6 amino acid histidine tag and a T7 tag fused in-frame to the amino terminus of CG53135. After transformation of pETMY-FGF-20 into BL21 E.coli (Novagen, Madison, WI), followed by T7 RNA
polymerase induction, CG53135-01 protein was detected in the soluble fraction of the cells.

In order to express FGF-20 without tags, CG53135-05 (a codon-optimized, full-length FGF-20 gene) and CG53135-02 (a codon-optimized deletion construct of FGF-20, with the N-terminal amino acids 2-54 removed) were synthesized. For the full-length construct (CG53135-05), an Nde I
restriction site (CATATG) containing the initiator codon was placed at the 5' end of the coding sequence. At the 3' end, the coding sequence was followed by 2 consecutive stop codons (TAA) and a Xho restriction site (CTCGAG). The synthesized gene was cloned into pCRScript (Stratagene, La Jolla, CA) to generate pCRScript-CG53135-05. An Nde I-Xho I
fragment containing the codon-optimized CG53135-05 gene was isolated from the pCRscript-CG53135-05 and subcloned into Nde I-Xho I-digested pET24a to generate pET24a-CG53135-05 (construct 3). The full-length, codon-optimized version of FGF-20 is referred to as CG53135-05.

To generate a codon-optimized deletion construct for CG53135, oligonucleotide primers were designed to amplify the truncated FGF-20 gene from pCRScript-CG53135-05.
The forward primer contained an Nde I site (CATATG) followed by coding sequence starting at amino acid 55.
The reverse primer contained a Hindlll restriction site. A single PCR product of approximately 480 base pairs was obtained and cloned into pCR2.1 vector (Invitrogen) to generate pCR2.1-CG53135del. An Nde I-Hind III fragment was isolated from pCR2.1-53135del and subcloned into Nde I-Hind III-digested pET24a to generate pET24a-CG53135-02 (construct 4).

The plasmids, pET24a-CG53135-05 (construct 3) and pET24a-CG53135-02 (construct 4) have no tags. Each vector was transformed into E.coli BLR (DE3), induced with isopropyl thiogalactopyranoside. Both the full-length and the N-terminally truncated CG53135 protein was detected in the soluble fraction of cells.

6.2 EXAMPLE 2: MANUFACTURE OF CG53135-05 AND PHARMACEUTICAL
FORMULATIONS
Aiming for a construct that would be suitable for clinical development, untagged molecules were generated in a phage-free bacterial host. The codon-optimized, full-length, untagged molecule (CG53135-05) has the most favorable pharmacology profile and was used to prepare product for the safety studies and clinical trial.

6.2.1 PRODUCTION PROCESS AND PHARMACEUTICAL FORMULATIONS
(PROCESS 1) CG53135-05 was expressed in Escherichia coli BLR (DE3) using a codon-optimized construct, purified to homogeneity, and characterized by standard protein chemistry techniques.
The isolated CG53135-05 protein migrated as a single band (23 kilodalton) using standard SDS-PAGE techniques and stained with Coommassie blue. The CG53135-05 protein was electrophoretically transferred to a polyvinylidenefluoride membrane and the stained 23 kD band was excised from the membrane and analyzed by an automated Edman sequencer (Procise, Applied Biosystems, Foster City, CA); the N-terminal amino acid sequence of the first 10 amino acids was confirmed as identical to the predicted protein sequence.

Fermentation and Primary Recovery Recombinant CG53135-05 was expressed using Escherichia co/i BLR (DE3) cells (Novagen).
These cells were transformed with full length, codon optimized CG53135-05 using pET24a vector (Novagen). A
Manufacturing Master Cell Bank (MMCB) of these cells was produced and qualified. The fermentation and primary recovery processes were performed at the 100 L (i.e., working volume) scale reproducibly.

Seed preparation was started by thawing and pooling of 1- 6 vials of the MMCB
and inoculating 4 - 7 shake flasks each containing 750 mL of seed medium. At this point, 3-6 L of inoculum was transferred to a production fermentor containing 60-80 L of start-up medium. The production fermentor was operated at a temperature of 37 C and pH of 7.1.
Dissolved oxygen was controlled at 30% of saturation concentration or above by manipulating agitation speed, air sparging rate and enrichment of air with pure oxygen. Addition of feed medium was initiated at a cell density of 30-40 AU (600 nm) and maintained until end of fermentation. The cells were induced at a cell density of 40-50 AU (600 nm) using 1 mM isopropyl-beta-D-thiogalactoside (IPTG) and CG53135-05 protein was produced for 4 hours post-induction. The fermentation was completed in 10-14 hours and about 100-110 L of cell broth was concentrated using a continuous centrifuge. The resulting cell paste was stored frozen at -70 C.

The frozen cell paste was suspended in lysis buffer (containing 3M urea, final concentration) and disrupted by high-pressure homogenization. The cell lysate was clarified using continuous flow centrifugation. The resulting clarified lysate was directly loaded onto a SP-sepharose Fast Flow column equilibrated with SP equilibration buffer (3 M urea, 100 mM sodium phosphate, 20 mM
sodium chloride, 5 mM EDTA, pH 7.4). CG53135-05 protein was eluted from the column using SP
elution buffer (100 mM sodium citrate, 1 M arginine, 5 mM EDTA, pH 6.0). The collected material was then diluted with an equal volume of SP elution buffer. After thorough mixing, the SP
Sepharose FF pool was filtered through a 0.2 pm PES filter and frozen at -80 C.

Purification of the Drug Substance The SP-sepharose Fast Flow pool was precipitated with ammonium sulfate. After overnight incubation at 4 C, the precipitate was collected by bottle centrifugation and subsequently solubilized in Phenyl loading buffer (100 mM sodium citrate, 500 mM L-arginine, 750 mM
NaCI, 5 mM EDTA, pH 6.0). The resulting solution was filtered through a 0.45 uM PES filter and loaded onto a Phenyl-sepharose HP column. After washing the column, the protein was eluted with a linear gradient with Phenyl elution buffer (100 mM sodium citrate, 500 mM L-arginine, 5 mM EDTA, pH
6.0). The Phenyl-sepharose HP pool was filtered through a 0.2 pm PES filter and frozen at -80 C in 1.8 L
aliquots.

Formulation and Fill/Finish Four batches of purified drug substance were thawed for 24 - 48 hours at 2 - 8 C and pooled into the collection tank of tangential flow ultrafiltration (TFF) equipment. The pooled drug substance was concentrated -5-fold via TFF, followed by about 5-fold diafiltration with the formulation buffer (40 mM sodium acetate, 0.2 M L-arginine, 3% glycerol). This buffer-exchanged drug substance was concentrated further to a target concentration of >10 mg/mL. Upon transfer to a collection tank, the concentration was adjusted to - 10 mg/mL with formulation buffer. The formulated drug product was sterile-filtered into a sterile tank and aseptically filled (at 10.5 mL per 20 mL vial) and sealed. The filled and sealed vials were inspected for fill accuracy and visual defects. A specified number of vials were drawn and labeled for release assays, stability studies, safety studies, and retained samples. The remaining vials were labeled for the clinical study, and finished drug product was stored at -80 15 C.

The finished drug product is a sterile, clear, colorless solution in single-use sterile vials for injection. CG53135-05 E. coli purified product was formulated at a final concentration of 8.2 mg/mL
(Table 3).

Table 3: Composition of Drug Product Component Grade Final concentration Amount per Liter CG53135-05 E. coli NA 8.2 mg/ml 8.2 g purified product Formulation Buffer Sodium acetate USP 40 mM 5.44 g (trihydrate) L-arginine HCI USP 200 mM 42.132 Glycerol USP 3% v/v 30 mL
Acetic acid USP NA QS to H 5.3 Water for injection USP NA QS to 1 L

The pharmacokinetics of optimally-formulated CG53135-05 E. coli purified product was assessed in rats following intravenous, subcutaneous, and intraperitoneal administration to compare exposure at active doses in animal models and predict exposure in humans.
Intravenous administration of CG53135-05 E. coli purified product resulted in high plasma levels (maximum plasma level = 19,680-47,252 ng/mL), which rapidly declined within the first 2 hours to 30-70 ng/mL;
decreased exposure was observed following the third daily dose (maximum plasma level = 5373-7453 ng/mL). Subcutaneous administration of CG53135-05 E. coli purified product resulted in slow absorption (maximum plasma level at 10 hours) and plasma levels of 40-80 ng/mL
up to 48 hours after dosing; some accumulation in plasma was seen following the third daily dose. Intraperitoneal administration of CG53135-05 E. coli purified product resulted in slow absorption (maximum plasma level at 2-4 hours) and plasma levels of 40-70 ng/mL up to 10 hours after dosing; decreased exposure was seen following third daily dose. No significant gender differences were observed by any route of administration.

Safety of intravenous administration of CG53135-05 E. coli purified product (0.05, 5 or 50 mg/kg/day (Bradford) for 14 consecutive days) was assessed in a pivotal toxicology study in rats.
There were no treatment-related findings in rats administered 0.05 mg/mL
(Bradford) CG53135-05 E. coli purified product for 14 days. In rats administered 5 mg/kg (Bradford) CG53135 for 14 days, food consumption was reduced and body weight was decreased; while there were no treatment-related changes in organ weights, urinalysis, ophthalmology, or histopathology parameters in this dose group, there were treatment-related changes in hematology and clinical chemistry parameters in this treatment group. In rats administered 50 mg/kg (Bradford) CG53135-05 E. coli purified product for 12 days (estimated maximum plasma level of 20-30 fold higher than active dose), food consumption was reduced and body weight was markedly decreased; while there were no treatment-related changes in ophthalmology, there were significant treatment-related changes in organ weights, urinalysis, hematology, clinical chemistry, and histopathology in this treatment group.

Safety of intravenous administration of CG53135-05 E. coli purified product (0 or 10 mg/kg/day (Bradford) for 7 consecutive days) was further assessed in a safety pharmacology study in rhesus monkeys. There were no treatment-related clinical observations in animals administered 1 mg/kg (Bradford) CG53135-05 E. coli purified product for 7 days. In animals administered 10 mg/kg (Bradford) CG53135-05 E. coli purified product for 7 days, minor effects on body weight were noted and associated with qualitative observations of lower food consumption. There were no apparent treatment-related effects on hematology, clinical chemistry, ophthalmology, or electrophysiology in either dose group.

Stability of CG53135-05 Drug Substances Stability studies on the CG53135-05 E. coli purified product produced during cGMP
manufacturing were performed. The analytical methods used as stability indicating assays for purified drug substance are listed in Table 4.

Table 4. Stability Assays for Drug Substance Assay Stability Criteria SDS-PAGE (Neuhoff stain) >98% pure by densitometry (reduced and nonreduced) RP-HPLC Peak at 5.5 t 1.0 min relative retention time SEC-HPLC >90% mono-disperse peak Total protein by Bradford method >0.2 mg/mL
Bioassay (BrdU) P1200 > 0.5 ng/mL and <20 ng/mL
pH 5.8 0.4 Visual appearance Clear and colorless P1200 = concentration of CG53135-05 that results in incorporation of BrdU at 2 times the background The SDS-PAGE, RP-HPLC, and Bradford assays are indicative of protein degradation or gross aggregation. The SEC-HPLC assay detects aggregation of the protein or changes in oligomerization, and the bioassay detects loss of biological activity of the protein. The stability studies for the purified drug substance were conducted at -80 to 15 C with samples tested at intervals of 3, 6, 9, 12, and 24 months.

In one experiment, stability studies of finished drug product were conducted by Cambrex at -80 t15 C and -20 t 5 C with samples tested at intervals of 1, 3, 6, 9, 12, and 24 months. Stability data collected after 1 month indicate that finished drug product is stable for at least 1 month when stored at -80 t15 C or at -20 5 C (Table 5).

Table 5. Stability Data for Drug Product after 1-month interval Assay Stability Criteria Initial -80 t15 C -20 t5 C
RP-HPLC Major peak Major peak Major peak Major peak retention time 0.2 retention time t retention time t retention time t min relative to 0.2 min relative 0.2 min relative 0.2 min relative Reference Standard to Reference to Reference to Reference Standard Standard Standard SDS-PAGE Major band Pass Pass Pass migrates at about 23 kDa;
nonreduced minor band below major band SEC-HPLC >90% mono-disperse 100% 100% 100%
peak Bradford 10 t 0.2 m/mL 8.2 8.6 8.3 Bioassay Pl2oo > 0.5 ng/mL 4.14 ng/mL 2.98 ng/mL 1/45 ng/mL
and <20 ng/mL
Sterility Pass (ie., no Pass NT NT
growth) pH 5.3t0.3 5.4 5.5 5.4 Visual Clear and colorless Pass Pass Pass ap earance solution Lot # 02502001 was stored at -80 t15 C or at -20 t 5 C at Cambrex and tested after 1 month; P1200 = concentration of CG53135-05 that results in incorporation of BrdU at 2 times the background; Pass = results met stability criterion; NT = not tested In another experiment, samples of finished drug product were stored at -80 t15 C or stressed at 5 t3 C, 25 t2 C, or 37 t2 C and tested at various intervals for 1 month. Stability data indicate that finished drug product showed no significant instability after 1 month of storage at -80 t150C or 5 t3 C. When stressed at 25 t2 C, finished drug product was stable for at least 48 hours;
degradation was apparent after I week at this temperature. When stressed at 37 t2 C, degradation of finished drug product was apparent within 4 hours.

6.2.2 IMPROVED PHARMACEUTICAL FORMULATIONS AND PRODUCTION
PROCESS OF CG53135-05 (PROCESS 2) An improved manufacture process for producing a drug product comprising one or more CG53135 proteins for clinical uses has been developed. FIG. I shows the steps involved in the improved manufacturing process of CG53135. The codon-optimized, full-length, untagged molecule of CG53135-05 (construct 3 in Example 1) was used. The process steps for the improved manufacture process are described below.

Cell Bank: a Manufacturing Master Cell Bank (MMCB) in animal component free complex medium was used in an earlier Process. A second Manufacturing Master Cell Bank (MMCB) in animal component free chemically defined medium was derived from the first MMCB and a Manufacturing Working Cell Bank (MWCB) was made from the second MMCB. This MWCB was used in the manufacturing process as described in FIG. 1.

Innoculum Preparation: the initial cell expansion occurs in shake flasks. Seed preparation is done by thawing and pooling 2 -3 vials of the MWCB in chemically defined medium and inoculating 3 - 4 shake flasks each containing 500 mL of chemically defined seed medium.

Seed and Final Fermentation: the shake flasks with cells in exponential growth phase (2.5 - 4.5 OD600 units) are used to inoculate a single 25 L (i.e., working volume) seed fermenter containing the seed medium. The cells upon reaching exponential growth phase (3.0 - 5.0 OD600 units) in the 25 L seed fermenter are transferred to a 1500 L production fermenter with 780 - 820 L
of chemically defined batch medium. During fermentation, the temperature is controlled at 37 2 C, pH at 7.1 0.1, agitation at 150 - 250 rpm and sparging with 0.5 - 1.5 (vvm) of air or oxygen-enriched air to control dissolved oxygen at 25% or above. Antifoam agent (Fermax adjuvant 27) is used as needed to control foaming in the fermenter. When the OD (at 600 nm) of culture reaches 25-35 units, additional chemically defined medium is fed at 0.7 g/kg broth/min initially and then with feed rate adjustment as needed. The induction for expression of the CG53135-05 protein is started when OD at 600 nm reaches 135 - 165 units. After 4 hours post-induction the fermentation is completed. The final fermentation broth volume is approximately 1500 L. The culture is then chilled to 10 - 15 C.

Homogenization: the chilled culture is diluted with cell lysis buffer at the ratio of one part of fermentation broth to two parts of cell lysis buffer (50 mM sodium phosphate, 60 mM EDTA, 7.5 mM
DTT, 4.5 M urea, pH 7.2. Polyethyleneimine (PEI), a flocculating agent is added to the diluted fermentation broth to a final PEI concentration at 0.033% (WN). The cells are lysed at 10 - 15 C
with 3 passages through a high-pressure homogenizer at 750 - 850 bar.

Capture and Recovery: the chilled cell lysate is directly loaded in the upflow direction onto a pre-equilibrated Streamline SP expanded bed cation exchange column. During the loading, the bed expansion factor is maintained between 2.5 - 3.0 times the packed bed column volume. After loading, the column is flushed with additional Streamline SP equilibration buffer (100 mM sodium phosphate, 40 mM EDTA, 10 mM sodium sulfate, 3 M urea, pH 7.0) in the upflow direction. The column is then washed further with SP Streamline wash buffer (100 mM sodium phosphate, 5 mM
EDTA, 25 mM sodium sulfate, 2.22 M dextrose, pH 7.0) in the downflow direction. The protein is eluted from the column with Streamline SP elution buffer (100 mM sodium phosphate, 5 mM EDTA, 200 mM sodium sulfate, 1 M L-arginine, pH 7.0) in the downflow direction.

PPG 650M Chromatography: the SP Streamline eluate is loaded on to a pre-equilibrated PPG 650 M, hydrophobic interaction chromatography column. The column is equilibrated and washed with 100 mM sodium phosphate, 200 mM sodium sulfate, 5 mM EDTA, 1 M
Arginine pH 7Ø
The column is further washed with 100 mM sodium phosphate, 5 mM EDTA, 0.9 M
Arginine, pH 7Ø
The product is eluted with 100 mM sodium phosphate, 5 mM EDTA, 0.2 M Arginine, pH 7Ø

CUNO Filtration: the PPG eluate is passed through an endotoxin binding CUNO

depth filter. The filter is flushed first with water for injection (WFI) and then with 100 mM sodium phosphate, 5 mM EDTA, 0.2 M Arginine, pH 7.0 (PPG eluate buffer). After flushing, the PPG eluate is passed through the filter. Air pressure is used to push the final liquid through the filter and its housing.

Phenyl Sepharose Chromatography: the CUNO filtrate is then loaded on to a pre-equilibrated Phenyl Sepharose hydrophobic interaction chromatography column.
The column is equilibrated and washed with 100 mM sodium phosphate, 50 mM ammonium sulfate, 800 mM
sodium chloride, 0.5 M Arginine, pH 7Ø The product is eluted with 50 mM
sodium phosphate, 0.5 M Arginine, pH 7Ø

Concentration and Diafiltration: a 1% Polysorbate 80 is added to the Phenyl Sepharose eluate so that the final concentration in the drug substance is 0.01 %(w/v).
The eluate is then concentrated in an ultrafiltration system to about 2 - 3 g/L. The retentate is then diafiltered with 7 diafiltration volumes of 50 mM sodium phosphate, 0.5 M Arginine, pH 7.0 (Phenyl Sepharose elution buffer). After diafiltration the retentate is concentrated between 12 - 15 g/L. The retentate is filtered through a 0.22 pm filter and subsequently diluted to 10 g/L.

Bulk Bottling: the retentate from the concentration and diafiltration step is filtered through a 0.22 pm pore size filter into 2 L single use teflon bottles. The bottles are frozen at -70 C.

Drug Product / Vial: the Frozen Drug Substance is used for the manufacture of the Drug Product. The bottles of frozen Drug Substance are thawed at ambient temperature. After the Drug Substance is completely thawed, it is pooled in a sterile container, filtered, filled into vials, partially stoppered, and lyophilized. After completion of the freeze-drying process, the vials are stoppered and capped. The lyophilized Drug Product is stored at 2-8 C.

6.3 EXAMPLE 3: CHARATERIZATION OF REFERENCE STANDARD OF THE DRUG
PRODUCT
A protein reference standard was prepared using a 140L scale manufacturing process that was representative of the bulk drug substance manufacturing process as described in Section 6.2.2 (Example 2). The reference standard was stored as I mL aliquots in 2 mL
cryovials at -80 C t 15 C. The proposed specifications for the reference standard are listed in Table 6.

Table 6: Proposed Specifications of CG53135-05 Reference Standard Property Assay Description of Expected Results Purity = SDS-PAGE (reduced, colloidal => 98% pure by densitometry Coomassie stain) = SDS-PAGE (reduced, silver stain) = At 10 g load, less than 100 ng of impurities are detectable = SEC-HPLC = FIO*

= RP-HPLC = > 90 % main peak Property Assay Description of Expected Results = Host cell protein Western Blot = As found Identity = Western Blot = Major band - 23 kDa = N-Terminal amino acid sequencing = Consistent with predicted primary N-terminal amino acid sequence = LC and MS = HPLC profile shows 1 major peak which is confirmed by MS to be = Total amino acid analysis CG53135-related = Consistent with predicted primary = Peptide mapping amino acid composition = Fragment pattern consistent with predicted primary amino acid sequence Strength = Total protein by A280 = 10 1 mg/mL

Potency = Bioassay (Relative Potency) = 60 - 140 % relative to reference standard Secondary = Far-UV CD spectroscopy = As found Structure Tertiary = UV derivative spectroscopy = As found Structure = Near-UV CD spectroscopy = As found Quartenary . Differential Scanning Calorimetry = As found Structure = Light Scattering (SEC-HPLC) = As found Safety . Residual DNA =< 100 pg/mg = Endotoxin (USP <85> gel clot) = 2 EU/mg = Bioburden = < 1 cfu/mL
Other . pH = 7.0 0.5 = Osmolarity = As found = Sulfyhydryl content = As found = Visual inspection = Clear and colorless solution *FIO: for information only.

6.4 EXAMPLE 4: PURITY ANALYSIS OF THE DRUG PRODUCT
The drug products produced by the manufacturing process as described in Section 6.2 (Example 2) are analyzed for purity by the experiments described in this section.

6.4.1 Purity by SDS-PAGE Analyses Purified protein using the improved manufacturing process as described in Section 6.2.2 ("Process 2") and the purified protein using manufacturing process as described in Section 6.2.1 ("Process 1") were analyzed by loading increasing amounts of protein on a 4-12% gradient Bis-Tris NuPAGE gel and stained with the gel code blue stain to detect trace impurities (FIG. 2A). Purified protein (from both Process 1 and Process 2, respectively) migrated as a single major band under reducing conditions (-23 kDa). No impurities above the LOD (<28 ng) were detected.

Purified protein of Process 1 and Process 2 was also analyzed by loading increasing amounts of protein on a 4-12% gradient Bis-Tris NuPAGE gel and using silver stain to detect trace impurities (FIG. 2B). Purified protein (from both Process 1 and Process 2, respectively) migrated as a single major band (-23 kDa) at all loads under reducing conditions.

6.4.2 Purity by RP-HPLC
Purified drug product was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). Purified protein from Process 1 and Process 2 was loaded onto a Protein C4 column (Vydac, 5 pm, 150 mm X 4.6 mm) using a standard HPLC system in a mobile phase containing water, acetonitrile and trifluoroacetic acid. Purified protein from Process 1 elutes as a major peak at 24.0 min and additional peaks at 24.3 and 24.7 minutes.
These represent isoforms of CG53135-05. CG53135-05 obtained using Process 2 elutes as a major peak with a retention time of 24.0 minutes (FIG. 3). Characterization of these peaks is discussed further below in Section 6.7 (Example 7).

6.4.3 Purity by Size Exclusion-HPLC
Purified protein (from both Process 1 and Process 2, respectively) was analyzed by size exclusion chromatography (SEC-HPLC) with UV detection at 280 nm. Analysis was performed by injecting the protein onto a size exclusion HPLC column (Bio-Sil SEC-250, 0.78 cm X 30 cm, Bio-Rad) using a standard HPLC system with a mobile phase containing 100 mM sodium phosphate, 1 M arginine-HCI, pH 7Ø Purified protein eluted isocratically as a single mono-disperse peak with a retention time of 20.5 minutes (FIG. 4) for Process 1 and 2. This retention time corresponds to an apparent molecular weight of approximately 45 kilodaltons (when compared against a set of calibration standards run under identical conditions), which suggests that FGF-20 exists as a non-covalently linked dimer.

6.4.4 Host Cell Protein Determination via Western Blot The levels of host cell protein impurities in purified drug product were assessed qualitatively by Western blot analysis. The purified CG53135 protein was resolved by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. The membrane was incubated with a primary antibody (rabbit anti-E. coli, Dako Systems) followed by a secondary antibody (goat anti-rabbit Alkaline Phosphatase conjugated, Bio-Rad) and developed using standard techniques. No host cell protein impurities were visible for Process 1 and only one band (-70kDa) is apparent from Process 2 (FIG. 5).

6.4.5 Identity of CG53135 via Western Blot Purified protein (from both Process 1 and Process 2, respectively) was identified by Western blot using rabbit polyclonal anti-CG53135 sera (FIG. 6). Purified CG53135-05 was resolved by loading 10 pg of protein on a 4-12% gradient Bis-Tris NuPAGE gel and electrophoretically transferred to a nitrocellulose membrane. The membrane was incubated with a primary antibody (polyclonal anti-CG53135 sera) followed by a secondary antibody (goat anti-rabbit Alkaline Phosphatase conjugated, Bio-Rad) and then developed using standard techniques.
Purified protein (from both Process 1 and Process 2, respectively), which migrates as a single band of the expected molecular weight (molecular weight of FGF-20) under reducing and nonreducing conditions, is immunoreactive with CG53135-specific antiserum.

6.5 EXAMPLE 5: POTENCY OF CG53135 PRODUCT
The potency was measured by cell growth of NIH 3T3 cells in response to the purified protein from Process 1 and Process 2. Cell growth was measured indirectly using fluorescence by the conversion of resazurin (CeIlTiter Blue Reagent) into resorufin. Using DEV-10 (Process 1) as the reference standard, the Process 2 interim reference standard was found to have comparable potency at 101 %. Several lots manufactured by Process 2 were analyzed. These results are shown in Table 7.

Table 7: Potency of Lots from Process 2 using DEV-1 0 (Process 1) as Reference Standard Lot Number Potency Result (%) The average potency for all of the lots tested is 106.4 t 10.3. This indicates that the potency of lots from Process 2 are equivalent to lot DEV-1 0 made with Process 1. Residual DNA, endotoxin and bioburden in the drug substance can also be tested using qualified assays.

The biological activity of CG53135-05 related species collected from the 4 peaks identified by LC and MS was measured by treatment of serum-starved cultured NIH 3T3 murine embryonic fibroblast cells with various doses of the isolated CG53135-05 related species and measurement of incorporation of bromodeoxyuridine (BrdU) during DNA synthesis. For this assay, cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were grown in 96-well plates to confluence at 37 C in 10% C02/air and then starved in Dulbecco's modified Eagle's medium for 24 - 72 hours. CG53135-05-related species were added and incubated for 18 hours at 37 C in 10% C02/air. BrdU (10 mM final concentration) was added and incubated with the cells for 2 hours at 37 C in 10% C02/air. Incorporation of BrdU was measured by enzyme-linked immunosorbent assay according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN).

Peak 4 was not included in this assay since insufficient material was collected (Peak 4 is less than 3% of the total peak area for CG53135-05). CG53135-05 and material collected from all 3 remaining fractions (i.e., Peak 1, 2, and 3) induced DNA synthesis in NIH 3T3 mouse fibroblasts in a dose-dependent manner (Table 4). The P1200 was defined as the concentration of protein that resulted in incorporation of BrdU at 2 times the background. CG53135-05 and CG53135-05 related species recovered from all 3 measurable peaks demonstrated similar biological activity with a P1200 of 0.7 - 11 ng/mL (Table 8).

Table 8: Biological Activity of CG53135-05 E. coli purified product (DEV10):
Induction of DNA
Synthesis Pi200 (ng/mL) CG53135-05 (DEV 10) Peak 1 Peak 2 Peak 3 1.0 0.7 11 8.6 6.6 EXAMPLE 6: CHARACTERIZATION STUDIES FOR BIOCHEMICAL
COMPARABILITY
Characterization studies for comparison of the primary, secondary, tertiary and quaternary structure as stated below in Table 9 may also be done. The side-by-side results of reference standard (designated DEV10) from the Process 1 with reference standard obtained using Process 2 (interim reference material) can be used to further demonstrate the biochemical properties of the purified CG53135 protein.

Table 9: Biochemical Characterization of CG53135-05 Reference Materials Attributes Process 1 Process 2 SDS-PAGE (silver) SDS-PAGE (silver) Purity HCP Western (HCP Western - release assay) Western RP HPLC - peak identification Identity (Primary N-Terminal sequencing N-Terminal sequencing Structure) Total Amino acid analysis Total Amino acid analysis Peptide mapping Peptide mapping / LC-MS
MALDI-TOF MS
Far UV CD spectroscopy Secondary Structure UV-derivative spectroscopy Tertiary Structure Near UV CD spectroscopy Light Scattering (SEC-HPLC) Quartenary Structure Diff. scanning calorimetry pH pH
Osmolality Osmolality Other Sulfhydryl content Sulfhydryl content 6.7 EXAMPLE 7: CHARACTERIZATION OF PRIMARY STRUCTURE OF THE PURIFIED
PROTEIN

6.7.1 RP-HPLC Assay: Peak Identification Purified drug substance (by both Process 1 and Process 2, respectively) was further analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) with both UV and electrospray mass spectrometric detection. Purified protein from either Process 1 or Process 2 was loaded onto a Protein C4 column (Vydac, 5 pm, 150 mm X 4.6 mm) using a standard HPLC system in a mobile phase containing water, acetonitrile and trifluoroacetic acid. The elution gradient for this method was modified to resolve four distinct chromatographic peaks eluting at 26.6, 27.3, 28.5 and 30.0 min respectively (FIG. 7). These peaks were characterized by electrospray mass spectrometry. As can be observed from the chromatograms, the four equipotent peaks are present in the purified final product from Process 1 and 2. However, the proportion of these peaks (1, 3 and 4) is much lower in the final product purified by Process 2 with the predominant form being Peak 2.
The identities of each peak from the RP-HPLC separation are indicated in Table 10.

Table 10: Identity of peaks from the RP-HPLC separation of CG53135-05 based upon accurate molecular weight determination.

Peak # Retention Molecular Assignment (residue #) ID Number Predicted Time (min) Weight Molecular Observed Weight 1 26.6 21329.2 24-211 CG53135-17 21329.2 1 26.6 22185.1 15-211 CG53135-16 22185.1 1 26.6 22412.4 12-211 CG53135-15 22412.4 2 27.3 23296.5 3-211 CG53135-13 23296.4 3 28.5 23498.9 1-211 CG53135-05 23498.7 4 30.0 23339.3 3-211(carbamylated) CG53135-13 23339.4 (carbamylated) 4 30.0 23539.7 1-211(carbamylated) CG53135-05 23539.7 (carbamylated) All the variants/fragments in the final purified product have high activity in the proliferation assays. Thus these variants/fragments are expected to have same utility as that of FGF-20. For the purpose of convenience, the term "CG53135-05 E. coli purified product" is used hereon to refer to a purified protein product from E. coli expressing a CG53135-05 construct. For example, a CG53135-05 E. coli purified product may contain a mixture of the full length CG53135-05 protein (SEQ ID
NO:2), CG53135-13 (SEQ ID NO:24), CG53135-15 (SEQ ID NO:28), CG53135-16 (SEQ
ID NO:30), and CG53135-17 (SEQ ID NO:32), with the majority of the content being CG53135-13 (SEQ ID
NO:24).

6.7.2 Edman Sequencing and Total Amino Acid Analysis The experimental N-terminal amino acid sequence of the Process 1 reference standard, DEV10, and the Process 2 interim reference standard were determined qualitatively. The reference standards were resolved by SDS-PAGE and electrophoretically transferred to a polyvinylidenefluoride membrane; the Coomassie-stained -23 kDa major band corresponding to each reference standard was excised from the membrane and analyzed by an automated Edman sequencer (Procise, Applied Biosystems, Foster City, CA). A comparison of the two major sequences is shown in Table 11 below. The predominant sequence for each reference standard were identical and corresponded to residues 3-20 in the theoretical N-terminal sequence of CG53135-05.

Table 11: Edman sequencing data for the first 20 amino acids of CG53135-05 for Process 1 and 2.
Theoretical Amino Acid Residue Residue Position Process 1 Process 2 3 Pro Pro 4 Leu Leu Ala Ala 6 Glu Glu 7 Val Val 8 Gly Gly 9 Gly Gly Phe Phe 11 Leu Leu 12 Gly Gly 13 Gly Gly 14 Leu Leu Glu Glu 16 Gly Gly 17 Leu Leu 18 Gly Gly 19 GIn GIn Gln Gln The experimental amino acid composition of the DEV10 reference standard and the PX3536G001-H reference standard were determined in parallel. Quadruplicate samples of each reference standard were hydrolyzed for 16 hours at 115 C in 100 pL of 6 N HCI, 0.2% phenol containing 2 nmol norleucine as an internal standard. Samples were dried in a Speed Vac Concentrator and dissolved in 100 NL sample buffer containing 2 nmol homoserine as an internal standard. The amino acids in each sample were separated on a Beckman Model 7300 amino acid analyzer. The amino acid composition of both reference standards showed no significant differences as shown in Table 8 below. Note that Cys and trp are destroyed during acid hydrolysis of the protein. Asn and gln are converted to asp and glu, respectively, during acid hydrolysis and thus their respective totals are reported as asx and glx. Met and his were both unresolved in this procedure.

Table 12: Quantitive amino acid analysis for final purified protein from Process 1 and Process 2 Amino Acid Mole Percent Residue DEV10 PX3536G001-H
asx 7.1 7.0 thr 4.0 4.0 ser 6.3 6.1 glx 12.2 12.2 pro 6.0 6.0 gly 14.4 14.3 ala 5.8 5.6 val 5.3 5.3 ile 3.5 3.5 leu 13.6 13.6 tyr 4.6 4.6 phe 5.2 5.2 lys 3.7 3.7 arg 8.5 9.1 6.7.3 Tryptic Mapping by RP-HPLC
Purified drug substance from Process 1 and 2 was reduced and alklated with iodoacetic acid and then digested with sequencing grade trypsin. The tryptic peptides were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using both UV
and electrospray mass spectrometric detection. The tryptic digest from either Process 1 or Process 2 was loaded onto an ODS-1 nonporous silica column (Micra, 1.5 pm; 53 x 4.6 mm) using a standard HPLC system in a mobile phase containing water, acetonitrile and trifluoroacetic acid. The eluting peptides were detected by UV at 214 nm (FIG. 8) and by positive-ion electrospray mass spectrometry. The major difference between the two chromatograms for Process 1 and Process 2 is the reduction in peak area of a peak obvious in the Process I trace ( peak at 8.2 min; FIG. 8). This peak corresponds to the T1 peptide, residues 1-40. This observation is expected since the source of this peptide if from the intact CG53135-05, which is in greater abundance in the Process I
material (peak 3, FIG. 7).

6.8 EXAMPLE 8: CHARACTERIZATION OF SECONDARY STRUCTURE: CIRCULAR
DICHOROISM SPECTROSCOPY OF THE PURIFIED PROTEIN
The far UV circular dichroism spectrum of the purified protein (from both Process 1 and Process 2, respectively) is characterized by a broad maximum at 226-227 nm and a sharp minimum at approximately 206 nm. Both features are common in other fibroblast growth factors and suggest a secondary structure dominated by R-sheet and R-turns. The far UV circular dichroism spectra of the DEV10 reference standard and the PX3536G001-H reference standard both display these features and are nearly identical (FIG. 9). The small differences in the spectra are attributable to experimental error.

6.9 EXAMPLE 9: CHARACTERIZATION OF TERTIARY STRUCTURE

6.9.1 Near UV Circular Dichroism Spectroscopy of the Purified Protein The near UV Circular Dichroism (CD) spectrum of a protein reflects the number and orientation of the protein's aromatic amino acids. For proteins having identical numbers of aromatic amino acids any differences in their near UV CD spectra represent differences in the position and orientation of the aromatic amino acids. The position and orientation of the aromatic amino acids are a measure of a protein's tertiary structure. Hence, differences in the near UV CD spectra for proteins represent differences in tertiary structure.

The near UV CD spectra of the DEV10 reference standard and the PX3536G001-H
reference standard are shown in FIG. 10. There are no significant differences between these two spectra and suggest that both reference standards have no significant differences in their tertiary structure.

6.9.2 Second Derivative UV Absorbance Spectroscopy of the Purified Protein The UV absorbance of aromatic amino acids is influenced by the amino acid's microenvironment. Aromatic amino acids embedded within a protein are in a less polar microenvironment than surface exposed residues. This difference in polarity has a profound effect on the UV absorbance of an aromatic amino acid. Different microenvironments can shift the spectra of aromatic amino acids 4-6 nm in extreme cases. Monitoring these changes for individual proteins is done by calculating the second derivative of the protein's UV absorbance spectrum. The second derivative UV absorbance spectrum of a protein contains a number of minima that correspond to the individual aromatic amino acids. The wavelengths of these minima reflect the microenvironment of the amino acid. Therefore, changes in these minima are indicative of conformational (tertiary) changes in the protein.

The second derivative UV absorbance spectrum of the purified protein (from both Process 1 and Process 2, respectively) is characterized by seven minima between 250 and 300 nm. As shown in Table 13 below, and qualitatively in FIG. 11, the wavelengths of all seven minima for both the DEV10 reference standard and the PX3536G001-H reference standard are not significantly different. These data demonstrate that the microenvironment around the individual aromatic amino acids in both standards are highly similar and suggests significant tertiary differences do not exist between these two reference standards.

Table 13: Second derivative UV absorbance spectral data for Process 1 and 2 Sample Result Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 (Phe) (Phe) (Phe) (Phe/T r) (Tyr) (T r/Tr ) (Trp) DEV10 Average 252.70 258.60 264.60 268.90 277.95 285.38 293.50 DEV10 SD 0.00 0.00 0.00 0.00 0.05 0.04 0.07 PX3536G001-H Average 252.70 258.60 264.60 268.90 277.98 285.40 293.53 PX3536G001-H SD 0.00 0.00 0.00 0.00 0.04 0.00 0.08 Results represent the average of 5 replicates for each reference standard.

6.10 EXAMPLE 10: CHARACTERIZATION OF QUARTERNARY STRUCTURE

6.10.1 Differential Scanning Calorimetry of the Purified Protein Differential scanning calorimetric analysis is based upon the detection of changes in the heat content (enthalpy) or the specific heat of a sample with temperature. As thermal energy is supplied to the sample its enthalpy increases and its temperature rises by an amount determined, for a given energy input, by the specific heat of the sample. The specific heat of a protein changes slowly with temperature in a particular physical state, but alters discontinuously at a change of state, e.g. melting or denaturation of the protein.

As can be seen in FIG. 12 the melting curves for the purified protein are similar and the average Tm (melting temperature) is 62.25 C for Process 1 and 62.02 C for Process 2. These differences are within the experimental error of the instrument.

6.11 EXAMPLE 11: MEASURING SULFHYDRYL CONTATENT OF THE PURIFIED
PROTEIN
The sulfhydryl content of the purified protein from Process 1 and Process 2 were measured (Table 14). The purified protein (from Process I and Process 2, respectively) was analyzed for total sulfhydryl content spectrophotometrically using 5, 5'-dithio-bis (2-nitrobenzoic acid), commonly referred to as Eliman's reagent. The results indicate that the total number of measurable sulfhydryis in the final product is the same for Process 1 and Process 2 and are sufficient to account for all the theoretical sulfhydryls in the purified protein.

Table 14: Results from Other Characterization Assays Conducted on the purified protein (Process 1 and Process 2, respectively) Assay Process1 Process 2 Sulfh dr I(SH content 108.0 3.6 101.4 4.9 6.12 EXAMPLE 12: IMPROVED FORMULATIONS COMPRISING CG53135 A new formulation was developed to meet the three requirements for a commercial product:
(1) the minimal storage temperature should be 2-8 C for ease of distribution;
(2) product should be stable at the storage temperature for at least 18 months for a commercial distribution system; and (3) product should be manufactured by commercial scale equipment, and processes should be transferable to various commercial contract manufacturers.

The new formulation consists 10 mg/mL of the protein product produced by the process described in Section 6.2 ("Process 2 protein") in 0.5 M arginine as sulfate salt, 0.05 M sodium phosphate monobasic, and 0.01% (w/v) polysorbate 80. The lyophilized product is projected to be stable for at least 18 months at 2-8 C based on accelerated stability data. In contrast to the new formulation, the previous formulation as described in U.S. Application No.
10/435,087 is not possible to be lyophilized for the following reasons: firstly, the acidic component of the acetate buffer is acetic acid, which sublimes during lyophilization. This loss of acetic acid to lyophilization increases the pH
to > 7.5, which is far from the target pH of 5.3. Secondly, the glycerol has a collapse temperature of <-45 C, which renders this formulation not be able to be lyophilized commercially. Most of the commercial lyophilizers have a shelf temperature ranged from -45 C to -50 C
with temperature variation of 3 C.

Four unexpected properties of CG53135 were discovered and used to develop the new formulation: (1) high concentration of arginine, >0.4 M, increases the solubility to > 30 mg/mL; (2) the use of sulfate salt of arginine increases the solubility by at least 2-6 folds; (3) the optimal concentration of sodium phosphate as a buffering salt is 50 mM, with a solubility of at least 1-2 fold increase in comparison with concentrations at 25, 75, and 100 mM; and (4) adding a surfactant during the diafiltration/ultrafiltration step minimizes the formation of aggregates. In development the lyophilized formulation, each component of the new formulation was evaluated for solubility individually. CG53135-05 was precipitated using the precipitate buffer (50 mM
NaPi, 5 mM EDTA, 1 M L-Arginine HCI, 2.5 M(NH4)2SO4). The precipitate was washed with 25 mM
sodium phosphate buffer at pH 6.5 to remove the residual arginine and ammonium sulfate. The washed precipitate was then re-dissolved in the following respective buffers listed in the tables. The following are examples of data.

Table 15. High concentration of arginine, >0.4 M, increases the solubility to > 30 mg/mL
Concenctration of Solubilit of Process 2 protein in mg/mL
Arginine (M) Batch #1 Batch #2 Batch #3 Batch #4 Batch #5 0.05 0.7 0.6 0.5 ND ND
0.10 1.4 0.6 1.2 ND ND
0.15 2.2 1.6 2.2 ND ND
0.20 3.0 4.7 4.3 ND ND
0.30 ND ND ND 5.8 ND
0.35 ND ND ND 10.1 ND
0.40 ND ND ND 9.8 ND
0.45 ND ND ND 32.3 ND
0.50 ND ND ND 23.8* 37 *: The solubility was lower as there was not sufficient protein in the experiment to be dissolved.
Table 16. The optimal concentration of sodium phosphate as a buffering salt is 50 mM
Concentration of Solubility of Process 2 protein in mg/mL
sodium phosphate monobasic* Batch #A Batch #B Batch #C Batch #D Batch #E
100 mM 3.78 2.8 2.4 2.9 2.47 75 mM 4.06 2.5 2.6 3.0 2.38 50 mM 5.47 4.7 3.3 4.3 4.81 25 mM 4.01 2.4 2.6 2.4 3.59 All formulation contains 0.2 M arginine.

An optimal concentration of the sodium phosphate as a buffering salt was observed (Table 16). The optimal concentration of sodium phosphate is 50 mM with a solubility of at least 1-2 fold increase in comparison with concentrations at 25, 75, and 100 Mm.

Table 17. The use of sulfate salt of arginine increases the solubility by at least 1-3 folds Solubility Increament of Process 2 protein in using Arginine Sulfate vs Arginine Phosphate in mg/mL
Formulation Batch #K Batch # J
50mM sodium phosphate monobasic and 0.15M Arginine at pH 7 4.4 2.3 50mM sodium phosphate monobasic and 0.15M Arginine at pH 7 6.5 5.2 Table 18 shows a need to add a surfactant during the diafiltration/ultrafiltration step to minimize the formation of aggregates. The experiment was conducted by performing the ultrafiltration/diafiltration at 2.5 mg/mL CG53135-05 in 0.2M arginine and 0.05 M sodium phosphate buffer at pH 7Ø After exchanging with 7 volumes of the final buffer (0.5M
arginine and 0.05 M
sodium phosphate buffer at pH 7.0), the diafiltrate is concentrated to -20 mg/mL. The diafiltrate is then diluted with the final buffer to -12.5 mg/mL and lyophilized. Polysorbate 80 is added either before or after the diafiltration to a final concentration of 0.01%.

Table 18. Adding a surfactant during the diafiltration/ultrafiltration step minimizes the formation of aggregates.

Polysorbate added during Process 2 protein Concentration ultrafiitration/diafiltration (m /mL) Turbidity (NTU) Yes 12.5 20.9 No 13.0 4.6 All formulation contains 0.5 M arginine, 0.05 M sodium phosphate monobasic, and 0.01%
polysorbate 80.

The new formulation has the following advantages: (1) a lyophilized product with a storage temperature of 2-8 C; (2) a lyophilized product with a projected shelf-life of at least 18 months when stored at 2-8 C achieve the solubility of > 30 mg/mL; and (3) The lyophilized product has a collapse temperature of -30 C which can be easily lyophilized by the commercial equipment. The interactions between arginine, sulfate, phosphate, and surfactant and CG53135 were unexpected.
6.13. EXAMPLE 13: IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS

This example demonstrated how some of the single nucleotide polymorphisms (SNPs) of FGF-20 were identified. A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. SNPs occurring within a gene may result in an alteration of the amino acid encoded by the gene at the position of the SNP.
Intragenic SNPs may also be silent, when a codon including a SNP encodes the same amino acid as a result of the redundancy of the genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation of the expression pattern. Non-limiting examples include alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, and stability of transcribed message.

SeqCallingTM assemblies produced by the exon linking process were selected and extended using the following criteria: genomic clones having regions with 98% identity to all or part of the initial or extended sequence were identified by BLASTN searches using the relevant sequence to query human genomic databases. The genomic clones that resulted were selected for further analysis because this identity indicates that these clones contain the genomic locus for these SeqCallingTM assemblies. These sequences were analyzed for putative coding regions as well as for similarity to the known DNA and protein sequences. Programs used for these analyses include Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and other relevant programs.

Some additional genomic regions may have also been identified because selected SeqCallingTM assemblies map to those regions. Such SeqCallingTM sequences may have overlapped with regions defined by homology or exon prediction. They may also be included because the location of the fragment was in the vicinity of genomic regions identified by similarity or exon prediction that had been included in the original predicted sequence. The sequence so identified was manually assembled and then may have been extended using one or more additional sequences taken from CuraGen Corporation's human SeqCallingTM database.
SeqCallingTM
fragments suitable for inclusion were identified by the CuraToolsTM program SeqExtend or by identifying SeqCalling fragments mapping to the appropriate regions of the genomic clones analyzed.

The regions defined by the procedures described above were then manually integrated and corrected for apparent inconsistencies that may have arisen, for example, from miscalled bases in the original fragments or from discrepancies between predicted exon junctions, EST locations and regions of sequence similarity, to derive the final sequence disclosed herein.
When necessary, the process to identify and analyze SeqCallingTM assemblies and genomic clones was reiterated to derive the full length sequence (Alderborn et al., Genome Research 10 (8) 1249-1265 (2000)).

Variants are reported individually in Table 19, but any combination of all or select subset of the variants is also encompassed by the present invention.

Table 19. SNPs of CG53135-01 (SEQ ID NOs: I and 2) Nucleotides Amino Acids Variant Position Initial Modified Position Initial Modified 13377871 301 A G 101 IIe Val 13375519 361 A G 121 Met Val 13375518 517 G A 173 Gly Arg 13375516 523 C G 175 Pro Ala 13381791 616 G A 206 Asp Asn 6.14. EXAMPLE 14: STIMULATION OF BROMODEOXYURIDINE INCORPORATION

A vector expressing residues 24-211 of FGF-20 ((d1-23)FGF-20; See Table 1 and SEQ ID
NO:32 (CG53135-17) was prepared. The incorporation of BrdU by NIH 3T3 cells treated with conditioned medium obtained using the vector incorporating this truncated form was compared to the incorporation in response to treatment with conditioned medium using a vector encoding full length FGF-20. This experiment was carried out as following:

293-EBNA cells (Invitrogen) were transfected using Lipofectamine 2000 according to the manufacturer's protocol (Life Technologies, Gaithersburg, MD). Cells were supplemented with 10%
fetal bovine serum (FBS; Life Technologies) 5 hours post-transfection. To generate protein for BrdU
and growth assays, cells were washed and fed with Dulbecco's modified Eagle medium (DMEM;
Life Technologies) 18 hours post-transfection. After 48 hours, the media was discarded and the cell monolayer was incubated with 100 pM suramin (Sigma, St. Louis, MO) in 0.5 ml DMEM for 30 min at 4 C. The suramin-extracted conditioned media was then removed, clarified by centrifugation (5 min; 2000 X g), and subjected to TALON metal affinity chromatography according to the manufacturer's instructions (Clontech, Palo Alto, CA) taking advantage of the carboxy-terminal polyhistidine tag. Retained fusion protein was released by washing the column with imidazole.

FGF-20 protein concentrations were estimated by Western analysis using a standard curve generated with a V5-tagged protein of known concentration. For Western analysis, conditioned media was harvested 48 hours post transfection, and the cell monolayer was then incubated with 0.5 ml DMEM containing 100 pM suramin for 30 min at 4 C. The suramin-containing conditioned media was then harvested.

Recombinant FGF-20 and (d1-23)FGF-20 were tested for their ability to induce DNA
synthesis in a bromodeoxyuridine (BrdU) incorporation assay. NIH 3T3 cells (ATCC number CRL-1658, American Type Culture Collection, Manassas, VA), CCD-1070Sk cells (ATCC
Number CRL-2091) or MG-63 cells (ATCC Number CRL-1427) were cultured in 96-well plates to -100%
confluence, washed with DMEM, and serum-starved in DMEM for 24 hours (NIH 3T3) or 48 hours (CCD-1070Sk and MG-63). Recombinant FGF-20 or (dl-23)FGF-20 was then added to the cells for 18 hours. The BrdU assay was performed according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN) using a 5 hour BrdU incorporation time.

The results are shown in FIG. 13. It indicates that (d1-23)FGF-20 retains high activity at the lowest concentration tested, 10 ng/mL. At this concentration, the activity of full length FGF-20 has fallen considerably, approaching the level of the control. It is estimated that (dl-23)FGF-20 may be at least 5-fold more active than full length FGF-20.

6.15. EXAMPLE 15: CELLULAR PROLIFERATION RESPONSES WITH CG53135 (STUDIES L-117.01 AND L-117.02) Experiments were performed to evaluate the proliferative response of representative cell types to CG53135, e.g., a full-length tagged variant (CG53135-01), a deletion variant (CG53135-02), and a full-length codon-optimized untagged variant (CG53135-05).

Materials and Methods:

Heterologous Protein Expression: CG53135-01 (batch 4A and 6) was used in these experiments. Protein was expressed using Escherichia coli (E. coli), BL21 (Novagen, Madison, WI), transformed with full-length CG53135-01 in a pETMY-hFGF20X/BL21 expression vector. Cells were harvested and disrupted, and then the soluble protein fraction was clarified by filtration and passed through a metal chelation column. The final protein fraction was dialyzed against phosphate buffered saline (PBS) plus 1 M L-arginine. Protein samples were stored at -70 C.

CG53135-02 (batch 1 and 13) was also used in these experiments. Protein was expressed in E. coli, BLR (DE3) (Novagen), transformed with the deletion variant CG53135-02 inserted into a pET24a vector (Novagen). A research cell bank (RCB) was produced and cell paste containing CG53135-02 was produced by fermentation of cells originating from the RCB.
Cell membranes were disrupted by high-pressure homogenization, and lysate was clarified by centrifugation.
CG53135-02 was purified by ion exchange chromatography. The final protein fraction was dialyzed against the formulation buffer (100 mM citrate, 1 mM
ethylenediaminetetraacetic acid (EDTA), and 1 M L-arginine).

CG53135-05, DEV10, which were also used in these experiments, was prepared by Cambrex Biosciences (Hopkinton, MA) according to Process 1 as described in Section 6.18.1, infra.
BrdU Incorporation: proliferative activity was measured by treatment of serum-starved cultured cells with a given agent and measurement of BrdU incorporation during DNA synthesis.
Cells were cultured in respective manufacturer recommended basal growth medium supplemented with 10% fetal bovine serum or 10% calf serum as per manufacturer recommendations. Cells were grown in 96-well plates to confluence at 37 C in 10% C02/air (to subconfluence at 5% CO2 for dedifferentiated chondrocytes and NHOst). Cells were then starved in respective basal growth medium for 24-72 hours. CG53135 protein purified from E. coli or pCEP4/Sec or pCEP4/Sec-FGF
20X enriched conditioned medium was added (10 pL/1 00 pL of culture) for 18 hours. BrdU (10 pM
final concentration) was then added and incubated with the cells for 5 hours.
BrdU incorporation was assayed according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN).

Growth Assay: growth activity was obtained by measuring cell number following treatment of cultured cells with a given agent for a specified period of time. In general, cells grown to -20%
confluency in 6-well dishes were treated with basal medium supplemented with CG53135 or control, incubated for several days, trypsinized and counted using a Coulter Z1 Particle Counter.

Results:

Proliferation in Mesenchymal Cells: to determine if recombinant CG53135 could stimulate DNA synthesis in fibroblasts, a BrdU incorporation assay was performed using CG53135-01 treated NIH 3T3 murine embryonic lung fibroblasts. Recombinant CG53135-01 induced DNA
synthesis in NIH 3T3 mouse fibroblasts in a dose-dependent manner (FIG. 14(A)). DNA
synthesis was generally induced at a half maximal concentration of -10 ng/mL. In contrast, treatment with vehicle control purified from cells did not induce any DNA synthesis.

CG53135-01 also induced DNA synthesis in other cells of mesenchymal origin, including CCD-1070Sk normal human foreskin fibroblasts, MG-63 osteosarcoma cell line, and rabbit synoviocyte cell line, HIG-82. In contrast, CG53135-01 did not induce any significant increase in DNA synthesis in primary human osteoblasts (NHOst), human pulmonary artery smooth muscle cells, human coronary artery smooth muscle cells, human aorta smooth muscle cells (HSMC), or in mouse skeletal muscle cells.

To determine if recombinant CG53135-01 sustained cell growth, NIH 3T3 cells were cultured with I lag CG53135-01 or control for 48 hours and then counted (FIG.
14(B)). CG531 35 induced an approximately 2-fold increase in cell number relative to control in this assay. These results show that CG53135 acts as a growth factor.

Proliferation of Epithelial Cells: to determine if recombinant CG53135 can stimulate DNA
synthesis and sustain cell growth in epithelial cells, a BrdU incorporation assay was performed in representative epithelial cell lines treated with CG53135. Cell counts following protein treatment were also determined for some cell lines.

CG53135 was found to induce DNA synthesis in the 786-0 human renal carcinoma cell line in a dose-dependent manner (FIG. 14(C)). In addition, CG53135-01 induced DNA
synthesis in other cells of epithelial origin, including CCD 1106 KERTr human keratinocytes, Balb MK mouse keratinocytes, and breast epithelial cell line, B5589.

Proliferation of Hematopoietic Cells: no stimulatory effect on DNA synthesis was observed upon treatment of TF-1, an erythroblastic leukemia cell line with CG53135-01. These data suggest that CG53135-01 does not induce proliferation in cells of erythroid origin. In addition, Jurkat, an acute T-lymphoblastic leukemia cell line, did not show any response when treated with CG53135-01, whereas a robust stimulation of BrdU incorporation was observed with serum treatment.

Effects of CG53135 on Endothelial Cells: protein therapeutic agents may inhibit or promote angiogenesis, the process through which endothelial cells differentiate into capillaries.
Because CG53135 belongs to the fibroblast growth factor family, some members of which have angiogenic properties, the antiangiogenic or pro-angiogenic effects of CG53135 on endothelial cell lines were evaluated. The following cell lines were chosen because they are cell types used in understanding angiogenesis in cancer: HUVEC (human umbilical vein endothelial cells), BAEC

(bovine aortic endothelial cells), HMVEC-d (human endothelial, dermal capillary). These endothelial cell types undergo morphogenic differentiation and are representative of large vessel (HUVEC, BAEC) as well as capillary endothelial cells (HMVEC-d).

CG53135-01 treatment did not alter cell survival or have stimulatory effects on BrdU
incorporation in human umbilical vein endothelial cells, human dermal microvascular endothelial cells or bovine aortic endothelial cells. Furthermore, CG53135-01 treatment did not inhibit tube formation, an important event in formation of new blood vessels, in HUVECS.
This result suggests that CG53135 does not have anti-angiogenic properties. Finally, CG53135-01 had no effect on VEGF induced cell migration in HUVECs, suggesting that it does no play a role in metastasis.

The above described experiments were also performed using CG53135-02 and 05 protein products, and the results are summarized in the Conclusion section below.

Conclusions Recombinant CG53135-01 (which encode the same protein as CG53135-05) induces a proliferative response in mesenchymal and epithelial cells in vitro (i.e., NIH
3T3 mouse fibroblasts, CCD-1070 normal human skin fibroblasts, CCD-1106 human keratinocytes, 786-0 human renal carcinoma cells, MG-63 human osteosarcoma cells and human breast epithelial cells), but not in human smooth muscle, erythroid, or endothelial cells. Like CG53135-01 and CG53135-05, CG53135-02 also induces proliferation of mesenchymal and epithelial cells. In addition, CG53135-02 induces proliferation of endothelial cells.

6.16. EXAMPLE 16: ACTIVITY OF CG53135 IN HAMSTER MODEL OF
CHEMOTHERAPY-INDUCED ORAL MUCOSITIS (N-212 STUDY) CG53135 was evaluated for the treatment of chemotherapy-induced oral mucositis in male Golden Syrian hamsters (protein concentrations in this Example were measured by Bradford assay).
Materials and Methods CG53135-05 used in this study (batch 29-NB849:76) was expressed and purified as described in Section 6.5, with the exception that the final protein fraction was dialyzed against formulation buffer containing 30 mM sodium citrate, 2 mM EDTA, 200 mM
sorbitol, 50 mM KCI, 20%
glycerol (pH 6.1).

Male golden Syrian hamsters (Charles River Laboratories) age 5 to 6 weeks and with similar body weight in all groups at study commencement were used in this study. Sixty male hamsters were randomized into 6 groups of 10 animals each prior to irradiation. The treatment groups are outlined in Table 20.

Table 20. Treatment Groups Group No. Treatment (0.1 mL, IP) Dosing Schedule I Vehicle (Disease control) Day 1 to Day 18 2 CG53135-05 E. coli purified product, 12 Day 1 to Day 18 m /k /da 3 CG53135-05 E. coli purified product, 12 Day 6 to Day 14 m/k/da 4 CG53135-05 E. coli purified product, 12 Day I to Day 9 m /k /da 5 CG53135-05 E. coli purified product, 12 Day 1 to Day 6 m /k /da 6 CG53135-05 E. coli purified product, 12 Day 1 to Day 2 m/k/da Mucositis was induced using 5-fluorouracil, delivered as single bolus (60 mg/kg, IP) on Days -4 and -2. A single submucosatoxic dose of radiation (40 Gy/dose) was locally administered to all animals on Day 0. Animals were treated once daily with 0.1 mL vehicle or 12 mg/kg CG53135-05 IP
following mucosa toxic insult according to the schedule shown in Table 11.
Mucositis was scored visually as described in Section 6.5 (Table 9) on alternate days beginning on Day 6 and every second day until the conclusion of the experiment on Day 30 (i.e., Days 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30). Each hamster was weighed daily for the period of the study (i.e., Day 0 to Day 30). Weight and survival were monitored as indices for severity of mucositis or possible toxicity resulting from treatment.

The effect of each treatment on mucositis compared with the control group was assessed according to the parameters listed in Table 21. Statistical differences between treatment groups were determined using the Student's t-test, Mann-Whitney U test, and chi-square analysis, with a critical value of 0.05.

Table 21. Parameters for evaluation of Activity Parameter Description The difference in the number of days On each evaluation day, the number of hamsters in each group have severe animals with a blinded mucositis score of mucositis (score _3). _ 3 in each drug treatment group was compared to the vehicle control group.
Differences were analyzed on a cumulative basis. Treatment success was considered a statistically significant lower number of hamsters with this score in a drug treatment group, versus the vehicle control value, as determined by chi-square analysis.

The rank sum differences in daily mucositis For each evaluation day the scores of the scores. vehicle control group was compared to those of the treated group using the non-parametric rank sum analysis. Treatment success was considered as a statistically significant lowering of scores in the treated group on 2 or more days from Day 6 to Day 30.

Results There were no statistically significant differences in weight or survival over time between the vehicle control group (Group 1) and CG53135-05 E. coli purified product treatment groups (Groups 2-6).

In this model of mucositis primarily induced by chemotherapy, dosing schedule was important in the treatment of oral mucositis. Administration of CG53135-05 E.
coli purified product (12 mg/kg/day) from Day 6 to Day 14 or Day 1 to Day 9 did not result in significant improvement in the course or severity of mucositis (FIG. 15). Administration of CG53135-05 E.
coli purified product (12 mg/kg/day) from Day 1 to Day 18 or Day 1 to Day 6 resulted in significant improvement of the duration of severe mucositis (Chi-square analysis). However, these treatments did not result in significant improvement of daily mucositis scores (rank sum analysis).
Treatment with 12 mg/kg/day CG53135-05 E. coli purified product (Day 1 to Day 2) had a significant effect on both the course and severity of mucositis in this study (FIG. 15). These results suggest that a short-course of treatment with a CG53135-05 E. coll purified product immediately after a combined chemotherapy and radiation regimen improves the outcome of the disease in this model of mucositis.

In another experiment, treatment of hamsters with 12 mg/kg/day CG53135-05 E.
coli purified product starting after radiation (Day 1 to Day 18) resulted in a significant reduction of ulceration (p < 0.001) combined with 7 days of significant reduction in mucositis scores, as determined by rank sum analysis (N-198 study). This suggests that the administration of a CG53135-05 E. coli purified product results in a significantly beneficial treatment of radiation-induced oral mucositis when administered after mucosa toxic insult.

In yet another experiment, administration of 12 mg/kg/day of CG53135-05 E.
coli purified product (formulated in 40 mM sodium acetate, 0.2 M L-arginine, and 3%
glycerol) on Days I to 2 significantly reduced the severity of mucositis (N-237 study). These results confirm the findings presented above.

Conclusions The activity of CG53135 was evaluated in a model of mucositis induced in hamsters treated with 60 mg/kg 5-flourouracil on Days -4 and -2, followed by a single sub-mucosatoxic dose of radiation (- 30 Gy) on Day 0. Clinically relevant oral mucositis (mucositis score of _3) developed -Day 15. Intraperitoneal administration of CG53135 for 2, 6, or 18 days significantly reduced severity of mucositis.

6.17. EXAMPLE 17: EFFECT OF CG53135-05 ADMINISTRATION ON HAMSTER
EPITHELIAL PROLIFERATION IN VIVO (N-225 STUDY) The experiment described herein evaluated in vivo incorporation of BrdU into the gastrointestinal epithelium and bone marrow after a single dose of a CG53135-05 E. coli purified product (protein concentrations in this example were measured by Bradford assay).

Materials and Methods Male Golden Syrian hamsters (Charles River Laboratories or Harlan Sprague Dawley), aged to 6 weeks, with a mean body weight of 82 g at study commencement were used.
Twenty-five male hamsters were randomized into 5 groups of 5 animals each as outlined in Table 22.

Table 22. Treatment Groups Group No. of Euthanasia/ Volume (mL);
No. Animals Treatment Treatment Necropsy 1 5 males BrdU 50 mg/kg, IP, (0 hrs) 2 hrs Adjust by body weight 2 5 males 12 mg/kg CG53135-05 E. coli 2 hrs Adjust by body purified product, IP (0 hrs) + weight BrdU 50 mg/kg, IP, (0 hrs) 3 5 males 12 mg/kg CG53135-05 E. coli 4 hrs Adjust by body purified product, IP (0 hrs) + weight BrdU 50 mg/kg, IP, 2 hrs) 4 5 males 12 mg/kg CG53135-05 E. coli 8 hrs Adjust by body purified product, IP (0 hrs) + weight BrdU 50 mg/kg, IP, 6 hrs) 5 5 males 12 mg/kg CG53135-05 E. coli 24 hrs Adjust by body purified product, IP (0 hrs) + weight BrdU 50 mg/kg, IP, (22 hrs) A single dose of a CG53135-05 E. coli purified product at 12 mg/kg IP was administered and hamsters were sacrificed at 2, 4, 8 and 24 hours post-administration.

BrdU Administration and /mmunohistochemistrY: all animals received BrdU 50 mg/kg IP two hours before sacrifice, allowing for uptake of the reagent into proliferating tissues. At euthanasia, the following tissues were harvested: cheek pouch mucosa, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum and sternum. All tissue samples were fixed in 10% neutral buffered formalin for 24 hours and then transferred to 70% ethanol. Samples were trimmed, paraffin embedded, sectioned and mounted. Epithelial tissues were stained for incorporation of BrdU by immunohistochemistry using Oncogene Research products BrdU
Immunohistochemistry kit Catalog # HCS24 in accordance with the manufacturer's instructions.

Results The effect of CG53135-05 E. coli purified product on the incorporation of BrdU
into all tissues was essentially the same: a relatively small increase in the number of BrdU labeled nuclei was observed 2 hours after the administration of CG53135-05 E. coli purified product. This was followed by a decrease in the number of labeled nuclei at 4 hours after the administration of CG53135-05 E. coli purified product. All tissues showed a dramatic increase in BrdU labeling at 8 hours post administration. At 24 hours, all tissues except rectum showed a decrease in the number of labeled nuclei compared with the untreated controls, while the rectal tissue showed a slight increase over the controls. Since no labeled cells were seen in the rectal tissue samples from the untreated animals, the observation of 2 labeled cells in the 24 hour time point has to be regarded as observational error, or data scatter, since there must be a low level of cell replication in the tissue.
Conclusions The in vivo mechanistic activity of CG53135 was evaluated using bromodexoyuridine labeling in vivo to evaluate the effect of a single bolus dose (12 mg/kg) of CG53135-05 E. coli purified product on mucosal tissue over a 24-hour period. CG53135-05 E. coli purified product stimulated the division of the epithelial cells of the cheek pouch, jejunum and rectum as well as the hematopoietic cells of the bone marrow. Peak increases in BrdU incorporation in these tissues were seen at 8 hours after the administration of CG53135-05. All tissues showed the same time response to the administration of CG53135-05 E. coli purified product.

6.18. EXAMPLE 18: MODULATION OF INTESTINAL CRYPT CELL PROLIFERATION
AND APOPTOSIS BY CG53135-05 ADMINISTRATION TO MICE (N-342) This study evaluated the effect of CG53135 on small intestinal crypt cell turnover in order to discriminate stem cell versus daughter cell effects, and to draw insights regarding the mode of action of CG53135 in syndromes associated with gastrointestinal stem cell damage (e.g., mucositis).
Furthermore, the effect of CG53135 on stem cell radiosensitivity was also assessed. Protein concentrations in this example were measured by Bradford assay.

A"crypt" is a hierarchical structure with the stem cells towards the crypt base. As cells become more mature, they move progressively from the bottom of the crypt towards the top of the crypt. Therefore, changes that may be affecting stem cells versus their transit amplifying daughter cells can be detected by looking at changes in event frequency at each cell position. The cell positions are marked in FIG. 16. Thus, the effects of CG53135 on the crypt microarchitecture were analyzed in the context of crypt cellularity.

Experimental Design Animals were sacrificed at various times after a single 12 mg/kg (I P) dose of a CG53135-05 E. coli purified product. Just prior to sacrifice the mice were labeled with a single injection of bromodeoxyuridine to label S-phase cells and determine the effect of the drug on crypt cell proliferation / apoptosis. Mice were weighed and then dosed with a CG53135-05 E. coli purified product (12 mg/kg, single injection, ip). Groups of 6 animals were sacrificed 0, 3, 6, 9, 12, 24, 48 hours post injection with a CG53135-05 E. coli purified product. All received a single injection of bromodeoxyuridine 40 minutes prior to sacrifice (see Table 23).

An additional two groups of 6 mice were used to assess the effects of CG53135-05 E. coli purified product on stem cell radiosensitivity (groups 8 and 9, see Table 23).
One group was treated with a CG53135-05 E. coli purified product (12 mg/kg, single injection, ip) and another group was injected with a placebo control. Twenty-four hours post injection, animals were irradiated with 1 Gy X-ray (specifically to induce stem cell apoptosis) followed by routine in vivo BrdU labeling. Animals were sacrificed 4.5 hours later (at time of peak apoptosis).

Table 23. Study Design Group Number of Treatment Treatment Number Animals Schedule*
1 6 males CG53135-05 E. coli Injected and euthanize 3 hr later purified product, 40mg/kg BrdU 40 min prior to sacrifice 12 m/k ,IP
2 6 males CG53135-05 E. coli Injected and euthanize 6 hr later purified product, 40mg/kg BrdU 40 min prior to sacrifice mg /k ,IP
3 6 males CG53135-05 E. coli Injected and euthanize 9 hr later purified product, 40mg/kg BrdU 40 min prior to sacrifice 12 m/k ,IP
4 6 males CG53135-05 E. coli Injected and euthanize 12 hr later purified product, 40mg/kg BrdU 40 min prior to sacrifice 12m /k ,IP
6 males CG53135-05 E. coli Injected and euthanize 24 hr later purified product, 40mg/kg BrdU 40 min prior to sacrifice 12 m/k ,IP
6 6 males CG53135-05 E. coli Injected and euthanize 48 hr later purified product, 40mg/kg BrdU 40 min prior to sacrifice 12m /k ,IP
7 6 males Untreated 40m /k BrdU 40 min prior to sacrifice 8 6 males CG53135-05 E. coli Dose 24 hr prior to irradiation purified product, Euthanize 4.5 hr post irradiation 12 mg/kg, IP 40mg/kg BrdU 40min prior to sacrifice 1 Gy X ray 9 6 males PBS, IP Dose 24 hr prior to irradiation I Gy X-ray Euthanize 4.5 hr post irradiation 40mg/kg BrdU 40 min prior to sacrifice Intestinal Crypt Cell Proliferation and Apoptosis Modulation: Procedure All S-phase dividing cells incorporate the injected bromodeoxyuridine (BrdU) and hence are marked as cycling cells. Animals that were irradiated were placed, unanaesthetised, in a perspex jig and subjected to whole body radiation of 1 Gy X-ray at a dose rate of 0.7Gy/min. This low level of radiation induced apoptosis in the small intestinal stem cell population, but not in the more mature cells.

The small intestine was removed, fixed in Carnoy's fixative, and processed for histological analysis (paraffin embedded). One set of 3 mm sections were immunolabeled for BrdU and one set of sections were stained with H&E. Longitudinal sections of small intestinal crypts were analyzed for the presence of either BrdU or apoptotic/mitotic nuclei. Fifty half crypts were scored per animal.

Groups 1-7 (Group A in the results) were tested to determine the effect of CG53135-05 E.
coli purified product over a 48 hour period. Groups 8-9 (Group B in the results) were tested to determine whether CG53135-05 E. coli purified product changes the number of apoptotic cells generated after low dose irradiation, i.e., whether CG53135-05 E. coli purified product influences the radiosensitive stem cell population.

The results generated show a frequency distribution for the crypts in each group of animals that were further analyzed for statistical differences. Tissue samples were harvested at 3, 6, 9, 12, 24, and 48 hours after treatment with CG53135-05 E. coli purified product.
Apoptosis, mitotic index, and proliferation were the end points for this study.

Results:
Group A.

In groups 1-7 (Table 23), CG53135-05 E. coli purified product had no significant effect on spontaneous apoptosis. Similar results were obtained with the mitotic index (Table 24). However, results of BrdU uptake as in Table 24, revealed the following:

a) At 3 hour, there was extension/increase of proliferative region (positions 12-22).
b) By 9 hours, large proliferative effects were noted in many positions.

c) By 12 hours, only positions 4-8 showed increase in uptake (stem cells).
d) By 24 hours, there was a significant inhibition of proliferation.

e) By 48 hours, the uptake was comparable to control levels.

Table 24. Summary of significant cell positions in the crypt after assessment of apoptosis, mitosis, and proliferation Sample time Significant Cell Positions (hours) After treatment BrdU labeling Index A optotic Index Mitotic Index 3 12 to 22 None None 6 None None None 9 5 to 9& 11 to 20 to 21 None None 12 4 to 8 None None 24 4 to 8 None None 48 None None None The comparisons shown in Table 24 are between treated groups versus the untreated group. The cell positions shown are the ones that are significantly different from the untreated control (P<0.05).

Group B:
In Groups 8 and 9 (Table 23), stem cell radiosensitivity was assessed. As shown in Table 23, CG53135-05 E. coli purified product or PBS was administered one day before dosing with 1 Gy radiation. Tissues were harvested 4.5 hours after radiation dosing. There was no significant effect on both radiation-induced apoptosis and the mitotic index. However, increased uptake in positions 4-8 by 12 hours and significant inhibition of proliferation were seen in mice pretreated with CG53135-05 E. coli purified product and irradiated, consistent with the Group A results (Table 24).
6.19. EXAMPLE 19: EFFECT OF CG53135-05 PROPHYLACTIC ADMINISTRATION ON
MICE INTESTINAL CRYPT SURVIVAL AFTER RADIATION INJURY (N-343) The purpose of this study was to evaluate the efficacy of CG53135 against radiation-induced crypt cell mortality in vivo using the ClonoquantT"" assay. Protein concentrations in this example were measured by Bradford assay.

Mice were weighed and then dosed with a CG53135-05 E. coli purified product (12 mg/kg) or placebo. A single injection was given, intraperitoneally (ip), 24 hours prior to irradiation. Each group of 6 animals was irradiated as per table below. For each radiation dose, the response of a drug treated group and a placebo treated group was compared.

The small intestine was removed, fixed in Carnoy's fixative, and processed for histological analysis (paraffin embedded). H&E sections were prepared following conventional protocols. For each animal, ten intestinal circumferences were analyzed, the number of surviving crypts per circumference was scored, and the average per group was determined. Only crypts containing 10 or more strongly H&E stained cells (excluding Paneth cells) and only intact circumferences, not containing Peyers patches, were scored.

The average crypt width (measured at its widest point) was also measured in order to correct for scoring errors due to crypt size difference. The correction was applied as follows:

Corrected number of crypts per circumference = Mean number of surviving crypts per circumference in treatment group X (Mean crypt width in untreated control /
Mean crypt width in treated animal).

Table 25: Study design Group Number of Induction Treatment Treatment Number Animals Schedule*
1 6 males 10 Gy PBS Day -1 Da 0 2 6 males 11 Gy, PBS Day -1 Day 0 3 6 males 12 Gy, PBS Day -1 Day 0 4 6 males 13 Gy, PBS Day -1 Da 0 6 males 14 Gy, PBS Day -1 Da 0 6 6 males 10 Gy CG53135-05 E. Day -1 Day 0 coli purified product, 12 m/k ,IP
7 6 males 11 Gy, CG53135-05 E. Day -1 Day 0 coli purified product, 12 m /k , I P
8 6 males 12 Gy, CG53135-05 E. Day -1 Day 0 coli purified product, 12m /k ,IP
9 6 males 13 Gy, CG53135-05 E. Day -1 Day 0 coli purified product, 12m /k ,IP
6 males 14 Gy, CG53135-05 E. Day -1 Day 0 coli purified product, 12m /k ,IP
11 6 males Untreated Results:

The crypt survival following prophylactic CG53135-05 E. coli purified product administration showed inverse correlation to the irradiation dose, the lesser the irradiation dose, the higher was the crypt survival (FIGs. 17(A) and (B)). Prophylactic administration of CG53135-05 E. coli purified product significantly increased the number of crypts (P<0.001). Table 26 shows the protection factor achieved for the radiation doses following prophylactic administration of the protein (CG53135-05 E. coli purified product). Protection factor (Table 26) represents the ratio between treated and untreated cells. On average, 1.55 times as many cells survived irradiation dose of 12 Gy, when animals were administered with CG53135-05 E. coli purified product prior to the radiation insult.

Table 26:
Radiation dose (Gy) Protection Factor 1.29 11 1.21 12 1.55 13 1.71 14 1.73 6.20. EXAMPLE 20: EFFECT OF CG53135 ADMINISTRATION ON CHEMOTHERAPY-RADIATION MODEL OF ORAL MUCOSITIS (N-346 STUDY) Material and Methods Escherichia coli BLR (DE3) cells (Novagen, Madison, WI) were transformed with full-length, codon-optimized CG53135-05 using pET24a vector (Novagen), and a manufacturing master cell bank (MMCB) of these cells was produced. Cell paste containing CG53135-05 produced by fermentation of cells originating from the MMCB was lysed with high-pressure homogenization in lysis buffer and clarified by centrifugation. CG53135-05 was purified from clarified cell lysate by 2 cycles of ion exchange chromatography and ammonium sulfate precipitation. The final protein fraction was dialyzed against the formulation buffer (30 mM citrate, pH 6.0, 2 mM
ethylenediaminetetraacetic acid (EDTA), 200 mM sorbitol, 50 mM KCI, 20%
glycerol). Vehicle contains 30mM sodium citrate, pH 6.1, 2mM EDTA, 200mM sorbitol, 50mM KCI, 20%
glycerol.
Protein concentrations in this example were measured by Bradford assay.

Golden Syrian hamsters (Charles River Laboratories or Harlan), of age 5 to 6 weeks, and with an average body weight of 84 g at study commencement, were used in this study. Animals were individually numbered using an ear punch and housed in small groups of up to 7 animals per cage. Animals were acclimated prior to study commencement. During this period, the animals were observed daily in order to reject animals in poor condition.

Sixty (60) hamsters were randomized into six groups of ten animals each, prior to irradiation. Each group was assigned a different treatment as listed in Table 27. Animals were dosed with 60 mg/kg 5-FU on days -4 and -2 and were acutely irradiated on the left buccal mucosa on day 0. Animals were treated once daily with CG53135-05 E. coli purified product IP 6, 12, 24 or 48 mg/kg/day, on day I only or 12 mg/kg/day on days 1 and 2, following acute radiation. Mucositis was evaluated on alternate days beginning on day 6 and continued until the conclusion of the experiment on day 28 (i.e., days 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 & 28).

Table 27. Treatment Groups Group Number of Treatment Treatment Volume Number Animals Schedule*

1 10 males Untreated Control Day 1 2 10 males 6 mg/kg CG53135-05 E. coli Day I
purified product, ip 3 10 males 12 mg/kg CG53135-05 E. co/i Day 1 purified product ip 4 10 males 24 mg/kg CG53135-05 E. coli Day 1 purified product, ip 10 males 48 mg/kg CG53135-05 E. coli Day 1 purified product ip 6 10 males 12 mg/kg CG53135-05 E. co/i Day 1& 2 purified product, ip *Mucositis was evaluated on alternate days beginning on day 6 and every second day until the conclusion of the experiment on day 28 (i.e., days 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, & 28).
Chemotherapy/Radiation Model of Oral Mucositis: the 5-FU/acute irradiation model for oral mucositis in hamsters is an experimental model designed to extend the clinical observations made with the acute radiation model for mucositis (Oral Surg Oral Med Oral Pathol 69(4):437 (1990)). The earlier acute radiation model has proven to be an accurate, efficient and cost-effective technique to provide a preliminary evaluation of anti-mucositis compounds including growth factors and cytokines (see e.g., Oral Oncol 36(4):373-381 (2000), Cytokine 9(8):605-612 (1997); Oral Oncol 33(1):47-54 (1997)).

Mucositis was induced using 5-fluorouracil, delivered as intraperitoneal (IP) doses (60 mg/kg) on days -4 and -2. A single dose of radiation (30 Gy/dose) was administered to all animals on day 0. Radiation was generated with a 160 kilovolt potential (18.75-ma) source at a focal distance of 21 cm, hardened with a 3.0 mm Al filtration system. Irradiation targeted the left buccal pouch mucosa at a rate of 1.32 Gy/minute. Prior to irradiation, animals were anesthetized with an IP
injection of ketamine (160mg/kg) and xylazine (8mg/kg). The left buccal pouch was everted, fixed and isolated using a lead shield. This resulted in ulcerative oral mucositis that peaked around day 14.

Evaluation of Mucositis: for the evaluation of mucositis, the animals were anesthetized with an inhalation anesthetic, and the left pouch was everted. Mucositis was scored visually by comparison to a validated photographic scale, ranging from 0 for normal, to 5 for severe ulceration.
The scale is described in Table 28.

Table 28. Description of Mucositis Score Values Score Description:
0 Pouch completely healthy. No erythema or vasodilation.
1 Light to severe erythema and vasodilation. No erosion of mucosa.
2 Severe erythema and vasodilation. Erosion of superficial aspects of mucosa leaving denuded areas. Decreased stippling of mucosa.
3 Formation of off-white ulcers in one or more places. Ulcers may have a yellow/gray due to pseudomembrane. Cumulative size of ulcers should equal about'/ of the pouch. Severe erythema and vasodilation.

4 Cumulative size of ulcers should equal about'/ of the pouch. Loss of pliability.
Severe erythema and vasodilation.
Virtually all of pouch is ulcerated. Loss of pliability (pouch can only partially be extracted from mouth) A score of 1-2 is considered to represent a mild stage of the disease, whereas a score of 3-5 is considered to indicate moderate to severe mucositis. Following clinical scoring, a photograph was taken of each animal's mucosa using a standardized technique. At the conclusion of the experiment, all film was developed and the photographs randomly numbered for blinded scoring.
Two independent, trained evaluators graded the photographs in blinded fashion using the above-described scale. For each photograph, the final blinded score was the average of the score assigned by the two independent evaluators. The scores from the blinded photographic evaluation were statistically analyzed.

Weights and Survival: each hamster was weighed daily for the period of the study (i.e., day -4 to day 28). Weight and survival was monitored and recorded in order to assess possible differences amongst treatment groups as an indication for mucositis severity and/or possible toxicity resulting from the treatments. If appropriate, survival was analyzed using a Kaplan Meier log-rank analysis. Differences in weight gain were assessed using a One-Way ANOVA
analysis of the area under the curve (AUC) values for the percentage weight gain for individual animals, with a critical value of 0.05.

Evaluation of Activity: the effect of each treatment on mucositis compared to the control group was assessed using a Chi-squared (X2) analysis of the number of animal days with a score of three or higher, and by using the Mann-Whitney Rank Sum test to compare the blinded mucositis scores for each group on each day the evaluations were performed. In each case, treatment groups were compared to the control group, with a critical value of 0.05. For the Mann-Whitney Rank Sum test, two days of statistically significant improvement are generally regarded as the minimum improvement necessary for a positive result.

Results Mucositis: the mean daily mucositis scores were calculated for each group and are shown in Figure 18. The peak of mucositis in the control group was on day 14 when the mean score for this group reached 3.2. All of the groups treated with CG53135-05 E. coli purified product had their peak scores on day 16, which ranged from a high of 3.0 in the groups treated with CG53135-05 E.
co/i purified product at 24 mg/kg or 48 mg/kg on day I to a low of 2.63 in the group treated with CG53135-05 E. coli purified product at 12 mg/kg on day 1. To evaluate the mucositis scores, an analysis of the number of days with a score of 3 or higher was performed, using the Chi-squared test. The results of this analysis are shown in Table 29 and Figure 18.
Further, Figure 19 depicts the duration of severe mucositis in animals with a mucositis score of >3 as calculated by the chi-square analysis. Both groups treated with CG53135-05 E. coli purified product at 12 mg/kg showed a significant reduction in the number of days with a score of 3 or higher, with the group treated on day 1 only having slightly more significance (P=0.003) than the group treated on days 1 and 2 (P=0.01 8). The group treated with CG53135-05 E. coli purified product at 6 mg/kg on day 1 showed some improvement, but failed to reach significance (P=0.092). The groups treated with CG53135-05 E. coli purified product at 24 mg/kg and 48 mg/kg were essentially the same as controls in this test.

Table 29. Chi Squared analysis of number of days animals had a mucositis score of 3 or higher Group Days>=3 Days<3 Total Days % Days Chi Sq. P
>=3 vs control Value Untreated control 94 140 234 40.2 - -CG53135-05 E. coli 70 148 218 32.1 2.8330 0.092 purified product 6mg/kg IPDay1 CG53135-05 E. coli 52 148 200 26.0 9.0760 0.003 purified product 12mg/kg IP Day 1 CG53135-05 E. coli 80 136 216 37.0 0.3420 0.558 purified product 24mg/kg IP Day 1 CG53135-05 E. coli 94 126 220 42.7 0.2090 0.647 purified product 48mg/kg IPDay1 CG53135-05 E. coli 70 168 238 29.4 5.5590 0.018 purified product 12mg/kg IP Day 1 and 2 To examine the levels of clinically significant mucositis, as defined by presentation with open ulcers (score >3), the total number of days in which an animal exhibited an elevated score was summed and expressed as a percentage of the total number of days scored for each group.
Statistical significance of observed differences was calculated using chi-square analysis.

Further analysis of the significance of the differences in the mucositis scores was performed by using the Mann-Whitney Rank-Sum test to compare the test groups with the control group on each day of evaluation. The results of this analysis are shown in Table 30 which indicates that the group treated with CG53135-05 E. coli purified product at 6 mg/kg on day 1 only showed significant improvement relative to controls on days 14 (P=0.010) and 26 (P=0.031). The group treated with CG53135-05 E. coli purified product at 12 mg/kg on day 1 only showed significant improvement relative to controls on days 14 (P=0.01 1), 16 (P=0.031), 18 (P=0.005), and 20 (P=0.037). The group treated with CG53135-05 E. coli purified product at 24 mg/kg did not show any significant improvement relative to controls. The group treated with CG53135-05 E. coli purified product at 48 mg/kg showed significant improvement on day 12 (P=0.035) but also showed significant worsening on days 26 (P=0.036) and 28 (P=0.006). The group treated with CG53135-05 E.
coli purified product at 12 mg/kg/day on days 1 and 2 showed significant improvements relative to controls on days 14 (P=0.010) and 18 (P=0.045). Since the standard for meaningful improvement in this test is 2 days of statistically significant improvement in the mucositis score relative to controls, the groups treated on day 1 with CG53135-05 E. coli purified product at either 6 mg/kg or 12 mg/kg, and the group treated with CG53135-05 E. coli purified product at 12 mg/kg/day on days I and 2 showed meaningful improvements.

Table 30. Mucositis scores as performed by using the Mann-Whitney Rank-Sum test Da Group 6 8 10 12 14 16 18 20 22 24 26 28 Comparison CG53135-05 0.597 0.735 0.826 0.298 0.010 0.606 0.324 0.164 0.224 0.736 0.03 0.202 E. coli purified product 6 mg/kg day I
vs control CG53135-05 0.989 0.595 0.042 0.164 0.011 0.031 0.005 0.037 0.232 0.762 0.57 0.347 E. coli purified product 12 mg/kg day 1 vs control CG53135-05 0.781 0.129 0.736 0.104 0.104 0.553 0.115 0.988 0.298 0.356 0.29 0.388 E. coli purified product 24 mg/kg day 1 vs control CG53135-05 0.797 0.989 0.393 0.035 0.101 0.553 0.295 0.224 0.780 0.164 0.03 0.006 E. coli purified product 48 mg/kg day 1 vs control CG53135-05 0.284 0.161 0.284 0.106 0.010 0.144 0.045 0.260 0.163 0.424 0.98 0.456 E. coli purified product 12 mg/kg day 1 and 2 vs control Significance of Group Differences Observed in Daily Mucositis Scores (Rank Sum Test).
This nonparametric statistic is appropriate for the visual mucositis scoring scale. The p values for each calculation are shown. Significant improvement is highlighted.

Survival: five animal deaths occurred during the study. The deaths occurred on day 4 in the group receiving CG53135-05 E. coli purified product at 24 mg/kg, day 7 in the 6 mg/kg group, days 9 and 11 in the 12 mg/kg on day 1 only group and day 11 in the 48 mg/kg group.
No deaths were observed in either the control group or in the group receiving CG53135-05 E.
coli purified product at 12 mg/kg/day on days 1 and 2. Survival was consistent with the death rate usually observed in the chemotherapy/radiation model.

Weight Change: the mean daily percentage weight change for each group is shown in FIG.

20. The overall increase in weight during the course of this study for animals in the untreated control group was 47.5%, compared with 45.9% in the group treated with CG53135-05 at 6 mg/kg in day 1, 53.8% in the group treated with CG53135-05 E. coli purified product at 12 mg/kg in day 1, 41.2% in the group treated with CG53135-05 E. coli purified product at 24 mg/kg in day 1, 49.7% in the group treated with CG53135-05 E. coli purified product at 48 mg/kg in day 1, and 46.9% in the group treated with CG53135-05 E. coli purified product at 12 mg/kg on days 1 and 2. Analysis of group weight gain was done by calculation of the area under the curve (AUC) for each animal. One-Way ANOVA analysis of group AUC values for weight among all study groups indicated that there were no significant differences between any groups in the study (P=0.687). A
mean comparison of AUC values for each group in this study is shown in FIG. 21. This result indicates that the animals in groups treated with CG53135-05 E. coli purified product gained weight in a manner that was equivalent to those in the untreated control group.

6.21. EXAMPLE 21: EFFECT OF CG53135 ON TREATMENT OF ESTABLISHED ORAL
MUCOSITIS IN HAMSTER CHEMO/RADIATION MODEL (N-318) Animal, type and age, and the chemotherapy/radiation model are same as described in Section 6.20. Protein concentrations in this example were measured by Bradford assay.

Sixty (60) hamsters were randomized into six (6) groups of ten (10) animals each, prior to irradiation. Each group was assigned a different treatment as listed and treated with CG53135-05 E. coli purified product,12 mg/kg IP as indicated in Table 31. In this study, animals were dosed with 60 mg/kg 5-FU on days -4 and -2, followed by an acute radiation dose of approximately 30 Gy on Day 0 in order to produce severe mucositis around Day 15. The duration of this study was 35 days.
The treatment schedule and dosing started after animals reach an oral mucositis score of 2. In addition to the mucositis scoring, this study evaluated the occurrence of diarrhea, weight loss and death for each animal in the experimental groups.

Table 31. Treatment Groups Group Number of Treatment Treatment Schedule Number Animals 1 8 males Untreated Control None 2 8 males Vehicle control Once Daily (3x) on OM score of 2 3 8 males 12 mg/kg CG53135-05 E. coli Once Daily (lx) on OM score of 2 purified product, ip, once 4 8 males 12 mg/kg CG53135-05 E. coli Once Daily (2x) on OM score of 2 purified product, ip, twice 8 males 12 mg/kg CG53135-05 E. coli Once Daily (3x) on OM score of 2 purified product, ip, thrice 6 8 males 12 mg/kg CG53135-05 E. coli Once Daily (4x) on OM score of 2 purified product, ip, four times Mucositis was evaluated on alternate days beginning on day 6 and every second day until the conclusion of the experiment on day 28 (i.e., days 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, & 28).
Mucositis was induced in hamsters. The end points, mucositis, weights and survival, were evaluated. Statistics applied were Chi-squared analysis and Mann-Whitney Rank Sum test. All the three parameters are described in Section 6.20.

Results Mucositis: in the untreated control group, the peak of mucositis occurred on day 14 with a mean score of 3. In the vehicle control group the peak of mucositis occurred on day 16 with a mean score of 3.4. The groups receiving CG53135-05 E. coll purified product 12 mg/kg IP on the first and second days after reaching a score of 2 showed similar patterns of mucositis scores to the control groups (FIG. 22(A)). The groups that received CG53135-05 E. co/i purified product 12 mg/kg IP on the third and fourth days after reaching a score of 2 showed a reduction in mucositis scores relative to the control groups, predominantly after the peak of mucositis (FIG. 22(B)).

The differences in mucositis scores between the groups were evaluated by comparing the number of days with a score of 3 or higher using a Chi-Squared test. In the untreated control group, 32.3% of the animals days evaluated had a score of 3 or higher, compared with 41.1 % of animals days in the vehicle control group. As a result of this difference between the two control groups, two treated groups (groups receiving CG53135-05 E. coli purified product on 2 and 3 days after reaching a score of 2) showed a significant improvement when compared with the vehicle control, but not when compared to the untreated controls. For the group treated with CG53135-05 E. coli purified product for 2 days, the P values were 0.347 compared to untreated controls and 0.007 when compared to the vehicle controls, and for the group treated with CG53135-05 E.
coli purified product for 3 days the P values were 0.580 compared to untreated controls and 0.020 when compared to vehicle controls. The group treated with CG53135-05 E. coli purified product for four days after reaching a score of 2 showed significant improvement when compared with both the untreated (P=0.003) and vehicle (P<0.001) controls. The group treated with CG53135-05 E.
coli purified product on only one day after reaching a score of 2 did not show significance when compared with either control group.

Further evaluation of the significance of the differences seen between the control and treated groups was performed by using a Mann-Whitney rank sum test to evaluate the mucositis scores for each group on each day that scores were obtained. In this analysis, the different treatment groups were compared with either the untreated control group or the vehicle control group. The results of this comparison with the untreated control group showed that there was a statistically significant difference between the group treated for 2 days and the untreated control group on day 10 only (P=0.011). There were statistically significant differences between the group treated for 3 days and the untreated control group on days 14 (P=0.036) and 22 (P=0.013).
Statistically significant differences between the group treated for 4 days and the untreated control were seen on days 10 (P=0.009), 12 (P=0.029), 14 (P=0.002) 22 (P=0.021) and 24 (P=0.032). No statistically significant differences were seen between the group treated with CG53135 on a single day and the untreated control group.

The results of the rank sum comparison between the vehicle control group showed that there was a statistically significant difference between the group treated on 3 days and the vehicle control group on days 14 (P=0.020) and 22 (P=0.020). Statistically significant differences between the group treated on 4 days and the vehicle control were seen on days 10 (P=0.036), 14 (P<0.001), 18 (P=0.024), 22 (P=0.048), 24 (P=0.021), 26 (P=0.048) and 28 (P=0.004). No statistically significant differences were seen between the groups treated with CG53135 on a single day, or 2 days and the vehicle control group.

Weight Change: animals in the untreated control group gained an average of 50.5% of their starting body weight by the end of the study. The vehicle control group had the lowest mean gain in weight during the study, gaining an average of 41.1 %. The group that received four daily doses of CG53135-05 E. coli purified product had the largest gain in weight during the study at 53.4%, while the group that received one, two and three daily doses gained an average of 48.1%, 46.8% and 44.4% respectively. These differences were evaluated by calculating the area under the curve (AUC) for the percent daily weight gain for each animal and then evaluating the AUC values using a one way ANOVA test. No significant differences were seen between the groups (P=0.266). The mean AUC data is shown in FIG.23.

6.22. EXAMPLE 22: CG53135 CAN BE USED SAFELY AS A SINGLE DOSE THERAPY
FOR MUCOSITIS IN HUMAN PATIENTS (STUDIES C-214 AND C-325) Safety, tolerability, and pharmacokinetic assessment of a CG53135-05 E. coli purified product (in the formulation as described in Section 6.2.1, referred as "CG53135-05 drug substance"
in this example) administered intravenously in human patients with advanced (Stage 4) cancer (single rising-dose tolerance) was conducted. The goal of this dose-escalating tolerance study was to assess the safety, tolerability and pharmacokinetics of CG53135-05 drug substance in cohorts of four patients at 0.03, 0.1, 0.33, and 1 mg/kg (UV). Dose escalation was stopped due to tolerability information at 0.33 mg/kg delivered in 15 minutes (Phase I study C-325) and the protocol was amended to add a 0.2 mg/kg dose.

During the trial, oral mucosa was examined by experienced study staff and assigned a mucositis score by both the World Health Organization (WHO) OM scoring system and the Oral Mucositis Assessment Scale (OMAS). See, WHO Handbook 1979 WHO. WHO Handbook for Reporting Results for Cancer Treatment. In: WHO Offset Publication No. 48.
Geneva, Switzerland:
World Health Organization; 1979. The OMAS provides a more quantitative assessment of injury.

After discharge, patients were provided with diaries where they noted a single WHO score for each day. Study staff instructed patients on how to self-assess and assign a score for oral mucositis.
Table 32. WHO Scoring System:

Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Normal Erythema and Ulceration, but Ulceration, diet Ulceration of soreness can eat solid limited to liquids such severity foods that patient requires parenteral feeding A value of the OMAS system is obtained by summing the erythema and ulceration/pseudomembrane scores.

Table 33. OMAS Scoring System Erythema None Mild/ moderate Severe erythema erythema Ulceration/ No lesions Cumulative Cumulative Cumulative surface psuedomembrane surface area of surface area of area of lesion is > 3 lesion < 1 cm2 lesion > 1 cm2 cm2 and < 3 cm2 Eleven patients have received the CG53135-05 drug substance at 0.03 mg/kg (n=4), 0.1 mg/kg (n=6), and 0.2mg/kg (UV) (n=1) as a single 100-m1 intravenous infusion administered 3 days after completion of the CT. Tolerability information is available for all 11 patients. Full clinical data from nine patients are available.

Preliminary pharmacokinetic data demonstrated plasma exposure with an average Cmax of 564.3 ng/ml at the 0.03 mg/kg (UV) dose level (n=3; range 175.6- 1192.6 ng/ml) and 564.7 ng/ml at the 0.1 mg/kg (UV) dose level (n=3; range 420.9 - 797.5 ng/ml). After infusion, the CG53135-05 drug substance reached maximum plasma concentration within 1 hour (15 to 35 minutes after completion of infusion). The mean terminal exponential half-life was 49 minutes (range: 16.2-87 minutes, n=5). No patients discontinued the trial due to adverse experiences.
Adverse events (number of patients) that may be related to the study drug include: nausea (2); chills (2); fever (2);
vomiting (1); dizziness (1); photopsia (1) (vision- lights flashing" on day 15) and astigmatism (1) (mild astigmatism on day 28); neuropathy (1) (on soles of the feet on day 15);
tachycardia (1);
headache (1); and asymptomatic, single premature atrial complex noted on ECG
(1). All reported incidences were mild to moderate. No Grade 3 or 4 laboratory toxicity associated with the study drug was noted. Among the 11 patients who have received the drug through September 3, 2004, six serious adverse events determined to be unrelated to study drug were noted from 3 patients.
These events included cancer progression (n=2), catheter infection, small intestinal obstruction, esophagitis/mucositis and neutropenic fever.

Among the 11 patients that completed the study, six patients did not develop oral mucositis.
Four patients developed Grade 1(n=1) or Grade 2 (n=3) oral mucositis. One patient with a Grade 3 oral mucositis was observed. No patients required total parenteral nutrition.
Patients receiving CPT-1 1 typically have a high incidence of diarrhea. In this trial, 7 of the patients received CPT-11 as part of CT and only two patients (both received 0.03 mg/kg (UV) of CG53135-05 drug substance) experienced mild to moderate diarrhea and only I patient developed diarrhea immediately after receiving CG53135-05 drug substance treatment. We concluded that the CG53135-05 drug substance was well-tolerated with single dose administration at 0.03, 0.1,and 0.2 mg/kg (UV).

A concurrent single rising-dose, phase I trial (study C-325) in autologous stem cell transplant patients is ongoing and 27 patients have been treated with CG53135-05 drug substance.
22 patients (pts) (ages 25-75) undergoing HDCT with PBSCT have completed the study with escalating doses of CG53135-05 drug substance, including (number of patients):
0.03 mg/kg (2), 0.1 mg/kg (10), 0.2 mg/kg (8), and 0.33 mg/kg (2). Patients were treated for:
multiple myeloma (n=11), non-Hodgkin's lymphoma (n=9), acute myelogenous leukemia (n=1), and desmoplastic round cell tumor (n=1) and were treated with conditioning regimens including melphalan (Mel 200), cyclophosphamide, carmustine and etoposide (CBV), carboplatin and thiotepa (CT), and busulfan/cyclophosphamide (targeted BuCy). The primary objective of the trial was to evaluate safety, tolerability and pharmacokinetics of the CG53135-05 drug substance.
Patients were also scored daily for presence of OM using both the WHO and OMAS grading scales (Tables 32 and 33, supra). Among the 22 patients that completed the study, 8 patients experienced no OM (including 4 Mel 200 pts); 10 patients experienced only WHO grade 1(n=7) or grade 2(n=3) OM, while 4 patients experienced severe OM of Grade 3 (n=3) or Grade 4(n=1). I patient experiencing grade 4 OM required TPN for 4 days. Patients tolerated the study drug well with no significant side effects up to a dose of 0.33 mg/kg. At that dose, 2 patients experienced an infusional reaction consisting of fevers, nausea, and mild hypotension. Preliminary Pharmacokinetic results from 13 patients confirmed dose dependent plasma exposure with an average Cmax of 135.5 ng/ml, 343.3ng/ml, and 658.3ng/ml at dose levels of 0.1, 0.2, and 0.33 mg/kg, respectively. The median day of neutrophil engraftment (as determined by ANC>500/uL) occurred on day 13 after stem cell infusion.
Preliminary data suggest that CG53135-05 drug substance is well tolerated in PBSCT patients at doses up to 0.33 mg/kg with apparent clinical effects in ameliorating or preventing OM. 18/22 patients, thus, avoided (WHO Grades 3-4) mucositis following HDCT. A larger Phase II clinical trial will be initiated to evaluate the efficacy of CG53135-05 drug substance in preventing HDCT- induced OM.

In conclusion, the CG53135-05 drug substance was generally well tolerated among the 38 patients that were administered in the two phase I trials to date. The doses tested were 0.03, 0.1, 0.2 and 0.33 mg/kg (UV). Infusional reactions when the 0.33 mg/kg of drug was administered over 15 minutes coupled with apparent activity observed at lower doses led to a discontinuation of this dose level. No other consistent drug-related or apparent dose-related adverse events or laboratory abnormals have been observed. No study drug-related serious adverse events were observed.

Sufficient information on tolerability and preliminary activity is considered to be present to utilize the 0.03, 0.1 and 0.2 mg/kg (UV) doses in Phase II testing.

6.23. EXAMPLE 23: CG53135 REDUCES THE INCIDENCE. LENGTH AND SEVERITY OF
RADIATION-INDUCED DIARRHEA (N-438) This study was performed to evaluate the activity of CG53135 against gastrointestinal injury induced by whole body irradiation as measured by diarrhea incidence and gut morphology. Protein concentrations in this example were measured by UV absorbance.

Materials and methods:

Dosing: Mice were weighed and then dosed with CG53135-05 E. coli purified product (4 or 16 mg/kg) or untreated. Dosing occurred as described in Tables 34 & 35. Each group of 20 animals was irradiated as per table below. All dosing of CG53135-05 E. coli purified product on day 0 was immediately after irradiation. No anesthesia was administered.

Intestinal Crypt Cell Damage Induction: Mice underwent whole body irradiation at a dose of 14 or 14.5 Gy delivered at a dose rate of 0.7Gy/min. Animals were followed for diarrhea incidence throughout the study period. After 6 days, animals were sacrificed, and the intestinal tract of the mice was harvested for histological analysis.

Body Weight: Every day for the period of the study, each animal was weighed and its survival recorded, in order to assess possible differences in animal weight among treatment groups as an indication of response to exposure to ionizing radiation.

Animals Found Dead or Moribund: Animals were assessed 2x/day from Day 3 onwards in order to accurately assess diarrhea onset / progression and detect moribund animals prior to death.
Such moribund animals were sacrificed by cervical dislocation. The ileum and mid-colon were removed and fixed in formalin, embedded in paraffin (1 animal per block, two tissues per block) for storage and future analysis/IHC if required. No tissue was removed from animals found dead.
Table 34. Study Design Group Number of Induction Treatment Treatment Volume Number Animals Schedule* (mL) 1 20 males 14 Gy None None Adjust per body Day 0 weight 2 20 males 14 Gy CG53135-05 E. coli Day -1, 0, 1 Adjust per body Day 0 purified product, weight 4 mg/kg, IP
( dx3 3 20 males 14 Gy CG53135-05 E. coli Day -1, 0, 1 Adjust per body Day 0 purified product, weight 16 mg/kg, IP
( dx3 4 20 males 14 Gy CG53135-05 E. coli Day 1 Adjust per body Day 0 purified product, weight 4 mg/kg, IP
(q6h x 4) 20 males 14.5 Gy None None Adjust per body Day 0 weight 6 20 males 14.5 Gy CG53135-05 E. coli Day -1, 0, 1 Adjust per body Day 0 purified product, weight 4 mg/kg, IP
( dx3 7 20 males 14.5 Gy CG53135-05 E. coli Day -1, 0, 1 Adjust per body Day 0 purified product, weight 16 mg/kg, IP
(qd x 3 Table 35. Test Article Requirements Conc. of Desired Volume Volume of Dose stock Conc of of dosing stock (mg/kg) Mass of solution Dosing solution solution Type of from #of # of Animal Admin vol by AZBO solution required required for Solution Group# Conc Animals doses (kg) (mUkg) (mg/mL) (mg/mL) (mL) dilution(mL) CG53135 1 0 20 0 0.025 10 10.2 0.000 0.000 0.000 CG53135 2 4 20 3 0.025 10 10.2 0.400 18.750 0.735 CG53135 3 16 20 3 0.025 10 10.2 1.600 18.750 2.941 CG53135 4 4 20 4 0.025 10 10.2 0.400 25.000 0.980 CG53135 5 0 20 0 0.025 10 10.2 0.000 0.000 0.000 CG53135 6 4 20 3 0.025 10 10.2 0.400 18.750 0.735 CG53135 7 16 20 3 0.025 10 10.2 1.600 18.750 2.941 140 ~ Total 8.333 Results:

Excel spreadsheet attached with diarrhea scores and weights for animals irradiated with 14 or 14.5Gy. Because the data from each radiation dose were very similar, only the analysis of the animals irradiated with 14Gy is provided.

Weights: Mass specific growth rate was calculated by:

in(Nff)-hiqvl;) = ].1'ISGR
TrTi Significance was calculated using One-way ANOVA and Dunnett's Multiple Comparison Test. No significant differences were seen between the changes in weight during the study between the groups (FIGs. 24 (A) and 24(B)).

Diarrhea score: Mice were scored for severity of diarrhea on a scale of 0-3 twice a day for three days beginning at 4 days after irradiation. Average diarrhea score over three days as well as the sum of the diarrhea score over three days was measured and graphed.
Significance was obtained by one-way ANOVA and Tukey's Multiple Comparison Test. (FIGs. 25(A) and 25(B)) An analysis of for each day of observation was also made to determine differences at days of peak diarrhea. Significance was determined as described above (* - P<0.05, ** - P<0.01, *** -P<0.001). (FIG. 26) Conclusions:
Dosing animals with 16mg/kg CG53135 at days -1, 0 and +1 respective to radiation resulted in a highly significant reduction in the incidence, length and severity of radiation-induced diarrhea.
Dosing animals every 6 hours on day 1 with 4mg/kg CG53135 also resulted in significant decrease in diarrhea incidence. The day of peak diarrhea was 5 days after radiation, at which point only the 16mg/kg dose of CG53135 provided a significant decrease in diarrhea. There were no significant differences between the treatment groups in weight loss over the course of the study.

6.24. EXAMPLE 24: CG53135 REDUCES CPT-11 INDUCED DIARRHEA IN RATS
(STUDY N-392) Irinotecan (CPT-11) is a chemotherapeutic agent which is commonly used against solid tumors which causes gastrointestinal (GI) mucositis manifest by severe diarrhea. The primary aim of this study was to investigate whether CG53135 reduces CPT-1 1 -induced GI
mucositis in an in vivo animal model. The secondary aim was to test varying schedules of administration of CG53135.
Methodology:

Diarrhea was induced in tumor-bearing rats with a single intraperitoneal dose of CPT-11 (200 mg/kg). Animals were treated with 16 mg/kg CG53135-05 E. coli purified product according to Process 2 described in Section 6.18.2 below (in a vehicle of 0.5M arginine, 0.05M sodium phosphate monobasic, 0.01 % polysorbate 80, and pH adjusted with sulfuric acid to pH 7.0) intraperitoneally either prior to, prior to and during, or post chemotherapy treatment. Rats were monitored closely for the incidence, and severity of diarrhea as well as mo l'Laiity. Animals were euthanized 168 hours following diarrhea induction. At euthanasia, tissues were harvested for histopathological evaluation of the gastrointestinal tract.

esults:

Severe or moderate diarrhea occurred in approximately 40% of rats treated with lone. This was associated with a 50% mortality rate at day 4 following chemotherapy. Rats that :ceived CG53135 prior to, or prior to and during CPT-11 treatment developed severe or moderate iarrhea, however, it occurred with a lower incidence and was not associated with mortality. Other osing regimens were not as effective.

onclusions:
CG53135 pre-treated animals (16 mg/kg) demonstrated an improvement in gastrointestinal iucositis as measured by a reduction in the incidence of diarrhea. A reduction in overall mortality ras also noted in this group. This has important implications for the use of CG53135 in GI
lucositis in humans, and should be further studied.

6.25. Example 25: Prophylactic Effect of the CG53135-05 E. colf Purified Product on Mice After Exposure to Acute Ionizing Radiation (Study N-308) This study was performed to investigate the effect of the CG53135-05 E. coli purified roduct administered prophylactically to mice that later were exposed to various doses of total body anizing radiation. Male C3H/He mice with an average weight of 22.1 gram at study initiation were sed for treatment groups. Animals were fed with a standard commercial mouse diet. Food and iater were provided ad libitum.

Protein concentration was measured by Bradford assay. Mice were exposed to ionizing adiation without anesthesia at a dose range of 484 to 641 cGy on day 0.
Animals were dosed with BS (control) or the CG53135-05 E. coli purified product (12 mg/kg, Bradford, daily IP) on day -1, or ays -2 and -1 before radiation. The schedule is represented in Table 36. The endpoints for the.
tudy were survival and weight changes. Survival was followed for 30 days post-irradiation.

Table 36. Stud y Design Group Number of Induction Treatment Treatment Number Animals Schedule*
1 16 males 484 cGy Day 0 PBS Day -2, -1 2 16 males 534 cGy Day 0 PBS Day -2, -1 3 16 males 570 cGy Day 0 PBS Day -2, -1 4 16 males 606 cGy Day 0 PBS Day -2, -1 16 males 641 cGy Day 0 PBS Day -2, -1 6 16 males 484 cGy Day 0 CG53135-05 E. coli purified Day-I
product, 12 mg/kg, Bradford, IP
Day -1 7 16 males 534 cGy Day 0 CG53135-05 E. coli purified product, 12 mg/kg, Bradford, IP

8 16 males 570 cGy Day 0 CG53135-05 E. coli purified Day -1 product, 12 mg/kg, Bradford, IP
9 16 males 606 cGy Day 0 CG53135-05 E. coli purified Day -1 product, 12 mg/kg, Bradford, IP
16 males 641 cGy Day 0 CG53135-05 E. coli purified Day -1 product, 12 mg/kg, Bradford, IP
11 16 males 484 cGy Day 0 CG53135-05 E. coli purified Day -2, -1 product, 12 mg/kg, Bradford, IP
12 16 males 534 cGy Day 0 CG53135-05 E. coli purified Day -2, -1 product, 12 mg/kg, Bradford, IP
13 16 males 570 oGy Day 0 CG53135-05 E. coli purified Day -2, -1 product, 12 mg/kg, Bradford, IP
14 16 males 606 cGy Day 0 CG53135-05 E. coli purified Day -2, -1 product, 12 mg/kg, Bradford, IP
16 males 641 cGy Day 0 CG53135-05 E. coli purified Day -2, -1 product, 12 mg/kg, Bradford, IP
Results:

Survival decreased as radiation dose increased in all treatment groups. In animals receiving PBS, 30-day survival at the lowest dose of radiation (484 cGy) was 93.75%, and decreased to 50.0% at 534 cGy, 31.25% at 570 cGy, 12.5% at 606 cGy, and 6.25%
at 641 cGy (Figure 27). In animals receiving CG53135, 12 mg/kg IP on Day -1 only, the 30-day survival at the owest dose of radiation (484 cGy) was 87.5%, compared to 87.5% at 534 cGy, 81.25% at 570 cGy, 43.75% at 606 cGy, and 31.25% at 641 cGy (Figure 28). In animals receiving GC53135, 12 mg/kg P on Days -2 and -1, the 30-day survival at the lowest dose of radiation (484 cGy) was 87.5%, -ompared to 75.0% at 534 cGy, 37.5% at 570 cGy, 31.25% at 606 cGy, and zero at 641 cGy (Figure 29).

A multiple comparison test demonstrated a 4.8-fold increase in the odds of survival in animals treated on day -1 versus control animals (p=0.00016). LD5oi3o values were calculated using a probit plot of survivorship with 95% confidence intervals calculated by bootstrapping. However, he odds of survival in animals treated on day -2 and -1 versus control animals were not significant (p=0.4162). The results are indicative of the therapeutic effect of prophylactic administration of CG53135-05 in radioprotection. Further, one day treatment before radiation (day -1) also protected animals from weight loss in all but the highest radiation level (641 cGy). In this particular system, Mngle dose (on day -1) was significantly more effective than the two-dose regimen (on days -2 and -1, respectively) especially in higher radiation levels.

In addition to the above results, the invention could be extended to additional dose regimens of the CG53135-05 E. coli purified product, such as prophylactically and/or therapeutically administer the CG53135-05 E. coli purified product prior and/or after the radiation exposure, which could be tested in the same animal model following the same procedures as described herein, in order to define the range of therapeutic efficacy of this compound. The dose regimen for therapeutic treatment may include, but is not limited to, +1, +1, and +2 days after radiation exposure. For example, in another experiment, mice were dosed IP with 4 mg/kg (UV) CG53135-05 E. coli purified product 24 hours prior to whole-body irradiation at the indicated doses. The survival of the mice was then followed for 30 days. Figure 28B shows the Kaplan-Meier plots for survival at 570 cGy and 606 -Gy with statistically significant differences between the group treated with the CG53135-05 E. coli purified product and the control group, i.e., p=0.008 and p=0.015, respectively. Figure 28C shows probit analysis for survival over the range of doses. The LD5oi3o for control and animals treated with he CG53135-05 E. coli purified product is 552.4 cGy and 607.4 cGy, respectively, with a dose nodification factor (DMF) of 1.10.

6.26. Example 26: Effects of CG53135 Prophylactic Dose Schedule on Survival of Irradiated Intestinal Crypt Cells (N-375) The objective of this study was to evaluate the ability of CG53135 to protect against ~adiation-induced intestinal crypt cell mortality in vivo when administered once daily for 4 days prior :o irradiation. CG53135-05 E. coli purified product (12 mg/kg) or PBS was administered to BDF1 nice intraperitoneally (IP) once daily for 4 consecutive days prior to exposure to lethal radiation loses from 10-14 Gy on Day 0. The number of surviving regenerating crypt foci was measured 4 lays after irradiation. Protein concentrations in this example were measured by Bradford assay.

When animals received CG53135 once daily for 4 days, an overall increase in crypt cell survival was noted when compared to PBS-treated, irradiated animals (Table 37).

i'able 37: Intestinal Crypt Protection Factors Resulting from CG53135-05 E.
coli purified product Vlultiple-Dose Administration Prior to Irradiation Mean Crypt Mean Crypt Survival (#) Radiation Survival (#) CG53135-05 Protection Dose PBS (12 mg/kg) Factora Gy 32.7 32.2 0.98 11 Gy 13.8 19.8 1.43 12 Gy 6.6 8.9 1.35 13 Gy 2.3 4.8* 2.09 14 Gy 1.7 1.3 0.76 Protection factor value indicates the number of surviving crypts per circumference in the CG53135-)5-treated animals compared to PBS, expressed as a ratio. *P:50.05 versus corresponding value rom PBS-treated control animals by ANOVA. # = number of crypts.

The greatest level of radioprotection occurred following 13 Gy of radiation, with a protection actor of 2.09 (e.g., a 2-fold increase in the number of surviving crypt cells). The crypt survival urves indicated a significantly reduced sensitivity to the radiation following CG53135-05 treatment 'Figure 30). Thus, pretreatment with CG53135 for 4 consecutive days increased the overall crypt -ell survival. This study indicates the use of multiple-day prophylactic dosing with CG53135-05 as a 3chedule with radioprotective properties.

6.27 Example 27: Evaluation of Radioprotection Window (N-382) Having established the effect of CG53135 on crypt cell radioprotection after a single day lose or multiple once daily dosing, this study evaluated the activity of CG53135 when dosed in ntervals other than 24 hours prior to irradiation. CG53135-05 E. coli purified product (12 mg/kg) or 'BS was administered to BDF1 mice by IP injection 6, 12, 24, 36, or 48 hours prior to exposure to a 3ingle bolus radiation dose of 13 Gy, respectively. The number of surviving regenerating crypt foci roas measured 4 days after irradiation. Protein concentrations in this example were measured by 3radford assay.

Administration of a single dose of CG53135-05 E. coli purified product at 24 or 36 hours )rior to irradiation offered the highest level of the intestinal crypt cell protection. These schedules -esulted in increased crypt survival by 80% and 31 %, respectively (Table 38).
Dosing at 6, 12, or 48 i prior to irradiation resulted in dose modification factors of 0.78, 0.40, or 0.84 respectively.

1'able 38: Intestinal Crypt Protection Factors Resulting from CG53135-05 Administration 6-48 h 'rior to Irradiation Treatment Mean Crypt Mean Crypt Survival (#) Schedule Radiation Survival (#) CG53135-05 Protection Dose PBS (12 m/k ) Factor a -6 h 13 G4.5 3.5 0.78 -12 h 13 G6.3 2.5* 0.40 -24 h 13 Gy 4.1 7.4" 1.80 -36 h 13 Gy 4.9 6.4 1.31 -48 h 13 Gy 4.4 3.7 0.84 ' Protection factor value indicates the number of surviving crypts per circumference in the CG53135-)5-treated animals compared to PBS-treated control animals, expressed as a ratio. *P < 0.001 /ersus value from corresponding PBS-treated control animals by ANOVA. # =
number of crypts.

These results suggest that an optimal window for administration of a single dose of :;G53135-05 E. coli purified product occurs from 24 to 36 hours prior to irradiation.

6.28. Example 28: Effects of CG53135 Dose Schedule on Survival of Irradiated Intestinal Crypt Cells (N-416) The objective of this study was to establishing an optimal dosing schedule of administration to establish the levels of protection against radiation-induced crypt cell mortality.
'rotein concentrations in this example were measured by UV absorbance. CG53135-05 E. coli )urified product (4 mg/kg) or phosphate-buffered saline (PBS) was administered to BDFI male mice )y intraperitoneally (IP) once daily either for 1, 2, 3, 4 or 5 consecutive days (Days -1, 0, 1, 2 and/or 3) prior to, or post-irradiation (13 Gy). The number of surviving regenerating crypt foci was -neasured 4 days after irradiation and the dose modification factor (DMF) were calculated.

Single dose administration of CG53135-05 on Day -1 resulted in a DMF of 2.3 (Figure 31).
4dministration of CG53135-05 on Days -1, 0 and 1 relative to TBI on Day 0 resulted in a DMF of 3.0 (e.g., a 3-fold increase in the number of surviving crypt cells). These data suggest that CG53135 is a GI crypt cell radioprotectant with prophylactic and intervention (treatment) properties.

6.29. Example 29: Radioprotective Mechanisms of CG53135 Among the many changes that occur in a cell upon an attack of ionizing radiation, an increase of reactive oxygen species occurs via the ionization of H20. As this process produces the most reactive molecules within the cell, in order to reduce cellular damage, the nucleus increases transcription of enzymes that scavenge these radicals to less reactive intermediates. As CG53135 has been shown to be a radioprotectant, it is relevant to determine if treatment of cells with CG53135 upregulates any of the genes known to be involved in radioprotection in the interest of "pre-loading" the cells with oxygen radial scavenging pathways. Evaluation of the effect of CG53135 on the intricate pathways involving ROS scavengers and transcription factors will mechanistically describe the observed in vivo radioprotective effects of this agent. Thus, expression studies and survival studies were carried out at the cellular level.

Expression Studies:
To delineate the mechanism of radioprotection by CG53135, expression profile of free oxygen radical scavengers and transcription factor(s) were studied in fibroblast and endothelial cells.
NIH3T3 (murine fibroblast), CCD-1070sk (human foreskin fibroblast), CCD-18Co (human colonic Finbroblast), and HUVEC (human umbilical cord vascular endothelial cells) cells were transferred to basal medium containing 0.1 % FBS and the indicated concentration of the CG53135-05 E. coli purified product. After 18 hours incubation, cells were harvested for total RNA using RNEasy ;Qiagen, Valencia, CA). RNA was reverse transcribed using SuperScript First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) and amplified for the gene of interest using the arimers and cycles indicated below.

Table 39: Primers for RT-PCR (Human) Gene Name Primer SEQ ID NO No. of cycles COX2 5'- TTCAAATGAGATTGTGGGAAAATTGCT -3' 42 30 5'- AGATCATCTCTGCCTGAGTATCTT -3' TFF3 5'- GTGCCGGCCAAGGACAG -3' 44 40 5'- CGTTAAGACATCAGGCTCCAG -3' 45 SOD1/CuZn SOD 5'- TGGCCGATGTGTCTATTGAA -3' 46 30 5'- GGGCCTCAGACTACATCCAA -3' 47 SOD2/MnSOD 5'- CTGGACAAACCTCAGCCCTA -3' 48 28 5'- CTGATTTGGACAAGCAGCAA -3' 49 SOD3/ECSOD 5'- TCCATTTGTACCGAAACACCCCGCTCAC -3' 50 30 5'- CAAACATTCCCCCAAAGGAGCAGCTCTCAG 51 -3' Nrf2 5'- ATGGATTTGATTGACATACTT -3' 52 40 5'- CTAGTTTTTCTTAACATCTGG -3' 53 GPXI 5'- AAGGTACTACTTATCGAGAATGTG -3' 54 28 5'- GTCAGGCTCGATGTCAATGGTCTG -3' 55 Actin 5'- GGACTTCGAGCAAGAGATGG -3' 56 5'- AGCACTGTGTTGGCGTACAG -3' 57 Table 15: Primers for RT-PCR (Mouse) Gene Name Primer SEQ ID NO No. of cycles MnSOD 5'- GGGAATTCAGCGTGACTTTGGTCTTTT -3' 58 26 5'- GCGGATCCGAGCAGGCGGCAATCTGTAA -3' 59 Actin 5'- GCATCCATGAAACTACATT -3' 5'- 60 CACTTGCGGTGCACGATGG -3' 61 Results Expression results are summarized in Table 40 and shown in FIGs. 32 (A) - (F).
Table 40 Gene NIH 3T3 CCD 1070 CCD 18Co HUVEC
MnSOD Increased Moderately Increased No change Increased ECSOD Increased No change Moderately Absent Increased Cu, Zn-SOD No change No change Increased No change Nrf2 Increased Increased Increased Increased-repressed at higher doses COX2 No change No change Increased Moderately Increased Tff3 No expression Increased Increased Increased GPX1 No change Moderately No change No change Increased Among the radioprotective superoxide dismutases, MnSOD, the most radioprotective one, Nas found to be induced by the CG53135-05 E. coli purified product consistently among cell lines (Table 40, FIGs. 32(A) - (F)).

The Nrf2 transcription factor, which is involved in regulation of several antioxidants that were thought to be the "antioxidant response element" (also termed as ARE), was induced by CG53135 in all cell lines studied (Table 40, FIGs. 32(A) -(F)). The ERK and Akt kinases are also activated by CG53135. CCD18Co human colonic fibroblasts were starved for 18 hours in basal media containing D.1 % BSA or left in complete serum ("Comp") then stimulated with 100ng/ml FGF-20 and harvested at the time points indicated. Lysates were immunoblotted for human ERK or Akt or their indicated phosphorylated counterparts. Both ERK and Akt kinases were active by 2 minutes of treatment with the CG53135-05 E. coli purified product (FIGs. 32 (D) and (E)). The activation of these kinases, particularly Akt, has been associated with radioprotective events.

It is well established that one mechanism by which cells are protected from ionizing radiation is through the induction of oxygen radical scavenging pathways. The gene expression studies detailed herein indicate that this may be one of the pathways by which CG53135 modulates radioprotection. Furthermore, one of the primary target organs of total body irradiation (TBI) is the gastrointestinal tract. The data disclosed herein show that (1) the most responsive of all cell lines studied was a gastrointestinal fibroblast (CCD-18co); and (2) a well-characterized intestinal radioprotectant, Tff3, was strongly upregulated by CG53135. Considering that other radioprotectants known in the art strictly affect bone marrow survival and no other compartments, it is important to note that CG53135 is active in a tissue that is as equally affected as the hematopoietic stem cells, but no less important to the survival of the animal.

Survival Studies:
Cells that receive a certain dose of radiation will have to brace against the onslaught of ionized radicals, repair the damage that the radicals perform or delay the onset of apoptosis in the face of irreparable harm, or, likely, a combination of all these mechanisms.
Each of these pathways is thus important for the ultimate survival of a cell and its ability to proliferate. Cell survival was assessed by a clonogenic assay in which surviving cells can form colonies in vitro after irradiation.

Clonogenic assays were performed using CCD-18co cells, FaDu human squamous cell carcinoma cells, IEC6 and IEC18 rat colon crypt cells, and NIH 3T3 mouse fibroblast cells, to assess the effect of CG53135 on radiation protection. Cell culture conditions were as follows: NIH 3T3 cells were grown in DMEM + 10% Bovine serum + 50 lag/ml Pennicilin/Streptomycin; IEC6 and IEC18 cells were grown in DMEM + 10% FBS + 0.1 U/ml Insulin + 50 Ng/ml Pennicilin/Streptomycin;
FaDu cells were grown in MEM + 10% FBS + 1 mM Sodium Pyruvate + 50 pg/ml Pennicilin/Streptomycin + Non-essential amino acids. Cells were plated at a density of 5x105 per 10 cm dish (NIH3T3) or 5x105 per well of a 6-well dish (IEC18, IEC6, FaDu) and allowed to attach.
Cells were then treated with the CG53135-05 E. coli purified product at doses of 10 or 100 ng/ml (IEC18, IEC6, FaDu) or at 50 and 200 ng/ml (NIH 3T3) in basal media containing 0.1% serum (IEC18, IEC6, FaDu) or 1% serum (NIH 3T3) and incubated for 16 hours (IEC18, IEC6, FaDu) or I
hour (NIH 3T3). Cells were then irradiated using a Faxitron X-ray irradiator (Wheeling, IL) fitted with a 0.5 mm aluminum filter at 2.5, 5, 7.5, 10, 12.5 and 15 Gy at 130kVp, resulting in a radiation rate of 50 cGy/min. Immediately after irradiation, cells were trypsinized and plated in duplicate at densities of NIH 3T3: 250, 500, 1000, 2000, 5000 and 10,000 cells per 60mm dish; FaDu, IEC18 and IEC6:
500, 2500 cells per well of a 6-well dish. Cells were grown in complete growth medium for 1-2 weeks until colonies of average diameter of 2 mm, after which the colonies were stained with crystal violet and counted.

Results:
The number of surviving untreated or CG53135-treated cells was plotted as a function of radiation dose, giving rise to survival curves. The slopes of different parts of the survival curves describe different properties of radiation cell killing and can be described as follows:

DO is the slope of the curve between the final two points, indicating speed of cell killing at the higher doses of radiation. The value is interpreted to indicate the amount of radiation required to reduce the fraction of surviving cells by 37% of the previous value on the graph. A smaller number indicates a more rapid rate of cell killing.

Dl is the slope of the curve between the first two points, indicating the speed of cell killing at the lower doses of radiation. The value is interpreted as the amount of radiation required to reduce the fraction of surviving cells by 37% of the previous value on the graph. A
smaller number indicates a more rapid rate of cell killing.

Dq is the width of the shoulder of the curve before an exponential decrease in cell survival is seen. This is essentially the threshold amount of irradiation required before an incidence of cell killing is seen. A larger Dq value indicates that the cells are completely protected at the lower doses of radiation.

The effect of CG53135 treatment on survival of irradiated IEC18 cells is shown in Figure 33(A). While the DO and D1 values showed no obvious treatment-related trends, the Dq value indicates that IEC18 cells treated with CG53135 are more protected from killing at the lower doses of radiation compared to untreated cells (shoulder of survival curves of cells treated with 10ng/ml and 100ng/ml CG53135 is broader). Thus, treatment of IEC18 cells with the CG53135-05 E. coli purified product results in cell killing at a higher dose of radiation compared to untreated cells, indicating that CG531 35 acts as a radioprotectant in these cells.

The effect of CG53135 treatment on survival of irradiated NIH 3T3 cells is shown in Figure 33(B). The DO values for NIH 3T3 cells treated with 50 ng/ml or 200 ng/ml CG53135 appear larger than the DO value for untreated cells, and the difference approaches significance for the 50 ng/ml dose. These results suggest that CG53135 may act as a radioprotectant, promoting survival of NIH
3T3 cells at the higher doses of radiation. Furthermore, the D1 value for cells treated with 100 ng/mL CG53135 is smaller than for untreated cells, indicating a slower rate of the cell death at lower doses of radiation. No trend in Dq values of the survival curves could be determined, largely due to the variation of survival in untreated cells.

The effect of CG53135 treatment on survival of irradiated HUVEC cells is shown in Figure 34. The DO value for cells treated with 100 ng/mL CG53135 is higher than that for untreated HUVEC cells or cells treated with 10 ng/mi CG53135, indicating a slower rate of cell death at higher radiation doses. In addition, the Dq value for cells treated with 100 ng/mL
CG53135 suggests that there is a slower rate of cell death at low doses of radiation compared to untreated HUVEC cells or cells treated with 10 ng/ml CG53135. No obvious effects of treatment of CG53135 on Dl values were observed. Thus, treatment of HUVEC cells with 100 ng/ml CG53135-05 E.
coli purified product provides a significant decrease in the speed of cell killing at the high dose of radiation. The HUVEC cells treated with 100 ng/ml CG53135-05 E. coli purified product also appeared to be more protected from killing at low doses of radiation compared to untreated cells.

In contrast, irradiated FaDu and IEC6 cells did not show any obvious trends in DO, Dq or D1 values as a result of CG53135 administration.

In another study (L-411 and L-432), post-radiation cell survival was examined in 7 cell lines that were representative of different cell types in each layer of intestinal mucosa: epithelium (IEC6 and IEC18, rat intestinal epithelia), mesenchyme (NIH3T3, mouse fibroblast;
CCD-18Co, human colonic fibroblast), and hematopoietic (32D, murine hematopoietic cell line) using a clonogenic assay (as described above). Cells were irradiated, plated in complete growth media with or without 100 ng/ml CG53135-05 E. coli purified product, and allowed to form colonies for 10-14 days. The data were plotted and analyzed for rate of cell killing at high doses (DO) and low doses of radiation (D1, Dq) (Figure 35, Table 41).

Table 41: Survival Response Parameters for Cells Cell Type Treatment Do D D, 32D Cells Untreated 0.60 2.60 20.38 (Hematopoietic) 100 ng/mL CG53135-05 0.71 3.06 21.45 Post-irradiation IEC6 Cells Untreated 1.03 1.98 2.50 (Epithelial) 100 ng/mL CG53135-05 Post-irradiation 1.03 3.32 5.89 IEC18 Cells Untreated 1.27 0.68 1.61 (Epithelial) 100 ng/mL CG53135-05 0.95 3.15 5.88 Untreated 0.88 2.87 5.22 NIH3T3 Cells 100 ng/mL CG53135-05 (Mesenchymal) Prior to irradiation 1.18 2.15 2.72 100 ng/mL CG53135-05 Post-irradiation 0.75 3.46 ----CCD-18Co Untreated 1.23 0.13 1.29 (Mesenchymal) 100 ng/mL CG53135-05 Post-irradiation 0.95 1.68 2.13 Untreated 0.77 0.49 1.02 U2OS 100 ng/mL CG53135-05 (Bone) Prior to irradiation 0.79 0.76 1.21 100 ng/mL CG53135-05 Post-irradiation 0.80 1.60 2.03 Saos2 Untreated 0.74 0.95 1.32 (Bone) 100 ng/mL CG53135-05 Post-irradiation 0.74 1.37 1.73 I"he D, and Dq parameters are indicative of the rate of cell killing at low doses of radiation, whereas he Do parameter reflects the rate of killing at high doses of radiation. An increase in these )arameters in CG53135-05-treated cells as compared to untreated indicates a protective effect.
Significant protection was observed the 32D, NIH3T3, IEC18, IEC6 and U2OS cell lines, vhile more modest protection was seen in the CCD18Co and Saos lines (Figure 35, Table 41).
freatment of U2OS and NIH3T3 cells with FGF-20 after irradiation was more efficacious than )retreatment.

In summary, these results indicate that the CG531 35-05 E. coli purified product has a arotective effect against radiation in vitro.

6.30. Example 30: Effect of CG53135 on Cytokine Release Cytokines are important cell signaling proteins mediating a wide range of physiological *esponses. Ionizing radiation can trigger a series of changes in gene expression and cytokine )rofiles. The aim of this study was to evaluate the cytokine profile upon CG53135 treatment in cell ulture over a time course.

BioPlex cytokine assays, which are multiplex bead based assays designed to quantitate nultiple cytokines from tissue culture supernatants, were used for detecting the cytokines. The Drinciple of the assay is similar to a capture sandwich immunoassay. NIH 3T3 cells were plated in a a6 well plate. The cells were washed with DMEM+0.1 % Calf Serum (SFM). The CG53135-05 E.
>oli purified product, at 10 ng/ml or 100 ng/ml, was added to the cells. The cell supernatant was ollected after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours.
Fifty ng of TNF was ised as a positive control. Bioplex 18-Plex Cytokine Assay (BioRad Laboratories Inc, CA) was oerformed following the procedure of the manufacturer.

Results:

Figure 36 shows the effect of the CG53135-05 E. coli purified product on Mo KC
release.
Ulo KC is also known as the chemokine CXCL1 (which also has been described as Grol, Melanoma growth stimulatory activity (MSGA) or neutrophil-activating protein-3 (NAP3)).
It functions as a ahemoattractant for neutrophils, signalling through the CXCRI receptor. It has also been implicated n the response to whole body irradiation, raising the possibility that it possesses radioprotective qualities of its own (see Radiat. Res. 160:637-46, 2003). Figure 36 shows a consistent dose (p =
D.0085) and time dependent increase (p = 4.6x10"6) in the measured response.
In addition both the aoncentrations of the CG53135-05 E. coli purified product showed significantly higher response than the control (no CG53135).

IL-6 and IL-11 expression in response to CG53135 treatment was also examined.
Both IL-6 and IL-11 have recently been implicated in the response to total body irradiation. In addition, IL-11 has been used as an agent to combat thrombocytopenia following chemo- or radiotherapy.
CCD18Co cells were incubated with 100 ng/ml CG53135 in basal media containing 0.1% BSA for the indicated time periods. Conditioned media was removed and analyzed for IL-6 and IL-11 oncentration by Luminex or ELISA respectively. Figure 36B shows that IL-6 and IL-11 cytokines are induced upon exposure to the CG53135-05 E. coli purified product in vitro.

Additional experiments can be performed to determine CG53135-05 in combination with XCL1 acts synergistically in radioprotection, both in vitro and in vivo.
Furthermore, induction of )ther cytokines (e.g., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, MCP-1, GM-CSF, RANTES) can also be ested, as a skilled person in art would recognize, in the presence of CG53135-05 in different cell ines (e.g., HUVEC, CCD-18, NIH3T3).

6. 31. Example 10: Measurement of Scavengers of Reactive Oxygen Intermediates After Radiation Exposure M-H,DCFDA method Cells with increased reactive oxygen species, as a first step, upregulate the Superoxide )ismutases - Cu, Zn-SOD, Mn-SOD, and extracellular-SOD to scavenge the superoxide radical to iydrogen peroxide. Activity of these enzymes can be indirectly measured by their byproduct of -izO2 using an acetoxymethyl ester. A derivative of this class, 5-(and-6)-chloromethyl-2',7'-iichlorodihydrofluorescein diacetate, also known as CM-H2DCFDA, is efficiently retained within the ell and fluoresces green when oxidized by H202.

Cu, ZnSOD
Dze- H2O2 + CM-H2DCFDA po Fluorescein MnSOD
ECSOD
IEC1 8 (rat intestinal epithelial) and CCD-18Co (human colonic fibroblast) cells were plated o 60 mm dishes at a density of 1x105 cells per dish. After attachment, the cells were switched to nedium containing 0.1% serum and the indicated dose of CG53135. After 18 hours of incubation, he cells were then irradiated at 2 or 4 Gy with X-rays using a Faxitron X-irradiator (Wheeling, IL), ollowed by incubation with 5 mM CM-H2DCFDA (Molecular Probes, Eugene, OR) for 15 minutes.
rhe cells were then washed, trypsinized, and analyzed on a Becton Dickinson FACSCalibur (San )ose, CA) on the FL1 channel.

Results indicate that IEC18 and CCD18Co cells possess increased intracellular HZOZ after reatment with the CG53135-05 E. coli purified product in a dose responsive manner (FIGs. 37 (A)-C)). This is believed to be due to enhanced expression of Superoxide dismutases, predominantly vInSOD induced by the CG53135-05 E. coli purified product.

Production of intracellular H2O2 by the CG53135-05 E. coli purified product in IEC18 cells is :nhanced with increasing dose of ionizing radiation (FIG. 37(B)). This result reflects the increased )roduction of more reactive oxygen species such as superoxide and hydroxyl by radiation, thus ncreasing substrate for the Superoxide Dismutases induced by the CG53135-05 E.
coli purified )roduct.

2ed CC-1 method Upon ionizing radiation exposure, cells accumulate reactive oxygen species as a result of radiation ionizing H20 to the hydroxyl radical (OH), superoxide (02 ) or hydrogen peroxide (H202).
As these ions in abundance can have deleterious effects on the cell, pathways are upregulated to scavenge these molecules to more stable variants. It is hypothesized that CG531 35 may protect the -ell from ionizing radiation damage by upregulating pathways that reduce the redox capacity of the -ytosol. A dye called Redox Sensor 1(Red CC-1) can monitor the redox level of the cytosol upon :)xidation by changing to a red fluorescent agent that can be measured by FACS
on the FL2 :;hannel.

Cytosolic Reactive Oxygen Species + Red CC-1 --> Oxidized Red CC-1 IEC18 (rat intestinal epithelial) and CCD-18Co (human colonic fibroblast) cells were plated :o 60mm dishes at a density of 1x105 cells per dish. After attachment, the cells were switched to medium containing 0.1 % serum and the indicated dose of the CG53135-05 E. coli purified product.
4fter 18 hours of incubation, the cells were then irradiated at 2 or 4 Gy with X-rays using a Faxitron K-irradiator, followed by incubation with 5 mM Red CC-1 (Molecular Probes, Eugene, OR) for 15 ,ninutes. The cells were then washed, trypsinized, and analyzed on a Becton Dickinson FACSCalibur on the FL2 channel.

Results: IEC18 and CCD18Co cells were found to possess decreased cytosolic redox aotential after treatment with CG53135-05 in a dose responsive manner as shown in FIGs. 38 (A)-;C). The data shown herein is believed to be the result of enhanced expression of superoxide lismutases induced by the CG53135-05 E. coli purified product, which scavenge the more reactive 3pecies of superoxide and hydroxyl radicals. Also, the CG53135-05 E. coli purified product is shown :o increase expression of a key antioxidant-controlling transcription factor, Nrf2, which may ontribute to this reduction in reactivity in the cytosol in other ways.

6.32. Example 32: In vitro Radioprotection of the Myeloid Cell Line 32D
The radioprotection effect of CG53135 in myeloid cells was also studied by in vitro Bxperiment using the myeloid cell line 32D. 32D cells were irradiated at 0, 1, 2, 3, 4 or 5 Gy then Aated in methylcellulose-containing growth media including 10 ng/ml IL-3 with or without 100 ng/ml -G53135-05 E. coli purified product. Cells were allowed to form colonies for 10 days and were then Bcored. FIG. 39 shows increased survival of 32D cells upon exposure to the CG53135-05 E. coli :)urified product. The cell survival is plotted by the natural Log of the surviving fraction, and a linear uadratic equation was used to obtain the curve. The qualities of the curve, i.e., Dl, Dq and DO, ndicating at what points radioprotection are observed, were derived using the methods described in -lall et al., Radiat. Res. 114(3):415-424 (1988), the description of which is incorporated herein by 'eference in its entirety. An increase in the Dq value, indicating the amount of radiation required for ell death to start exponentially (or the width of the "shoulder" of the curve at low radiation doses) 'rom 2.10 Gy to 2.79 Gy was observed.

6.33. Example 33: Effect of CG53135 on Repopulation of Thymus Following Bone Marrow Ablation and Subseguent Bone Marrow Transplant Long-term effects of CG53135 specifically in the thymus microenvironment on reconstitution of the immune system were also examined. Protein concentrations in this example were measured by UV absorbance. The CG53135 E. coli purified product was tested in a bone marrow ablation and transplantation model and repopulation of the thymus with thymocytes was examined. Mice were irradiated with 9Gy to ablate the bone marrow, and subsequently underwent bone marrow transplantation. Prior to this, one group of mice was dosed with 16 mg/kg (UV) CG53135 (IP), once daily on days -3, -2, -1, 0 and +1 relative to the day of bone marrow ablation. Thirty days after bone marrow transplantation, the thymi of both untreated and treated mice were harvested and thymocytes collected. Cells were counted (A) as well as stained (B) for the T-cell specific markers CD4 and CD8.

FIG. 40 shows that the total thymocyte cell population, as well as mature CD4/CD8 positive T-cells within the thymus, was significantly increased in animals treated with the CG53135-05 E. coli purified product (p=0.00003).

6.34. EXAMPLE 34: EFFECTS OF CG53135 ON RADIATION INDUCED DIARRHEA
(STUDY N439) This study examined the ability of CG53135 to reduce the level and severity of diarrhea in mice exposed to a lethal dose of irradiation. A previous study (N-438) indicated that 16mg/kg CG53135 given in a triple dosing schedule on Day-1, 0 and +1 significantly reduced the diarrhea severity in mice exposed to 14Gy X- irradiation. Conversely, dosing 4mg/kg on Day 0, at 0, 6, 12, 18 hours post irradiation increased diarrhea severity. This study further tested these observations, and assessed whether other therapeutic CG53135 treatment schedules, when the drug was only given post radiation exposure, could reduce diarrhea severity.

Materials and Methods:

Test materials: The test material used in this study was CG53135-05 E. coli purified product, batch number PT0504A. This was supplied as a frozen stock at 9.9mg/mI
and each vial thawed from -80 C and diluted as required in Aminosyn to 1.6 or 0.4mg/mI in a laminar flow hood, and the Aminosyn was sterile filtered prior to the dilution. Animals were then injected ip with 0.1 ml/10g body weight. CG53135 was freshly prepared (diluted) each day.

Test animals and husbandry: 140 male BDF1 mice (Harlan, UK) aged 10-12 weeks at study initiation were individually numbered using an ear punch. Each treatment group consisted of 20 mice, housed in 4 cages of 5 mice. Animals were housed in individually ventilated cages with 27 air changes/hour. Animals were allowed to acclimatize for 14 days prior to study commencement. The rooms were on an automatic timer for a 12 hour light/dark cycle with no twilight. All cages were labeled with the appropriate information necessary to identify the study, dose, animal number, and treatment group. Animals were fed a standard rodent maintenance diet. Food and filtered water were provided ad libitum.

Experimental Procedures:

Mice were divided into 7 treatment groups of 20 animals/ group and ear punched for identification. Animals were then dosed with drug (CG53135) by IP injection or remained untreated according to the schedule in Table xx. Mice were exposed to a single dose of 14Gy X ray whole body irradiation using a Pantak PMC 1000, Model HF320 machine with radiation delivered at 0.7Gy/min. All irradiations were performed during 13:00-17:00 hours. Animals were restrained but unanaesthetised for the duration of the irradiation.

Table 42: Study Design:

Group Number Induction on Treatment Treatment Number of Day 0 Schedule Male Animals 1 20 14 Gy Day 0 Untreated Untreated 2 20 14 Gy Day 0 CG53135-05 E. coli purified Day-1, 0*, +1 product, 16m /k IP
3 20 14 Gy Day 0 CG53135-05 E. coli purified Day 0*
product, 16mg/kg IP
4 20 14 Gy Day 0 CG53135-05 E. coli purified Day 0*, +1 product, 16mg/kg IP
20 14 Gy Day 0 CG53135-05 E. coli purified Day 0*, +1, +2 product, 16m /k IP
6 20 14 Gy Day 0 CG53135-05 E. coli purified Day 0*, +1, +2, +3 product, 16mg/kg IP
20 14 Gy Day 0 CG53135-05 E. coli purified Day 0*, and +6, product, 16mg/kg IP +12, +18 hours Body weights and diarrhea: Animals were weighed daily to assess possible changes in animal weight among treatment groups. Animals losing more than 20% of their body weight and showing signs of distress were considered moribund and sacrificed. Animals were observed for diarrhea incidence and severity twice a day from Day 3 onwards. In addition to checking incidence, the severity of the diarrhea was recorded on a scale of 0-3 for each treatment group:

0 = no sign of diarrhea 1= loose stools/ mild diarrhea 2= diarrhoea, peri-anal staining/matting of fur 3= severe watery diarrhea with wide area of staining/matting of fur.
Individual animal diarrhea scores were recorded.

Histopathology: At the time of sacrifice the ileum and mid colon was removed and fixed in formal saline, prior to paraffin embedding. These embedded samples were then cut to produce 3pm sections that were stained with H&E and the level of intestinal damage evaluated by eye. The sections from each mouse were evaluated and assigned a score from 1-5:

1: Normal Histology 2: Regenerating epithelium, almost repaired 3: Ulceration 4: Severe ulceration, few crypts remaining 5: Totally Denuded Results:

The relative loss in body weight per group is shown in FIG. 41. The raw data for the diarrhea severity scores are summarized in FIGs. 42 (A) and (B).

Although all the animals lost weight, the CG53135-treated animals initially lost weight at a greater rate than the controls. This lost difference was greatest mid-term (Days 2 and 3) and was directly related to the dosing schedule (animals receiving the most doses losing the greatest weight).
However, by the latter days of the study there was no difference between the levels of weight loss among the groups. As previously, mice were deemed moribund and were culled if they had lost more than 20% of their body weight and were also displaying signs of distress.
If mice had lost more than 20% of their body weight but were active, they were spared and re-evaluated at the next time point. Occasionally mice that had been spared were found dead, and thus tissue was not taken and there are no histology scores.

Diarrhea incidence was measured twice daily from Day 3 onwards, although no diarrhea was observed until Day 4. It is immediately obvious that 16mg/kg CG53135-05 reduced diarrhea severity. Dosing using the previously efficacious protocol on Day-1, 0, +1 with respect to irradiation, again was the most effective protocol, with dosing 0, 6, 12 and 18 hours post irradiation the least effective protocol. A single therapeutic injection immediately following radiation exposure, or dosing immediately following radiation exposure and 24 hours later was also very effective. The former appeared to be have a lesser effect at the earlier time points, but reducing diarrhea severity by the evening of Day 5. During this mid-term phase, there was a correlation between dose frequency and severity, with animals receiving the more daily doses experiencing the more severe diarrhea.

If the total diarrhea scores per group are examined, the effect of dosing on Day -1, 0, and Day +1 is to reduce diarrhea severity to 60% of the untreated level (49% if moribund animals are assigned a severity score of 3). This increases to 68% for those given CG53135 immediately post irradiation (70% if moribund animals are given a severity score of 3). Animals given further doses have severities of 69 (dose Day 0, 1), 80 (dose Day 0, 1, 2) and 77% (dose Day 0, 1, 2, 3) of control (60, 56 and 63% if moribund animals are included).

The histology scores in the small intestine confirm the diarrhea data.
Examination of the total and average scores, with moribund mice assigned a score of 4, reveals the best response in mice dosed on day 0 only, and mice dosed -1, 0, +1. Again, CG53135 dosed 0, 6, 12 and 18 hours later was least effective.

An additional anecdotal observation during this study was that most of the mice receiving 16mg/kg CG53135 ejaculated immediately on injection. This is a new observation, but we have performed most of our previous work using 4mg/kg, so it may be related to the higher dose of drug.

In conclusion, 16mg/kg CG53135-05 consistently reduced the diarrhea severity in mice exposed to 14Gy X irradiation. Dosing day 0 only or days -1, 0, +1 yielded the best protection when examined by a variety of parameters. Further dosing (Days 0, 1; Day 0, +1, +2;
and Day 0, +1, +2, +3) provided no extra benefit. Dosing 0, 6, 12 and 18 hours post irradiation also failed to reduce the total diarrhea severity.

6.35. EXAMPLE 35: WOUND REPAIR TEST
In vitro cell culture: The human colon cancer cell line Caco2, HT29 and THP-1 cells were obtained from the American Type Culture Collection (Rockville, MD), HT-29 MTX
were provided by Dr. Lesuffler, INSERM, Dillejuis, France. These cell lines (Caco2, HT-29 and HT-29MTX) were grown as described previously. THP-1 cell lines were grown in RPMI-1640 medium (Life Technologies, Gaithersburg, MD) with 10% fetal bovine serum, 100 units /ml of antibiotics/antimycotics (Life Technologies, Gaithersburg, MD).

An in vitro healing assay was performed using a modified method. Briefly, reference lines were drawn horizontally across the outer bottom of 24-well plates. HT-29 and Caco-2 cells were seeded and grown to confluence, then incubated with media containing 0.1 % FBS
for 24 hours.

Linear wounds were made with a sterile plastic pipette tip perpendicular to the lines on the bottom of the well. Isolated CG53135-05 E. coli purified product (100ng/ml) was then added. The size of the wound was measured at three predetermined locations at various times after wounding (0, 6, 20 and 24 hours). The closure of the wounds was measured microscopically at 20x magnification over time, and the mean percentage of wound closure was calculated relative to baseline values (time 0). To investigate whether the effect of FGF-20 on cell restitution is involved with TGF-R and ITF pathway, anti-TGFR antibody (R&Dsystem, Minneapolis, MN) and polyclonal anti-ITF antibody (a gift from D K Podolsky, Harvard Medical School, Boston, MA) were used.

FIG. 43 shows the effect of FGF-20 in the closure of wounds in various human cell lines.
There is a dose dependent increase in the effectiveness of FGF-20 in the closure of wounds in all the cell lines tested, demonstrating the role of FGF-20 in wound repair.

6.36. EXAMPLE 36: REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION
(RT-PCR) Total RNA from cell lines and the colonic tissue was prepared using TRlzol reagent (Invitrogen) according to the manufacture's instructions. RNA was reverse transcribed using 2pg of total RNA, 15U of RNA inhibitors, lx first strand buffer (Life technologies, Long Island, NY), 5mM

dNTP (Pharmacia, Uppsal, Sweden), 125 pmol random hexamer primers (Pharmacia), and 125 U of Moloney murine leukemia virus RT (Life Technologies) in a final volume of 25u1. The reaction was carried out for 1 hour at 39 C followed by 7 minutes at 93 C and 1 minutes at 24 C and then slowly cooled to 4 C for 20 minutes. PCR was carried out in a volume of 50 NI
containing 5 pl of RT
mixture, 1X Thermos aquaticus (Taq) buffer, 5 pmol of each primer, 2.5mM dNTP, and 1 U of Taq polymerase.

The sequence of primers used were as follows:

human COX-2 sense; 5'-AGATCATCTCTGCCTGAGTATCTT-3' (SEQ ID NO: 62), human COX-2 antisense: 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3' (SEQ ID NO: 63), human Intestinal trefoil factor (ITF) sense: 5'-GTGCCAGCCAAGGACAG-3', (SEQ ID
NO: 64), human ITF antisense: 5'-CGTTAAGACATCAGCCTCCAG-3', (SEQ ID NO: 65), human PPAR-y sense: 5'-TCTCTCCGTAATGGAAGACC-3' (SEQ ID NO: 66), human PPAR-y antisense: 5'-GCATTATGAGACATCCCCAC-3' (SEQ ID NO: 67), human (3-actin sense: 5'-CCAACCGCAAGAAGATGA-3' (SEQ ID NO: 68), human R-actin antisense: 5'-GATCTTCATGAGGTAGTCAGT-3' (SEQ ID NO: 69), mouse COX-2 sense: 5'-GCAAATCCTTGCTGTTCCAATC-3' (SEQ ID NO: 70), mouse COX-2 antisense: 5'-GGAGAAGGCTTCCCAGCTTTTG-3' (SEQ ID NO: 71), mouse ITF sense: 5'-GAAGTTTGCGTGCTGCCATGGAG-3' (SEQ ID NO: 72), mouse ITF antisense: 5'-CCGCAATTAGAACAGCCTTGTG-3' (SEQ ID NO: 73), mouse IL-10 sense: 5'-CTCTTACTGACTGGCATGAGGATC-3' (SEQ ID NO: 74), mouse IL-10antisense: 5'-CTATGCAGTTGATGAAGATGTCAAATT-3' (SEQ ID NO: 75), mouse G3PDH sense: 5'-CGGTGCTGAGTATGTCGTGGAGTCT-3' (SEQ ID NO: 76), mouse G3PDH antisense: 5'-GTTATTATGGGGGTCTGGGATGGAA-3' (SEQ ID NO: 77).

PCR was carried out in a Perkin-Elmer 9600 cycler set for 20- 40 cycles to assess linearity of the amplification. The PCR products were electrophoresed on 2% tris-acetate and EDTA agarose gels containing gel star fluorescent dye (FMC Corporation, Philadelphia, PA).
A negative from the gels was taken with Alphalmager 2000 (Alpha Innotech Corporation, CA) and relative abundance of RT-PCR transcript was assessed by Adobe photoshop 3Ø4 soft ware, normalized to the density of (3-actin and G3PDH transcript.

Expression of some protective genes was also detected by mRNA expression in cell lines or cells isolated from mice (C57BL6) using standard procedures.

COX-2 gene expression in HT29 cell line in the presence of CG53135 was dose dependent, showing highest expression when induced by 100ng/ml of CG53135-05 E. coli purified product (FIG.
44). At this concentration, the gene expression was higher at 1 hour and 3 hour time periods of incubation and decreased thereafter at 6 hour and 24 hour.

COX-2 gene expression in Caco2 cell line was high when stimulated with 100ng/ml of CG53135 as seen in FIG. 45. Increased expression of COX2 was detected at 1, 3 and 6 hours after incubation with 100ng/ml of CG53135 E. coli purified product.

Expression of COX-2 in IEC-6 cell line showed a dose dependent increase in the presence of CG53135 (FIG. 46). Increased expression of COX-2 was detected at 1 hour after incubation with 100ng/ml of CG53135-05 E. coli purified product.

Expression of Intestinal Trefoil factor (ITF) in HT-29 and Caco2 cell lines, in the presence of CG53135, is shown in FIG. 47. Results show dose and time dependent increase in expression of ITF in both HT-29 and Caco2 cells when stimulated by FGF-20. FIG. 48 reiterates that COX-2 is expressed in HT-29 cells. In addition, TGF-(3, ITF, PPAR-y expression is also shown in FIG. 48.

The results presented suggest that CG53135 plays a key role in mucosal repair possibly by inducing COX-2 and ITF genes. Based on the data, that FGF-20 induces TGF-(3 expression, wound repair in Caco-2 cells was tested as described in Example 35, in the presence of anti-TGF-(3 antibody (20 pg/ml). FIG. 49 shows that Epithelial Restitution by XG53135 is mediated in part by TGF-P pathway (p<0.05 vs CG53135 E. coli purified product + anti-TGF-(3).

6.37. EXAMPLE 37: TRANSCRIPTION PATHWAY ASSAYS
Signal transduction was considered a possible mechanism for inducing COX-2 expression in epithelial cells, upon stimulation with CG53135. Various kinases were tested for their expression in the presence of 100ng/ml of CG53135 E. coli purified product, in Caco2 cells. The results indicated that phosphorylated MAPK (p-p38MAPK) was induced in the presence of CG53135, while no other kinase tested, showed any significant induction (FIG. 50(A)). Also IkBa expression demonstrated moderate degradation in the presence of CG53135. In addition, FIG. 50(B) demonstrates that inhibitors of Erk and MAPK decreased COX-2 expression in the presence of FGF-20, in Caco2 cells. Furthermore, expression of kinases was analyzed in THP-1 macrophage cell line, in the presence of 100ng/ml of CG53135-05 E. coli purified product (FIG. 50(C)). The results demonstrated increased expression of phosphorylated STAT3, p-p38MAPK
and SOCS-3 genes. Also FIGs. 50(D)-(E) show increased expression of phosphorylated Elk-1, ATF-2 and minimal induction of phosphorylated Protein Kinase C in Caco-2 cells in the presence of CG53135.
In HT-29 cells, C-Fos and C-Jun were induced, when cultured with CG53135 (FIG.
50(D)-(E)).

6.38. EXAMPLE 38: DOSE RESPONSIVE EFFECTS OF CG53135 IN FEMALE SWISS
WEBSTER MICE WITH DEXTRAN SULFATE-INDUCED COLITIS
The experiments reported in this Example report the results of dose titration experiments in an animal model of inflammatory bowel disease.

Introduction and General Methods Colitis Study Design: Normal female Swiss-Webster mice (Harlan Labs), 6 - 8 weeks old weighing approximately 20 g, were acclimated for 4 days (Day -4 through Day -1) and then given water orally (po) ad libitum containing 5% dextran sulfate sodium (DSS) or control water ad libitum for 7 days (Day 0 through Day 6). DSS (Spectrum Chemicals, Gardena CA) was made as a 5%
solution in tap water; DSS was made every other day and stored at 4 C. Mice were divided into 8 treatment groups including QD doses of 0.3, 1, 3 and 10 mg/kg, and a BID dose regimen of 5 mg/kg per dose (Table 43). On Day 0, daily intraperitoneal (ip) treatments with vehicle (1 M L-arginine in phosphate buffered saline) or CG53135 protein in vehicle were initiated and continued through Day 6. On Day 7, mice were sacrificed with COa.

Table 43: Treatment Groups Treatment Normal Disease CG53135 CG53135 CG53135 CG53135 Disease CG53135 Group Controla ControlbQD QD QD QD QD BiDtrol BID
Group # 1 2 3 4 5 6 7 8 CG 53135 0 0 10 3 1 0.3 0 5 (mg/kg) Number of Test 4 10 10 10 10 10 10 10 Animals a normal control = vehicle only; b disease control = 5% DSS + vehicle Protein production: The CG53135 protein was produced in E. coli as follows:
The cDNA for CG53135-01 was identified and cloned into the pRSET vector (Invitrogen) to provide the vector pETMY-CG53135-01. The gene product of this construct provides a polypeptide incorporating (His)6-(enterokinase cleavage site)-(multicloning site) at the N-terminal end of the polypeptide; in addition, in this construct, the CG53135 sequence begins with the Ala at position 2 FGF-20 (SEQ ID
NO:2). This vector was transformed into Escherichia co/i. The E. coli cells were grown up to 10 L
scale and infected with CE6 phage to produce the recombinant CG53135. The recombinant protein was purified by disrupting the E. coli cells (resuspended in a 1 M L-arginine solution) in a microfluidizer, followed by multiple metal affinity chromatography steps. The final purified protein was dialyzed into phosphate buffered saline containing 1 M L-arginine.

Colon content was scored at necropsy according to the following criteria:
0 = normal to semi-solid stool, no blood observed 1= normal to semi-solid stool, blood tinged 2 = semi-solid to fluid stool with definite evidence of blood 3 = bloody fluid Pathology Methods: Three sections equidistant apart from the distal one third of the colon (area that is most severely affected in this model) were processed for paraffin embedding, sectioned and stained with hematoxylin and eosin for pathologic evaluation.

For each section, submucosal edema was quantitated by measuring the distance from the muscularis mucosa to the internal border of the outer muscle layer.
Inflammation (foamy macrophage, lymphocyte and PMN infiltrate) was assigned severity scores according to the following: Normal = 0; Minimal = 1; Mild = 2; Moderate = 3; Marked = 4; and Severe = 5. Splenic lymphoid atrophy was also scored by the above criteria. The parameters reflecting epithelial cell loss/damage were scored individually using a % area involved scoring method:
None = 0; 1-10% of the mucosa affected = 1; 11-25% of the mucosa affected = 2; 26-50% of the mucosa affected = 3;
51-75% of the mucosa affected = 4; and 76-100% of the mucosa affected = 5.

. Parameters that were scored using % involvement included: (1) Colon glandular epithelial loss - this includes crypt epithelial as well as remaining gland epithelial loss and would equate to crypt damage score; and (2) Colon Erosion - this reflects loss of surface epithelium and generally was associated with mucosal hemorrhage (reflective of the bleeding seen clinically and at necropsy). For each animal, 3 proximal (less severe lesions) and 3 distal (most severe lesion) areas were scored and the mean of the scores for each of the regions was determined.
Group means and % inhibition from disease control were determined. By doing it this way (rather than summing the scores from various sections) one can look at the mean SE for in individual parameter (represented by 3 sections) and equate it to a delineated severity. As an example, if the mean is 4 for gland epithelial loss one knows that 51-75% of the mucosa was devoid of epithelium.

The three important scored parameters (inflammation, glandular epithelial loss, erosion) were ultimately summed to arrive at a sum of histopathology score which indicates the overall damage and would have a maximum score of 15.

One final summation of proximal + distal summed scores was done to reflect the overall total colonic severity score.

Statistics: The mean and standard error (SE) for each treatment group was determined for each parameter scored; the data were compared to the data for the disease controls (Group 2) using a 2-tailed Student's t test with significance at p<_ 0.05.

Live Phase, Necropsy and Organ Weight Results Four animals died during the course of the study (#10 in vehicle control group 2 on day 7, #3 in group 6, 0.3 mg/kg on day 6, #5 in group 8 vehicle control BID on day 7, and #6 in group 7 5 mg/kg BID on day 6).

DSS treatment-related changes in body weight were obvious by day 5 in all DSS
treated mice and ultimately were most severe in animals treated with vehicle (FIG.
51(A)). At study termination, DSS+vehicle controls had a 28% decrease in body weight. A
significant beneficial effect on DSS induced weight loss was seen in mice given AB020858 (CG53135) QD
at all doses (FIG. 51(B)).

Clinical evidence of bloody diarrhea was evident in all DSS+vehicle animals.
At necropsy all DSS controls had blood or blood tinged fluid in the colon. In contrast, mice treated QD with 10 mg/kg AB020858 (CG53135) generally had semi-solid stool and less blood (except animals #5).
Clinical benefit was also evident but less impressive in those given doses of 3 or 1 mg/kg QD and absent in those treated with 0.3 mg/kg (FIG. 51(C)). Mice treated BID with 5 mg/kg had the most impressive clinical benefit (68% inhibition) and clinically these mice had the best overall improvement.

Absolute colon lengths were decreased 41 % in mice treated with vehicle.
Treatment with AB020858 (CG53135) QD at 10 mg/kg resulted in significant (21 %) inhibition of the DSS-induced changes in colon length. Treatment with AB020858 (CG53135) BID at 5 mg/kg reduced the colon length decrease 36%.

Absolute colon weights were decreased approximately 26% in mice treated with DSS in vehicle. Treatment with AB020858 (CG53135) at 10 mg/kg QD or 5 mg/kg BID
resulted in significant reduction of the DSS-induced changes in colon weights.

Absolute spleen weights were increased approximately 40% in mice treated with DSS+vehicle (due to extreme extramedullary hematopoiesis). Spleen weights were significantly greater in all DSS treated animals vs. normal.

Histopathology Findings Significant reduction of colonic inflammation, gland loss, erosion and total histopathology scores occurred in mice treated with AB020858 (CG53135) QD (10 mg/kg) and BID
(5 mg/kg) and was of approximately equal magnitude (FIGS. 52 (A) - (D)).

Splenic lymphoid atrophy (an indication of stress) was inhibited in these same animals 47%
and 46% respectively (FIG. 53). Inhibition of induction of splenic extramedullary hematopoiesis was greater in mice treated BID vs. QD and occurred in all treatment groups (FIG.
54).

Discussion and Conclusions The experiments reported in this Example provide dose-response information for the administration of AB020258 (CG53135). The results indicate that simultaneous administration of AB020258 (CG53135) is effective in inhibiting the appearance of markers of DSS-induced inflammatory bowel disease, especially with the highest doses used.

An additional experiment was carried out in which mice were also treated subcutaneously with CG53135. These studies demonstrate that prophylactic administration of CG53135 at doses of or 10 mg/kg ip and 5 or 1 mg/kg sc significantly reduce the extent and severity of mucosal damage induced by dextran sulfate sodium in a murine model of colitis.

6.39. EXAMPLE 39: EFFECTS OF ADMINISTERING CG53135 TO INDOMETHACIN-TREATED RATS
Treatment of rats with indomethacin results in gross and histopathologic intestinal alterations that are similar to those occurring in Crohn's Disease. The experiments provided in this Example report on the efficacy of CG53135 in treating the rat model of indomethacin-induced intestinal injury. The efficacy of this protein in an alternate model of intestinal injury adds support to the therapeutic potential of CG53135 in treatment of inflammatory bowel disease.

Materials & Methods Protein production: Preparation of CG53135 protein was the same as described in Example 38.

Study Design: Female Lewis rats (Harlan, Indianapolis, IN) weighing 175-200 g were acclimated for 8 days (Day -8 through Day -1). Rats were divided into 8 treatment groups: four groups receiving CG53135 (three groups iv and one group sc), two iv controls for normal and the disease model, and two sc controls for normal and the disease model. On Day -1, treatments with CG53135 or vehicle were initiated and continued through Day 4. CG53135 was injected iv (tail vein) at doses of 5, 1 or 0.2 mg/kg, or 1 mg/kg sc; vehicle controls were injected with BSA (5 mg/mL in PBS + 1 M L-arginine). On Days 0 and 1 rats were treated with indomethacin (Sigma Chemical Co., St. Louis, MO; 7.5 mg/kg doses) in order to induce gross and histopathologic intestinal alterations similar to those occurring in Crohn's Disease. Indomethacin was prepared in 5%
sodium bicarbonate. On Day 5, rats were injected with a single ip dose of 50 mg/kg 5-bromo-2'deoxyuridine (BrdU, Calbiochem, LaJolla, CA) 1 hour prior to necropsy in order to pulse label proliferating cells in the intestine and spleen. Following termination, a 10 cm section of jejunum in the area at risk for lesions was weighed, given a gross pathology score, and then collected into formalin for histopathologic evaluation and scoring of necrosis and inflammation. Blood was collected for CBC
analysis.

Observations and Analysis of Markers of Pathology Gross Observations: Body weight was measured daily beginning on Day 0. At necropsy, liver and spleen weights were measured, and a 10 cm section of jejunum in the area at risk was weighed, scored for gross pathology, and collected into formalin for histopathologic evaluation and scoring of necrosis and inflammation. The area at risk for indomethacin-induced injury was scored at necropsy according to the following criteria: 0 = normal; 1= minimal thickening of the mesentery/mesenteric border of the intestine; 2 = mild to moderate thickening of the mesentery/mesenteric border of intestine, but no adhesions; 3 = moderate thickening with 1 or more definite adhesions that are easily separated; 4 = marked thickening with 1 to numerous hard to separate adhesions; and 5 = severe intestinal lesions resulting in death.

Histopathology: Five sections (approximately equally spaced) taken from the weighed 10cm area at risk of small intestine for indomethacin-induced lesions were fixed in 10% neutral buffered formalin, processed for paraffin embedding, sectioned at 5 pm and stained with hematoxylin and eosin for histopathologic evaluation. Necrosis was scored according to the percent area of the section affected in the same way as described in Example 38 for scoring epithelial cell loss.

Inflammation was scored according to the following criteria: 0 = none; 1=
minimal inflammation in mesentery and muscle or lesion; 2= mild inflammation in mesentery and muscle or lesion; 3 = moderate inflammation in mesentery and muscle or lesion; 4 =
marked inflammation in lesion; and 5 = severe inflammation in lesion.

The means for inflammation and necrosis were determined for each animal, and then the means for each group were calculated.

Statistics. The mean and standard error (SE) for each treatment group were determined for each parameter scored; the data were compared to the data for the disease controls using a 2-tailed Student's T test with significance at p < 0.05.

Results Weight loss was observed in all animals treated with indomethacin. A slight, but significant reduction in weight loss was observed in animals treated with CG53135 (0.2 mg/kg iv) as compared with disease controls (iv). Other doses of CG53135 (both iv and sc routes of administration) provided diminished, but not statistically significant, indomethacin-induced weight loss.

At necropsy, a 10 cro section of jejunum in the area at risk from each animal was weighed.
Indomethacin treatment resulted in an elevation in small intestine weight as compared with normal iv and sc controls, consistent with edema and inflammation associated with this model of intestinal injury. Treatment with CG53135 (1 mg/kg or 0.2 mg/kg iv) resulted in significant reductions in small intestine weight as compared with disease controls (FIG. 55(A)). A slight reduction in the small intestine clinical score was observed, with the greatest benefit occurring with the 1.0 mg/kg iv dose (38%) and the 0.2 mg/kg iv dose (25%); these benefits, however, were not statistically significant.
Relative spleen and liver weights were increased in animals treated with indomethacin.
Administration of CG53135 produced moderate additional increases in these weights.

Hematology: Administration of 2 doses of indomethacin to rats increased the total white blood cell count as a result of increased neutrophils and lymphocytes.
Reductions in red blood cell count, hematocrit, and hemoglobin concentration were also observed. Treatment with CG53135 (5 mg/kg and 0.2 mg/kg iv) resulted in significant reductions in absolute neutrophil counts as compared with disease controls (FIG. 55(B)). Hemoglobin concentration was diminished in the indomethacin controls compared to normal controls, and slightly further diminished in rates treated with CG53135.

Histopathology: Evaluation and scoring of 5 sections of intestine were conducted for each animal. Histologic evidence of a protective effect on the intestine was observed only in animals treated with CG53135 (0.2 mg/kg iv). A 53% reduction in jejunal necrosis and 38% reduction in inflammation score were observed for the 0.2 mg/kg iv CG53135 dose as compared with disease controls iv (FIG. 56). Photomicrographs of affected small intestine are shown in FIG. 57 for a normal and disease control, and a rat treated with 0.2 mg/kg CG53135. Panel A
shows the small intestine from a normal control animal treated iv with vehicle (BSA). Normal villous architecture and mesentery (arrow) are apparent. Panel B presents a photomicrograph of the small intestine from an indomethacin- treated rat, with vehicle (BSA) iv. Focal mucosal necrosis extending across most of the area associated with attachment of the mesentery is apparent (see, for example, the asterisks at upper right intestinal wall and lower right intestinal wall). Marked inflammatory cell infiltrate is present in the mesentery (arrow). Panel C shows the image of the small intestine from an indomethacin-treated rat further treated with CG53135, 0.2 mg/kg iv. There is no apparent necrosis, in contrast to the disease control shown in Panel B. There is a focal area of attenuated villi and cellular infiltration into muscle layer (see, for example, the three asterisks at the upper right, right and lower right of the intestinal wall). Mesentery (arrow) is infiltrated by inflammatory cells. The photomicrographs in FIG. 57 provide further support for the protective effect of 0.2 mg/kg iv CG53135.

BrdU labeling was carried out by injecting 50 mg/kg 1 hour prior to necropsy.
In the small intestine from a normal control animal, normal pattern of crypt labeling is seen at 100X (FIG. 58, Panel A). BrdU incorporation in the disease model was decreased or absent in epithelial cells in mucosal areas of necrosis, but increased in subajacent inflammatory tissue in which fibroblast labeling was prominent (FIG. 58, Panel B, visualized at 50X). Focal mucosal necrosis (arrow) is delineated by an absence of BrdU immunostaining as well as severe infiltration of inflammatory cells and fibroblast proliferation. Small intestine from a rat treated with indomethacin + CG53135 0.2 mg/kg iv shows an absence of crypt labeling, but relatively intact mucosa (arrow in FIG. 58, Panel C, visualized at 50X). Subadjacent smooth muscle and mesentery is only mildly infiltrated with inflammatory cells, compared with that seen in the disease control (Panel B).
In certain animals treated with CG53135, in which preservation of mucosal integrity occurred, increased crypt labeling was also observed; this is in the direction found in the normal control.

The results of the experiments in this Example may be summarized as follows:
treatment of rats with indomethacin results in gross and histopathologic intestinal alterations that are similar to those occurring in Crohn's Disease. Administration of CG53135 (0.2 mg/kg iv) to indomethacin-treated rats resulted in significant reductions in weight loss, small intestine weight, absolute neutrophil counts, and jejunal necrosis and inflammation scores. Higher doses of CG53135 (5, 1 mg/kg iv and 1 mg/kg sc) were less efficacious. The morphological appearance of tissues collected from animals injected with BrdU 1 hour prior to necropsy suggested that the beneficial effects of CG53135 in this model of intestinal injury were the result of mucosal protection rather than a proliferative effect on target cells.

6.40. EXAMPLE 40: THERAPEUTIC ADMINISTRATION OF CG53135 ENHANCES
SURVIVAL IN THE MURINE DSS MODEL.
In the experiments described above, DSS exposure and CG53135 administration were initiated simultaneously on day 0. In the present Example, the effect of CG53135 administered after the initiation of DSS treatment was examined. CG53135 was prepared as described in Example 38.
Balb/c mice were exposed to DSS for 7 days (day 0 to day 6). The mice were injected daily subcutaneously with various concentrations of CG53135 (5, 1 and 0.2 mg/kg) beginning on the fifth day of DSS exposure (i.e. day 4) and ending 3 days after the termination of DSS exposure (i.e. day 9), or with vehicle only. Animal survival was recorded on a daily basis and the experiment was concluded on day 10.

As shown in FIG. 59, therapeutic administration of CG53135 at 5 mg/kg enhanced survival relative to the disease control group. Thus, while only 44% (4 of 9) of the animals in the disease control group survived until the end of the study, 89% (8 of 9) of the animals treated with CG53135 at 5 mg/kg survived.

6.41. EXAMPLE 41: EFFECTS OF CG53135 IN AN IMMUNE-MEDIATED MODEL OF
INFLAMMATORY BOWEL DISEASE IN IL-10 DEFICIENT MICE (IL-10 KNOCK-OUT
MICE) The objective of the study was to assess the ability of CG53135 to therapeutically inhibit the inflammation that occurs in IL-10 deficient mice when transferred from a germ free to a specific pathogen free environment. As inflammatory bowel disease is thought to have an immune component, this study evaluated the efficacy and safety of CG53135 in this immune-mediated model of IBD when dosed therapeutically at the time of significant disease.

Table 44: Materials and Methods Species/strain: IL-10 Knock-out Mice (mixed C57BL X 129 Ola background) Ph siolo ical state: Germ-free Age/weight range at start of stud : -8-12 weeks old weighing a proximatel 20-25 g Test Article: CG53135-05 (FGF-20) protein (purity >97%) in 20% glycerol buffer.

Storage Conditions of test article: All tubes were stored at -70 C until ready for use.

Vehicle: Glycerol buffer: 20% Glycerol, 200mM Sorbitol, 1 mM EDTA, 100mM
Citrate, 50mM KCL
Storage Conditions of vehicle: All tubes were stored at -70 C until ready for use.

Table 45: Administration of Test Article Route and method of administration: Intraperitoneal (ip) Justification for route of administration: This route and dose has been used in previous studies with CG53135 in other murine models of colitis.
Frequency and duration of dosin : Once daily Administered doses of CG53135-05: 5.0 mg/kg in 20 % glycerol buffer Administered volume: 0.3 mL per mouse Justification for dose levels: Similar doses have been used in other efficacy models.
Table 46: Experimental Study Design Group Number of Animals Dose Volume Number Treatment Females Males (mg/kg) mUk 1 Normal controla 4 4 0 10 2 Disease controlb 4 4 0 10 3 5 mg/kg CG53135 (14d 4 4 5 mg/kg 10 therapeutic) a Normal control: mice are untreated and maintained in germ free conditions throughout study b Disease control: vehicle administered ip, once daily using the therapeutic dosing regimen.

Table 47: Study Schedule Stucdy Schedule Event Day of Study Transfer from germ free cages x Fecal slurry~ ~o x Therapeutic CG53135 (iR) x x x x x x x x x Therapeutic Vehicle (io x x x x x x x x x Body weight x x xx x x x x x x xx x xx xxx Serum collected for CG53135 x x antibodies Tissues collected (including x x colon assessments) Scheduled terminations x x a Mice are dosed orally with a slurry of fecal contents solubilized in PBS
from donor SPF
documented free of Heliobacter.
b CG53135-05 or vehicle will be administered daily through to day prior to scheduled termination.
Experimental Procedures Mice were acclimated for 2 days before bacterial colonization and given autoclaved food and tap water ad libitum during this time. Mice will be treated with CG53135-05 E. coli purified product or buffer for 2 days beginning the day of transfer, then colonized with specific pathogen free bacteria by swabbing their mouth and rectum with solubilized fecal material.
Animals were examined prior to initiation of the study to assure adequate health and suitability. Animals that were found to be diseased or unsuitable will not be assigned to the study.

This study was performed in two segments of approximately 20 animals each due to animal availability and the tedious collections of cells and tissues at necropsy for T cell stimulation and colonic strip culture. The two study segments had mice evenly assigned to all dose groups (e.g., 2 animals per sex per treatment group). If the number of available animals at the time of initiation is not evenly divided between males and females, animals were assigned to groups to balance males and females as best as possible.

Clinical Observations/Signs Mice were observed daily for significant clinical signs of toxicity, moribundity and mortality approximately 60 minutes after dosing.

Body Weight: Individual body weights of all mice were recorded pretest (for randomization) and daily through Day 10. Body weights taken on the day of necropsy for animals scheduled for termination was used for determination of organ to body weight ratios.
Following are the organs/tissues measured.

Table 48: Organs/Tissues For Weight Measurement Cecum Kidneys Rectum Colon Liver Spleen Histopathology: All animals surviving to scheduled termination (Day 10) will be terminated using CO2 with assessment of gross observations, organ weights and collection of all scheduled tissues into 10% neutral buffered formalin for histopathologic evaluation.

Special colon assessments: From the areas at risk (cecal tip, transverse colon and rectum), 3 sections approximately 1 cm apart in length will be collected, preserved in formalin, and stained to quantitate inflammation (hematoxylin & eosin), mucin (periodic acid schiff), and collagen (trichrome).
All 3 sections should be representative of the affected area.

Tissues taken from the colon, will be collected and processed for paraffin embedding, sectioned and stained as noted above. Histopathology will be performed in a blinded manner on the tissue samples of cecum, transverse colon and rectum with assignment of an inflammation score ranging from 0 to 4, where: 0 = no inflammation; 1= mild inflammation with increased mononuclear cells infiltrating, mild crypt hyperplasia; 2 = more active inflammation with increased infiltrating mononuclear cells, mild goblet cell depletion, and mild crypt hyperplasia; 3 = active inflammation with crypt hyperplasia, goblet cell depletion and marked mononuclear cell infiltration;
and 4 = severe active inflammation characterized by widespread infiltrate of neutrophils, ulceration, crypt abscesses and marked mucosal hyperplasia.

Following were the organs/tissues considered for macroscopic examination and histopathology.

Table 49: Organs/Tissues For Histopathology Evaluation*

Cecum Eyes Rectum Colon Kidneys Spleen Liver *AII tissues except eyes to be fixed in 10% neutral buffered formalin, eyes to be fixed in 6%
glutaraldehyde.

Preparation of cells and cell cultures: Colonic strip cultures were established from the remaining colon fragments, pooled from segments of proximal, middle and distal colon. Colon segments were flushed with phosphate-buffered saline (PBS) to remove fecal contents, opened lengthwise and cut into 0.5 to1.0 cm pieces and shaken vigorously in PBS.
Approximately 50 to100 mg of tissue was then distributed per well of a 24 well tissue culture plate in duplicate and cultured in 1 mL of complete medium containing antibiotics and an antimycotic agent (Veltkamp et al, 2001, Gastroenterology, Vol 120(4):900-913). After incubation at 37 C for 18 - 24 hours, culture supernatants were collected in aliquots and frozen at -70 C for cytokines and possibly immunoglobulin measurements. IgG2a and IL-12 in the supernatant were measured by ELISA.

Mesenteric lymph nodes (MLN) was mechanically dispersed, washed, counted and used for cecal bacterial lysate-stimulated interferon gamma measurement as described by Veltkamp et al, 2001, Gastroenterology, Vol 120(4):900-913. Briefly, CD4+ T lymphocytes were isolated by negative MACS selection and incubated with antigen-pulsed antigen presenting cells derived from wild type mice splenocytes after T cell removal. Alternatively, unfractionated MLN cells were incubated with antigen.

Cytokine assays: IL-12 (Pharmingen, San Diego, CA), TNF-a and IFN-y (R&D
systems, Minneapolis, MN) was measured in MNL cell and splenocyte culture supernatants by ELISA.
Moreover, IL-12 and PGE2 (Assay Design, Ann Arbor, MI) was measured in supernatants of colon cultures using standard ELISA protocol. Concentrations of these cytokines and PGE2 were measured in duplicate culture supernatants by comparison with standard curves generated using recombinant cytokines.

Results Body Weight and Histopathology - Prophylactic: Weight change in the prophylactic group was assessed. FIG. 60(A) shows weight change when challenged with FGF-20 in IL-10 knock-out (KO). FIG. 60(A) also shows the histopathology of the colon when the mice are challenged with different concentrations of FGF-20 (0.2, 1, 5 mg/kg). The results indicate that, administration of FGF-20 had a protective effect as compared to the vehicle control. FIG. 60(B) further demonstrates that, upon administration of FGF-20, there is a dose-dependent decrease in the total Cecal Histologic score, as compared to the vehicle (12.2 2.3 vs. 2.5 0.6;
p<0.001).

Cytokine Production - Prophylactic: Cytokine production was assayed by ELISA.
FIGS.
61(A) (IL-12), 61(B) (IFNy) and 61(C) (PGE2) indicate that FGF-20 altered cytokine production in MLN, colonic strip culture, Spleen cell culture, which were prepared from the IL-10KO mice as described above. FIG. 62 also shows FACS analysis of total MLN number (32 3.4 vs. 23 2.5;
p<0.05), CD4+ and CD8+ and CD4+CD69+ cells (3.2 0.3 vs. 1.67 t 0.1 ; p<0.05).

Body Weight and Histopatho%gy- Treatment: Study protocol for the treatment group was established by treatment of established colitis in ex-germ free IL-10 -/- mice colonized with SPF
bacteria on day 1. On day 10, treatment was started by intraperitoneally administering either Vehicle or FGF-20 (5mg/kg) and necropsy was performed on day 17.

FIG. 63 shows the Weight change in the treatment study, where FGF-20 (5mg/kg) was administered to IL-10 KO mice. Histology of the cecum and rectum are respectively shown in FIGS.

64 and 685, that demonstrates protective effect of FGF-20. Cecal histologic score shows that FGF-20 decreased as compared to the vehicle control (13.1 1.8 vs. 5.9 1.4;
p<0.006, FIG. 66).

Cytokine production in treatment group as assayed by ELISA demonstrated that administration did not significantly alter the cytokine production in Gut culture and unseparated splenocytes of IL-10 fCO mice. IL-12, IFN-y, TNF-a and PGE2 were the cytokines that were assayed. FIG. 67 shows FACS analysis of MLN number, CD4+ and CD8+ and CD69+
cells, all of which were decreased in FGF-20 treated group as compared to the vehicle treatment.

Results in normal mice: Expression of COX-2, IL-10, ITF, TGF-(3 were analyzed in normal wild type (WT) C57BL6 mice following 7 days of injection FGF-20 (5mg/kg). RT-PCR was performed (as described in Example 36) in colonic tissue and unseparated MLN
to study the expression of the above list of genes. COX-2, IL-10, ITF and TGF-(3 are upregulated in the colonic tissue of WT mice, upon administering FGF-20. In unseparated MLN, IL-10 expression is found to be upregulated as compared to vehicle.

6.42. EXAMPLE 42: EFFECT OF CG53135-05 IN A CHRONIC 2 WEEK MURINE MODEL
OF DSS-INDUCED ULCERATIVE COLITIS (N-404) Female Swiss Webster mice exposed to dextran sulfate sodium (DSS) for 7 days develop inflammation and gland loss with erosion in the colon. Gross and histopathologic changes resulting from this treatment resemble those occurring in human ulcerative colitis (UC), a subset of inflammatory bowel disease (IBD) (see Animal Models of Intestinal Erosion in "Inflammatory Bowel Disease" ed. MacDermott RP and Stenson WF. Elsevier, New York (1992); Okayasu et al., Gastroenterology 98:694-702 (1990); and Cooper et al., Lab Invest. 69:238-249 (1993)).
Compounds that are effective in the treatment of human IBD have activity in this model and it is being used to investigate potential new therapies (see Axelsson et al., Aliment Pharmacol Ther.
12:925-934 (1998); Egger et al., Digestive Dis Sci. 44:436-444 (1999); Miceli et al., J Pharmacol Exp Ther. 290:464-471 (1999); and Jeffers et al., Gastroenterology 123 (4):1151-62 (2002)). However, the 7-day version of the model, in which exposure to DSS is continuous, is only useful for evaluating the effects of agents on the acute phase of mucosal inflammation and damage. A
more chronic model of ulcerative colitis, with less potential for lethality, was developed to test CG53135-05 for its capacity to enhance mucosal repair. The objective of this study was to assess the potential therapeutic activity of CG53135-05 in the chronic 2 week murine model of ulcerative colitis induced by administration of 3% (DSS) for 5 days and 1% DSS for 9 days.

Materials and Methods:

Colitis was induced in female Swiss Webster mice by exposure to 3% DSS in the drinking water (ad libitum) on study Days 1-5 and maintained by exposure to 1% DSS in the drinking water (ad libitum) on study Days 6-15. Mice were randomly assigned to 4 groups of 15 animals (Table 50). Mice were exposed to 3% DSS in drinking water on test Days 1 through 5 and 1% DSS on test Days 6-15, with concurrent intraperitoneal (IP) treatments of vehicle or CG53135-05 at 0.33, 1.67 and 3.33 mg/kg (UV) Days 6-14 after disease was established. Mice were weighed daily. On Day 15,animals were terminated by cervical dislocation, the colon length was measured, and colon content was scored. Tissues were collected into 10% neutral buffered formalin and processed routinely for histopathology.

Table 50:Study Design 3% DSS 1% DSS
Treatment Treatment CG53135-05 Group Number of (water po), (water po), Treatment CG53135-05 Treatment Number Animals* Days 1-5 Days 6-15 (mg/kg, UV) Schedule (IP) 1 15 females Yes Yes 0 Vehicle only, Days 6-14 2 15 females Yes Yes 0.33 Once daily, Days 6-14 3 15 females Yes Yes 1.67 Once daily, Days 6-14 4 15 females Yes Yes 3.33 Once dail , Days 6-14 *Acclimation of animals at least I week prior to randomization and start of treatment Clinical Parameters and Gross Pathology: Only animals that survived the duration of the study were included in the analysis of body weight change, terminal colon lengths, and colon content scores. At necropsy, the colon length was measured and assessed for evidence of stool consistency changes. Colon content was scored at necropsy according to the following criteria: 0 Normal (firm, well formed stool); 1= Semi-solid stool; 2 = Semi-solid to fluid stool; and 3 = Semi-solid with definite evidence of blood, bloody fluid or no content.

Histopathology: At necropsy, the colon was harvested and divided into 2 approximately equal segments, proximal and distal, to assess regional changes induced in this model. Distal ends were marked to maintain orientation. These colon segments were collected, preserved in 10%
neutral buffered formalin, and routinely processed for histopathologic evaluation. During processing for histology, the proximal and distal colon segments were each trimmed to obtain 4 equally spaced segments, and hematoxylin and eosin (H&E) stained slides were prepared for each. Both proximal and distal tissues were examined as these tissues are affected to different degrees of severity in this model (more severe and less variable symptoms predominate in the distal colon).

For each colon section, submucosal edema was quantified by measuring the distance (mm) from the muscularis mucosa to the internal border of the outer muscle layer.
The extent of inflammation (foamy macrophages, lymphocytes and polymorphonuclear cell infiltrate) was assigned severity scores according to the following ranking: 0 = Normal; 1 = Minimal; 2 = Mild; 3 = Moderate;
4 = Marked; and 5 = Severe.

The parameters reflecting epithelial cell loss/damage were scored individually using a percent area involved scoring method. Parameters that were scored using the percent involvement scale included colon glandular loss and colon erosion.

0 = None 1= 1-10% of the mucosa affected 2 = 11-25% of the mucosa affected 3 = 26-50% of the mucosa affected 4 = 51-75% of the mucosa affected 5= 76-100% of the mucosa affected Mucosal epithelial hyperplasia (basophilia, mitotic figures, multilayered on basement membranes, absence of goblet cells) was scored 0-5 based on the following criteria O=Normal 1=Minimal-small foci generally adjacent to the inflammatory changes 2=Mild-11-25 /a of mucosa affected 3=Moderate-26-50% of mucosa affected 4=Marked-51-100% of mucosa affected 5=Severe-51-100% of mucosa affected plus papillary proliferation into lumen Mucosal thickness (an indicator of proliferative changes or edematous inflammatory mucosal expansion) was measured by placing an ocular micrometer at the base of the glands and the overlying surface epithelium in a non-tangential area of section representative of the thickest areas of mucosa.

The scores for proximal tissues and distal colon were averaged to determine a score for the entire colon for each parameter evaluated. The 3 scored parameters (i.e., inflammation, glandular loss, and surface epithelial erosion) were combined to arrive at a sum of overall histopathology scores for proximal or distal sections of the colon, and then proximal and distal overall scores were averaged to arrive at an overall histopathology score for the entire colon.
These summations indicate the overall damage in the distal, proximal, or entire colon and would have a maximum severity score of 15.

Additional tissues (cheek, tongue, esophagus, mid colon and rectum) were collected and divided into two equal sections. One section was snap frozen in liquid nitrogen, the other was placed in 10% neutral buffered formalin for paraffin embedding and immunohistochemical staining with Ki67 antibody. The formalin preserved tissues were trimmed into approximately 0.5 cm sections (1 section/tissue), processed through graded alcohols and a clearing agent, infiltrated, embedded in paraffin and sectioned. Slides were then re-hydrated and stained with mouse Ki67 antibody (Dako) at 1:70 dilution followed by streptavidin/HRP detection system with a DAB
peroxidase indicator and counterstained with Gill's Hematoxylin. The slides were exposed to diaminobenzadine (DAB) for four minutes to provide optimal specific staining with minimal background nonspecific staining.

Statistics: Differences in body weight changes between treatment groups were analyzed with repeated measures ANOVA with a Greenhouse-Geisser correction for sphericity, followed by pair-wise repeated measures analysis to identify the source of any variation.
These pair-wise measures were performed between each day and the normal control. Quantitative measurements of pathology (colon length, edema) were analyzed with a one-way analysis of variance (ANOVA) followed by a linear contrast. Qualitative measures of pathology (histology scores) were analyzed as follows: Pathological evaluations were done at several points along the proximal and distal colon. These replicates were converted to a mean pathology score and a maximal pathology score for each individual to correct for problems of pseudo-replication. The mean and maximal scores were analyzed with a Kruskal-Wallis test followed by a Dunn's multiple comparison test between each day and normal control animals. In all other cases, multiple comparison test results were adjusted for the number of comparisons being performed using a Bonferonni correction. In the case of pathology scores, the comparisons were effectively doubled, by analyzing the mean and the maximum values.

Results:

Fifty-nine of the sixty mice survived the duration of the study and were included in the analysis of body weight loss, terminal colon lengths, and colon content.
Decreased body weight gain was seen in all groups with the most severe effects occurring on Day 9 and recovery of gain beginning on Day 10. A dose-dependent trend in inhibition of DSS-induced body weight loss was observed, corresponding to 19, 35 and 67% reduction in weight loss for groups receiving IP
injections of 0.33, 1.67 or 3.33 mg/kg (UV) CG53135-05, respectively. Gross pathology evaluation indicated that colon length decreases and colon content score increases were greatest in the vehicle-treated disease control group. Dose-responsive increases in colon length were seen in all groups treated with CG53135-05 IP, with the greatest effect (29% inhibition, statistically significant) occurring in mice administered 3.33 mg/kg (UV) CG53135-05. Colon scores were also significantly improved (21-29%) in mice treated with 1.67 or 3.33 mg/kg (UV) CG53135-05.

Disease vehicle-treated control mice all had lesions of colitis with minimal to severe inflammation and gland loss. Erosion and gland hyperplasia were evident in 14 of 15 mice with these changes being greatest in the distal colon. Edema was sporadically seen, primarily in the distal colon. Distal colon mucosal thickness was increased approximately 130%
in diseased controls as compared to normal controls, as a result of inflammation and hyperplasia. In contrast, dose-responsive inhibition of proximal, distal, and total colon inflammation and gland loss as well as summed total colon histopathologic scores was seen. Furthermore, mice that received 3.33 mg/kg (UV) CG53135-05 showed significant improvements in on (35%) and total (34%) inflammation, distal (38%) and total (37%) gland loss as well as distal (38%) and total (37%) summed histopathologic scores. Protective effects (46-62% inhibition, non-significant) of CG53135-05 were also seen on proximal, distal and total colon erosion in the mid and high dose groups.
Beneficial effects (dose responsive, non-significant) on inhibition of mucosal thickness changes and hyperplasia scores occurred with 39% inhibition of the total mucosal thickness in mice treated with 3.33 mg/kg (UV) and 31 % improvement in the hyperplasia score.

Conclusions:
In summary, treatment with CG53135-05 IP, once daily at doses of 1.67 or 3.33 mg/kg (UV) resulted in mild to moderate, dose-responsive, inhibitory effects on gross and histopathologic parameters in the 15-day chronic model of DSS-induced colitis, when treatment was initiated after disease was established. Colon length, mean total gland loss, distal colon mean histology summed score, max distal and total inflammation, and max distal and total gland loss showed a significant linear dose response and were significantly different between animals in the vehicle-treated control group and the group treated with 3.33 mg/kg (UV) CG53135-05.

In another experiment, administration of 3.33 mg/kg (UV) of CG53135-05 IP, once every other day on Days 6, 8, 10, 12, and 14 (q2d) significantly reduced the severity of chronic DSS-induced ulcerative colitis in female Swiss Webster mice (N-405 study). These results confirm the findings presented above.

6.43. EXAMPLE 43: TREATMENT OF STROKE
Thirty male Sprague Dawley rats were allocated to treatment groups as indicated in the study design Table 51 below.

Table 51: Experimental Design F__7 Number of Dose * Volume *
Animals Treatment Males (pg) (uL) Vehicle 1 10 0 50 CG53135-05 E. 10 1 50 coli purified product CG53135-05 E. 10 2.5 50 coli purified product *Administered dose and volume is based on an average bodyweight of 330 g.
Experimental Procedures Middle cerebral artery (MCA) Surgery and Intracisternal Injections: Animals were handled for 7 days prior to surgery. Cefazolin sodium (40 mg/kg, i.p) was administered on the day before surgery and just after surgery. At the time of surgery, the rats were anesthetized with 2% halothane in a 2:1 N20 :02 mixture. Body temperature was maintained at 37 0.50 C. The proximal right MCA was electrocoagulated from just proximal to the olfactory tract to the inferior cerebral vein and was then transected. For intracisternal injections, animals were re-anesthetized as above and placed in a stereotaxic frame. Rats were given CG53135-05 E. coli purified product or vehicle (40mM acetate, 200 mM mannitol (pH 5.3)) by percutaneous injection into the cisterna magna, once at 1 day, (approximately 24 hours) and once at 3 days, (approximately 72 hours) after MCA.
Animals were given test article (2 dose groups) or vehicle treatment according to the study design.
Clinical Observations/Signs Animals were observed immediately over a 1 hour period following injections for signs of seizure (indicated by tremor and violent motion about the cage), pain (indicated by loud vocalization), and lethargy. Animals were also observed daily for mortality and moribundity.

Body Weight: Animals were weighed on Days 1, 3, 7, 14 and 21.

Limb Placing Test: limb placing tests were carried out on all animals on Day -1 (pre-operation), Day 1 Qust prior to injection), Day 3 and then every 7 days thereafter (Days 7, 14, 21).
Forelimb Placing TestAssessment Score: The forelimb placing test measures sensorimotor function in each forelimb as the animal places the limb on a table top in response to visual, tactile, and proprioceptive stimuli. The forelimb placing test consists of the following evaluations and scoring, where the combined total score for the forelimb placing test reflects a range from 0 (no impairment) to 10 (maximal impairment):

visual placing (forward, sideways): 0 - 4 tactile placing (dorsal, lateral): 0 - 4 proprioceptive placing: 0 - 2 Total score for all forelimb tests: 0 - 10 Hindlimb Placing TestAssessment Score: Similarly, the hindlimb placing test measures sensorimotor function of the hindlimb as the animal places it on a tabletop in response to tactile and proprioceptive stimuli. The hindlimb placing test consists of the following evaluations and scoring, where the combined total score for the hindlimb placing test reflects a range from 0 (no impairment) to 6 (maximal impairment):

tactile placing (dorsal, lateral): 0 - 4 proprioceptive placing: 0 - 2 Total score for all hindlimb tests: 0 - 6 Body Swing Test the body swing test was carried out on all animals on Day -1 (pre-operation), Day I Qust prior to injection), Day 3 and then every 7 days thereafter (Days 7, 14, 21).
The body swing test examines side preference as the animal is held approximately one inch above the surface of the table, and swings to the right or the left side.
Thirty swings are counted, and the score is then calculated based on the percentage of swings to the right. (score range =
-50% right swing (no impairment) - 0% right swing (maximal impairment)) Cylinder Test: the cylinder test was carried out on all animals on Day -1 (pre-operation) and 7 days thereafter (Days 7, 14, 21). The cylinder test measures spontaneous motor activity of the forelimbs. Animals are placed in a narrow glass cylinder (16.5 x 25 cm) and videotaped for 5 min on the day before stroke surgery and at weekly intervals thereafter.
Videotapes are then scored independently by one experienced observer and up to 50 spontaneous movements will be counted min per rat per day). Spontaneous movements include those made by each forelimb to initiate rearing, to land on or to move laterally along the wall of the cylinder, or to land on the floor after rearing.

Macroscopic and Histomorphology. on the day of scheduled termination (Day 3), animals were euthanized by an intraperitoneal injection of Chloral hydrate (500mg/Kg).
Brains were examined grossly and removed, postfixed in formalin, dehydrated and embedded in paraffin.
Coronal sections (5 mm) will be cut on a microtome mounted on to glass slides, and stained with hematoxylin/eosin (H&E). The area of cerebral infarcts on each of seven slices (+4.7, +2.7, +0.7, -1.3, -3.3, -5.3, and -7.3 mm compared with Bregma) was determined using a computer interface imaging system using the indirect method (area of the intact contralateral hemisphere - area of the intact ipsilateral hemisphere) to correct for brain shrinkage during processing. Infarct volume was then expressed as a percentage of the intact contralateral hemispheric volume.
Volumes of the infarction in the cortex and striatum were also determined separately using these same methods.
H&E stained section was examined for histological changes such as hemorrhage, abscess or tumor formation.

Statistical Analysis: all intracisternal injections, behavioral testing, and subsequent histological analyses were done by investigators blinded to the treatment assignment of each animal. Data are then expressed as means +/- SEM, and will be analyzed by one or two way (ANOVA) followed by appropriate pairwise post hoc tests with correction for multiple comparisons.
Results Forelimb Placing Test: on days-1, 1, 3, 7, 14, and 21 relative to MCA
occlusion, animals were examined by using a limb placing test to assess sensorimotor function in the forelimb in response to visual, tactile and proprioceptive stimuli (Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. & Finklestein, S. P. (1997) Proc.
Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H., Van Deuren, B. &
Clincke, G.
(1992) Brain Res. 573, 44-60.) Visual placing (scored 0-4), tactile placing (scored 0-4), and proprioceptive placing (scored 0-2) were summed to generate a range of potential total scores from 0 to 12, with 12 representing maximal impairment (FIG. 68 (A)).

Hindlimb Placing: on days -1, 1, 3, 7, 14, and 21 relative to MCA occlusion, animals were examined by using a limb placing test to assess sensorimotor function in the hindlimb in response to tactile and proprioceptive stimuli (Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R.
R., Benowitz, L. I. & Finklestein, S. P. (1997) Proc. Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res. 573, 44-60). Tactile placing (scored 0-4), and proprioceptive placing (scored 0-2) were summed to generate a range of potential total scores from 0 to 6, with 6 representing maximal impairment (FIG. 68(B)).

Body Swing Test. On days -1, 1, 3, 7, 14, and 21 relative to MCA occlusion, animals were examined by using a body swing test to assess side preference as the animal is held approximately one inch above the surface of the table, and swings to the right or the left side. (Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. &
Finklestein, S. P. (1997) Proc.
Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res. 573, 44-60.) Thirty swings were counted, and the score calculated based on the percentage of swings to the right (FIG. 68(C)).

Cylinder Tesfi. On days -1, 1, 3, 7, 14, and 21 relative to MCA occlusion, animals were examined by cylinder test to assess spontaneous motor activity of the forelimbs (Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. &
Finklestein, S. P. (1997) Proc.
Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res. 573, 44-60.) Briefly, animals are placed in a narrow glass cylinder (16.5 x 25 cm) and videotaped for 5 min on the day before stroke surgery and at weekly intervals thereafter. Videotapes are then scored independently by one experienced observer and up to 50 spontaneous movements will be counted (- 5 min per rat per day).
Spontaneous movements include those made by each forelimb to initiate rearing, to land on or to move laterally along the wall of the cylinder, or to land on the floor after rearing (FIG. 68 (D)).

Body Weight: animals were weighed on days -1, 1, 3, 7, 14, and 21 relative to MCA
occlusion and the results indicate no significant difference between the vehicle and CG53135-05 treatment (FIG. 68(E)).

Conclusion Administering CG53135 following MCA occlusion suggested that both the low and high doses produced a significant enhancement of recovery on forelimb (FIG. 68(A)) and hindlimb placing tests (FIG. 68(B)) for the contralateral (affected) limbs, and improvement on the body swing test (FIG. 68(C)). This pattern of activity with other therapeutics in this model has generally been shown to reflect improvement in cerebrocortical and subcortical (striatal) function, respectively (Dijkhuizen RM, Ren J, Mandeville JB, Wu 0, Ozdag FM, Moskowitz MA, Rosen BR, Finklestein SP.
2001, Proc Nati Acad Sci U S A 98(22):12766-71). No apparent differences were seen on the cylinder test (FIG. 68(D)) of spontaneous limb use or on animal body weight (FIG. 68(E)).

Therefore, CG53135 administration will be useful in the treatment of pathological conditions including ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning and neurodegenerative diseases (such as Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease).

6.44. EXAMPLE 44: MATRIX METALLOPROTEINASE PRODUCTION ASSAY
The matrix metalloproteinases (MMPs) are a family of related enzymes that degrade the extracellular matrix in bone and cartilage. These enzymes operate during normal development in tissues differentiation and remodeling. In arthritic diseases, such as Osteoarthritis (OA) and Rheumatoid Arthritis (RA), elevated expression of these enzymes contributes to irreversible matrix degradation. Thus, effect of CG53135 on MMP production was assayed.

The activity of CG53135 on matrix metalloproteinase (MMP) production was assessed using the SW1353 chondrosarcoma cell line (ATCC HTB-94). This cell line is a well-established chondrocytic cellular model for matrix metalloproteinases (MMP) production.
SW1353 cells were plated in a 24-well plate at I x105 cells/mI (1 ml) in DMEM medium-10 % FBS.
Following overnight incubation, the medium was replaced with DMEM + 0.2 % Lactabulmin serum.
CG53135-05 E. coli purified product was added to the wells at doses ranging from 10 to 5000 ng/ml, in the absence or presence of IL-1 beta (0.1 to I ng/ml, R&D systems Minneapolis, MN), TNF-alpha (10 ng/ml, R&D
systems) or vehicle control to a final volume of 0.5 ml. IL-1 beta and TNF-alpha are both potent stimulators of MMP activity. All treatments were done in triplicate wells.
After 24 hours, the supernatants were collected and Pro- MMP-1, and -13, as well as TIMP-1 (tissue inhibitor of matrix metalloproteinase), a natural inhibitor of MMP activity, was measured by ELISA
(R&D systems). The measurements were normalized to the number of cells by an MTS assay.

Results CG53135 significantly decreased MMP-13 production in the presence of either IL-1 beta or TNF-alpha as demonstrated in FIG. 69(A) and FIG. 69(B), respectively. IL-1 beta and TNF-alpha are both potent stimulators of MMP activity. MMP-13 affinity for type II
collagen, the main collagen that is degraded in OA, is ten times higher that of MMP-1. Since MMP-1 3 expression increases in OA and RA, the decrease of MMP-1 3 observed with addition of CG53135 indicates that the protein can be used as an OA and RA therapeutic. Furthermore, CG53135 up-regulated the production of TIMP-1, a natural inhibitor of MMP activity (FIG. 69(C)). This enhancement of TIMP-1 production by CG53135 is beneficial in reducing the matrix breakdown by MMP-1 and -13 observed in OA and RA. In addition, CG53135 had no effect on MMP-3 production constitutively or after IL-1 induction.
Similarly, CG53135-05 E. coli purified product showed increase in basal expression of MMP-1 in SW1353 cells.

6.45. EXAMPLE 45: EFFECT OF CG53135 ON NORMAL RATS: PROOF OF PRINCIPLE
TO THE MENISCAL TEAR MODEL
The effect of CG53135 on the normal rats was studied as a proof of principle to drive further studies in disease model (ex: meniscal tear model of osteoarthritis in rats).
The effect of CG53135 on synovium and cartilage was assessed by injecting the protein into normal male Lewis rats.

Effects of Intra-articular iniection of CG53135-05 E. coil Purified Product in Normal Rats The rats were injected intra-articularly three times per week for 2 weeks with vehicle solution (8mM acetate, 40 mM arginine, and 0.6% glycerol (pH 5.3) in approximately 1%
hyaluronic acid), 10 pg CG 53135-05 E. coli purified product or 100 pg CG 53135-05 E. coli purified product.

Study Design: Male Lewis rats weighing 293-325 grams on day 0 were obtained from Harlan Sprague Dawley (Indianapolis, Indiana) and acclimated for 8 days. The rats were divided into three treatment groups with three animals in each group: two groups received CG53135 and one received only the vehicle control. The rats were anesthetized with isoflurane and injected through the patellar tendon into the area of the cruciate attachments of both knees. CG53135 was injected at doses of 0.1 mg/ml (0.01 mg/joint) or 1.0 mg/ml (0.1 mg/joint).
Controls were injected with the vehicle solution as described above. Injections were done Monday, Wednesday and Friday for 2 weeks. The animals were terminated on day 15 at which time they were injected ip with BRDU
(100 mg/kg) in order to pulse label proliferating cells.

Observations and Analysis of Markers of Pathology Gross observations: Rats were observed daily for abnormal swelling or gait alterations and were weighed weekly.

Histopathology: Preserved and decalcified (5% formic acid) knees were trimmed into 2 approximately equal longitudinal (ankles) or frontal (knees) halves, processed through graded alcohols and a clearing agent, infiltrated and embedded in paraffin, sectioned, and stained with toluidine blue (knees). Multiple sections (3 levels) of right knee were analyzed microscopically with attention to the parameters of interest listed below. Each parameter was graded as normal, minimal, mild, moderate, marked or severe. Evaluation of the cartilage was done using descriptive parameters rather than the scoring criteria generally used in the osteoarthritis model because of the type of alterations generated by the repetitive injection of the protein.
Although animals were injected with BRDU prior to termination, the proliferative changes were readily observed in toluidine blue stained sections.

Results Table 52: Microscopically Monitored Parameters Synovial Alterations Cartilage Central Cruciate Chondrogenesis Alterations Attachment Area Alterations -hyperplasia -cartilage -inflammation and -marginal zone or -infiltration of synovium with proteoglycan fibroplasia periosteal macrophages loss -bone or cartilage chondrogenesis -fibroplasia -cartilage damage -matrix (proteoglycan fibrillation deposition in fibrotic synovium) Live Phase Parameters Body weights were similar in vehicle and protein injected animals throughout the study (Table 52). Knees injected with 100 pg of protein had some evidence of fibrosis clinically during the injection process beginning with the 3rd injection.

Morphologic Pathology: Vehicle injected rats had minimal to mild synovial hyperplasia, inflammation and fibroplasia with none to minimal matrix deposition in fibrotic synovium. Articular cartilage had no proteoglycan loss or fibrillation. The central area of the joint where the cruciates attach and in which the intra-articular injections are made had none to minimal fibroplasia and cartilage/bone damage. No marginal zone chondrogenesis was present.

Knees injected with 10 pg CG 53135-05 E. coli purified product had mild to moderate synovial hyperplasia, inflammation and fibroplasia with minimal to moderate matrix deposition in fibrotic synovium. Articular cartilage had no proteoglycan loss or fibrillation. The central area of the joint where the cruciates attach and in which the intra-articular injections are made had none to minimal fibroplasia and cartilage/bone damage. One knee had minimal marginal zone chondrogenesis.

Knees injected with 100 pg CG 53135-05 E. coli purified product had moderate to marked synovial hyperplasia, inflammation and fibroplasia with moderate matrix deposition in fibrotic synovium. Articular cartilage had none to minimal proteoglycan loss or fibrillation. The central area of the joint where the cruciates attach and in which the intra-articular injections are made had minimal to marked fibroplasia and cartilage/bone damage. All knees had mild to moderate marginal zone chondrogenesis. One animal had chondrogenesis in areas associated with articular cartilage.
Conclusion These results demonstrate that repetitive intra-articular injection of CG53135 induces synovial fibroplasia and chondrogenesis. Vehicle injections resulted in mild inflammation and fibroplasia thus suggesting that this vehicle has some irritant potential.
Concentration responsive increases in synovial proliferative response as well as marginal zone chondrogenesis occurred in animals injected with protein. The area of the cruciate attachment where injections occurred had areas of bone resorption and fibroplasia which also increased in severity with increasing concentrations of the protein as did the synovial inflammation. The potentially adverse effects of observed synovial fibroplasia and bone resorption could have been due to either FGF-20 activity or endotoxin levels within the non-clinical grade hyaluronic acid used to formulate the protein. In addition, inflammation in the joint can induce bone resorption and marginal zone chondrogenesis so these results need to be interpreted in light of the possibility that the inflammatory response to the protein injection contributed to the proliferative response. The morphologic appearance of the proliferative changes and chondrogenesis clearly indicates that the biological activity of this protein (CG53135) is important in generating the response.

The results of the experiments reported herein indicate repetitive intra-articular injection of CG53135 induces synovial fibroplasia and chondrogenesis.

6.46. EXAMPLE 46: INTRA-ARTICULAR INJECTION OF CG53135-051N MENISCAL
TEAR MODEL OF RAT OSTEOARTHRITIS: PROPHYLACTIC AND THERAPEUTIC
DOSING
Example 45 utilized CG53135 administration into the joints of normal rats to identify effects on relevant cell populations by histomorphometric analysis. At the dose of 100 ug/joint, CG53135 induced significant marginal zone chondrogenesis similar to that seen with other growth factors such as TGF-beta, suggesting an effect on pluripotent stem cells within the marginal zone.
There was no apparent effect on mature chondrocytes as evidenced by the lack of a response in the mature cartilage areas of the joints. The potentially adverse effects of observed synovial fibroplasia and bone resorption could have been due to either FGF-20 activity or endotoxin levels within the non-clinical grade hyaluronic acid used to formulate the protein.

Further studies in osteoarthritic animals performed addressed the following:
(1) synergy with an anti-inflammatory drug (standard approach for osteoarthritis patients); (2) whether CG53135 can induce functional repair or protection of joint cartilage layers; and (3) whether synovial fibroplasia and bone resorption were CG53135-induced or due to contaminating endotoxin within the formulation.

Thus one aspect of this study was to evaluate the protective and therapeutic effects of intra-articular injection of CG53135 on joint damage in osteoarthritis in the meniscal tear model of rat osteoarthritis. This relatively new model of OA has been shown to have morphologic alterations of cartilage degeneration and osteophyte formation that resemble changes occurring in spontaneous disease and surgically induced disease in other species (Bendele, A.M., Animal Models of Osteoarthritis. J. Musculoskel. Neuron Interact. 2001; 1:363-376, Bendele, A.
M. and Hulman, J. F.
Spontaneous cartilage degeneration in guinea pigs. Arthritis Rheum. 1988;
31:561-565). The model can be used to evaluate potential beneficial effects of anti-degenerative as well as regenerative therapies.

Experimental Design Animals (10/group), housed 2/cage, were anesthetized with isoflurane and the right knee area is prepared for surgery. A skin incision was made over the medial aspect of the knee and the medial collateral ligament was exposed by blunt dissection, and then transected. The medial meniscus was then reflected medially with a fine scissor and a cut was made through the full thickness to simulate a complete tear. The skin was closed with suture.

Prophylactic Dosing: intra-articular dosing (CG53135-05 E. coli purified product) of the right knee joint was initiated on the day of surgery and is continued for 2 weeks post-surgery with intra-articular injections given Thursday, Saturday, and Monday (day 0, 2, 4, 7, 9, and 11) with rats under Isoflurane anesthesia. Indomethacin, a nonsteroidal anti-inflammatory drug, was dosed (1 mg/kg/day) daily by the oral route starting on the day of surgery to reduce any potential inflammation due to the injection. Body weights were recorded on days 0, 7 and 14. After animal termination on day 14 post-surgery, both knees were collected for histopathologic evaluation. The study design is shown in Table 53.

Table 53. Prophylactic Dosing Study Design Group CG53135-05 Co-therap~ Number of Animals Treatmenta Treatment Males Vehicle Vehicle 10 I intra-articular) 2 Vehicle T___Ifndomethacin 10 (intra-articular) 3 CG53135-05 E. coli Vehicle 10 purified product (intra-articular) 4 CG53135-05 E. coli Indomethacin 10 purified product (intra-articu lar) None None 10 aAdministration 3 times per week for 2 weeks (100 pg/joint, intra-articular) bAdministration daily for 2 weeks (0.5 mg/kg, PO) Therapeutic Dosing: intra-articular dosing (CG53135-05 E. coli purified product) of the right knee joint is initiated on day 21 of post-surgery and is continued for 2 weeks with intra-articular injections given Friday, Sunday, and Tuesday (day 22, 25, 27, 29, 32, and 34) with rats under isoflurane anesthesia. Indomethacin is dosed daily by the oral route starting on the day of surgery.
Body weights are recorded on days 0, 7, 14, 21, 28, and 35. On day 35, both knees are collected for histopathologic evaluation. The study design is shown in Table 54.

Table 54. Therapeutic Dosing Study Design CG53135-05 E. coli b Number of Animals Group purified product Co-therapy Treatment Males Treatmenta 1 Vehicle Vehicle 10 (intra-articular) 2 Vehicle Indomethacin 10 (intra-articular) 3 CG53135-05 E. coli Vehicle 10 purified product (intra-articular) 4 CG53135-05 E. coli Indomethacin 10 purified product (intra-articular) 5 None None 10 aAdministration 3 times per week for 2 weeks (100 pg/joint, intra-articular) bAdministration daily for 2 weeks (0.5 mg/kg, PO) Results of prophylactic dosing study. Observations made include the standards followed for this model. Multiple sections (3 levels) of right knee were analyzed microscopically and scored according to the following methods. In scoring the 3 sections, the worst case scenario for the 2 halves on each of the 3 slides representing 3 levels was determined for cartilage degeneration and osteophyte formation. This value for each parameter for each slide was then averaged to determine overall subjective cartilage degeneration scores for tibia and femur and osteophyte scores for tibia.

Cartilage degeneration was scored none to severe (numerical values 0-5) for depth and area (surface divided into thirds) using the following criteria:

0=no degeneration 1=minimal degeneration, chondrocyte and proteoglycan loss with or without fibrillation involving the superficial zone 2= mild degeneration, chondrocyte and proteoglycan loss with or without fibrillation involving the upper 1/3 3=moderate degeneration, chondrocyte and proteoglycan loss with fibrillation extending well into the midzone and generally affecting 1/2 of the total cartilage thickness 4=marked degeneration, chondrocyte and proteoglycan loss with fibrillation extending well into the deep zone but without complete (to the tidemark) loss of matrix 5=severe degeneration, matrix loss to tidemark Strict attention to zones (outside, middle, inside thirds) was adhered to in this scoring method and the summed scores reflect a global summation of severity of tibial degeneration.
In addition to this overall subjective analysis of cartilage degeneration, an additional subjective assessment was done using similar criteria to evaluate severity of degeneration but with attention to specific regional differences across the tibial plateau. In this OA model, generally the outside 1/3 of the tibia is most severely affected by the meniscal tear injury with lesions often extending to the tidemark by 3 weeks post-surgery. The middle 1/3 is usually a transition zone where severe or marked change becomes moderate or mild and the inner 1/3 seldom has changes greater than mild or minimal. In an attempt to determine potential differences of treatment on the severe lesion of the outside 1/3 vs. the milder lesions of the middle 1/3 and inside 1/3, these regions were each scored separately. The sum of the regional values was calculated and expressed as sum of 3 zones.

In addition to the above subjective scoring, a micrometer measurement of total extent of tibial plateau affected by any severity of degeneration (Total Tibial Cartilage Degeneration Width pm) extended from the origination of the osteophyte or marginal zone if no osteophyte was present with adjacent cartilage degeneration (outside 1/3) across the surface to the point where tangential layer and underlying cartilage appeared histologically normal.

An additional measurement (Significant Cartilage Degeneration Width pm) reflected areas of tibial cartilage degeneration in which chondrocyte and matrix loss extended through greater than 50% of the cartilage thickness.

Finally, a micrometer depth of any type of lesion (cell/proteoglycan loss, change in metachromasia, but may have good retention of collagenous matrix and no fibrillation) expressed as a ratio of depth of changed area vs. depth to tidemark was included and taken over 4 equally spaced points on the tibial surface. These measurements were taken (1 st) matrix adjacent to osteophyte (2nd) 1/4 of the distance across the tibial plateau (3rd) 1/2 of the distance across the tibial plateau (4th) 3/4 of the distance across the tibial plateau. This measurement was the most critical analysis of any type of microscopic change present. The depth to tidemark measurement (denominator) also gives an indication of cartilage thickness across the tibial plateau and therefore allows comparisons across groups when trying to determine if hypertrophy or hyperplasia has occurred.

A single tibial growth plate measurement was taken for each section in an area thought to best represent the overall width in the non tangential plane of the section.

Scoring of the osteophytes and categorization into small, medium and large was done with an ocular micrometer.

None = 0 no measurable proliferative response at marginal zone Small osteophytes =1 (up to 299 pm) Medium osteophytes=2 (300-399 pm) Large osteophytes=3 (>400pm) The score (0-3) was included in the overall joint score. In addition, the mean SE for the actual osteophyte measurement (average for 3 sections) was also determined.

Generally, in doing the surgery, attempts were made to transect the collateral ligament at a location that results in the meniscus being reflected proximally toward the femur. The cut was then made by inserting the scissors tip toward the femur rather than the tibia.
Some mechanical damage may then be detected in the femoral condylar cartilage but is rarely encountered on the tibia, thus making the tibia the most appropriate site for assessment of chondroprotection.

Focal small areas of proteoglycan and cell loss that were likely a result of physical trauma to the femoral cartilage were described but not included in the score with larger more diffuse areas receiving subjective scores according to methods described for the tibia.
These larger areas were more consistent with non traumatic degeneration. Because of the possibility of iatrogenic lesions on the femur, overall joint scores were expressed both with and without femoral cartilage degeneration scores.

Damage to the calcified cartilage layer and subchondral bone was scored using the following criteria:

O=No changes 1=lncreased basophilia at tidemark: no fragmentation of tidemark or marrow changes 2=lncreased basophilia at tidemark: minimal to mild fragmentation of calcified cartilage of tidemark, mesenchymal change in marrow involves 1/4 of total area but generally is restricted to subchondral region under lesion 3=lncreased basophilia at tidemark: Mild to marked fragmentation of calcified cartilage, Mesenchymal change in marrow is up to 3/4 of total area, Areas of marrow chondrogenesis may be evident but no collapse of articular cartilage into epiphyseal bone 4=lncreased basophilia at tidemark: Marked to severe fragmentation of calcified cartilage, Marrow mesenchymal change involves up to 3/4 of area and articular cartilage has collapsed into the epiphysis to a depth of 250pm or less from tidemark 5=lncreased basophilia at tidemark: Marked to severe fragmentation of calcified cartilage, Marrow mesenchymal change involves up to 3/4 of area and Articular cartilage has collapsed into the epiphysis to a depth of greater than 250pm from tidemark Descriptive comments were made on degree of synovial inflammation, synovial fibrosis, marginal zone chondrogenesis, bone resorption, fibrous overgrowth with or without chondrogenesis/incorporation into existing cartilage Statistical Analysis: statistical analysis of histopathologic parameters was done by comparing group means using the Student's two-tailed t-test with significance set at p:50.05.
Because of the nature of the data, a non Parametric ANOVA (Kruskal-Wallis test) was used to analyze the scored parameters and a parametric ANOVA was used to analyze the measurements.
The appropriate post test used was Dunnett's multiple comparisons test on the parametric data and a Dunn's test was used on the non parametric data. Significance was set at p:50.05 for all parameters.

Results: intra-articular injection of 100 pg CG53135-05 E. coli purified product with or without concurrent indomethacin administration resulted in significant inhibition (39%) of tibial cartilage degeneration on the middle 1/3 (40-43% for zone 1) and an overall insignificant inhibition of the summed 3 zones of 41% (FIG. 70(A)). Total cartilage degeneration width was significantly decreased 35-37% (FIG. 70(B)) and significant degeneration was reduced 70-89%
with this inhibition being significant only in the group treated with protein and indomethacin (FIG. 70(C)).

Results of the prophylactic dosing study: the data described indicate that intra-articular injection of 100 pg of CG53135-05 E. coli purified product in knee joints of rats with medial meniscal tear results in chondroprotective effects as a result of both inhibition of cartilage degeneration and stimulation of cartilage repair. Some joints had layering of proliferated new cartilage over existing normal appearing or damaged cartilage. This observation is particularly exciting as it demonstrates the potential for resurfacing to occur.

These beneficial effects were always associated with diffuse synovial fibroplasia, bone resorption and increased synovial inflammation. Concurrent indomethacin treatment (1 mg/kg/day) had minimal if any effect on the disease process in knees injected with Synvisc alone or the disease process and reaction to the protein in knees injected with Synvisc containing protein. The single exception to this statement is reflected in the data for osteophyte measurements where all groups had similar measurements except the group treated with protein and vehicle po.
This group had greater measurements thus suggesting greater marginal zone stimulation, not an uncommon occurrence in inflamed joints.

The morphologic changes induced by injection of 100 pg of this protein demonstrate the potential for CG53135 to be effective in cartilage repair processes. It has the capacity to induce fibrous tissue proliferation with differentiation to cartilage and importantly, integration of that newly proliferated tissue. The proliferative processes are somewhat disorganized and counter productive in areas such as the marginal zone and subchondral bone. However, rodents definitely have much greater propensity to exhibit marginal zone, periosteal and marrow proliferation from a variety of stimuli including inflammatory mediators so some of the excessive and counter productive responses seen in rats might not occur in dogs or primates. Also, there may have been some induction of an antibody response thus leading to enhanced knee inflammation that would not occur in humans or other animals that did not have an antibody response.

Additional studies that are useful in delineating the potential efficacy of CG53135 in osteoarthritis include: (1) evaluation in an animal model, e.g., a rabbit or a dog model of OA-this would allow evaluation in a larger joint with cartilage and bone structure that is more similar to humans and species that has less tendency to exhibit hyperproliferative responses such as those that occur in rodents; and (2) evaluation of ia injections for 3-4 weeks, possibly with more aggressive anti-inflammatory systemic therapy followed by a recovery period to see how the new tissue remodels would be interesting. It may be that allowing the joint to remodel with no further proliferative stimulus would result in a more pleasing morphologic endpoint.
Alternatively, the time point of which the immune response that would clear may result in a endpoint.
Cycles of treatment with periods of remodeling might be the way to achieve the most satisfactory repair. Studies such as these would also answer the question of whether the repair tissue will hold up long term. Generally fibrocartilage has less of a tendency to do this.

Results: Intra-articular injection of 100 pg CG531 35-05 E. coli purified product with or without concurrent oral indomethacin administration did not result in significant inhibition of tibial cartilage degeneration scores (FIG. 71(A)). Total or significant cartilage degeneration width was not decreased (FIGS. 71(B) and (C)).

Results of the therapeutic dosing study: The data described demonstrated the potential chondroproliferative activities of CG53135 administered intra-articularly.
However, protein injected joints had markedly increased inflammation, fibroplasia and connective tissue resorptive process. -The most important difference between the prophylactic and therapeutic dosing studies was the nature of the OA lesion at the time of initiation of dosing. Rats in the therapeutic dosing study had an area of severe matrix loss in the outer to middle 1/3 of the cartilage thus exposing the calcified cartilage/subchondral bone to the protein. Effective repair thus required filling of this defect with newly proliferated tissue coming from the marginal zone or exposed marrow pleuripotential cells. In the prophylactic dosing study, beneficial effects required inhibition of matrix degradation and stimulation of repair on a degenerating scaffold with repair tissue originating from the marginal zone only. Since the filling of a defect would be much more difficult than repairing a damaged scaffold, it may be that a longer duration of treatment would be required in a therapeutic model in order to see beneficial effects.

Indomethacin treatment was not effective in reducing the inflammatory changes and it had no beneficial effects on inhibiting the resorptive processes occurring in bone. In order to achieve effective proliferation and differentiation to cartilage in the absence of inflammation and tissue destruction, following modification to the therapeutic dosing study can be attempted: Increasing the dosing interval to once or twice weekly and/or increasing the study duration to allow time for the proliferative tissue to fill the large cartilage defects induced by this disease process. Another possibility is to investigate the effects of CG53135 in a larger species such as the dog as dogs have less of a tendency to proliferate connective tissue and resorb bone in response to various stimuli than rodents.

The results detailed herein (both prophylactic and therapeutic dosing studies) indicate that CG53135 has specific utility in severely osteoarthritic joints that are destined for joint replacement.
These types of agents would be injected into joints that have little or no normal cartilage remaining and are in need of resurfacing. In this situation, repair could originate from pleuripotential cells in the marginal zones or bone marrow. Repair originating from these locations will likely result in production of fibrocartilage rather than hyaline cartilage. However, some cartilage would be preferable to no cartilage and it may be that an injectable method of sustaining a cartilage surface would be acceptable even though treatments would likely have to be repeated over time to sustain the repair. Treatments with injectable anabolic agents will likely require some kind of cyclical process in conjunction with continuous passive motion rather than sustained active load bearing motion.

6.47. EXAMPLE 47: CG53135 RESCUES NEURONAL PC12 CELLS FROM SERUM-STARVATION INDUCED CELL DEATH
To assess the trophic (neuroprotective) qualities of CG53135 and compare to the action of Nerve Growth Factor (NGF) and Epidermal Growth factor (EGF), the following experiment was performed.

Materials and Method:

Materials: PC12 cells, tissue culture plates and medium (DMEM +/- 10% FBS), NGF, EGF, CG53135-05 E. coli purified product (by Process 1).

Method: Plate PC12 cells at low density on poly-lysine coated tissue culture plates in DMEM
+ 10% FBS. Culture 24 hours. Administer serum-free media containing NGF or CG53135 at a range of doses or no growth factor supplements. Photograph at 72 hours to visualize cell survival and proliferation.

Results:

CG53135 prevented cell death in a dose-dependent fashion. The maximal trophic activity was achieved at 50 ng/ml. The potency of CG531345 was approximately 20% of the potency of NGF, however the maximal extent of trophic action by both growth factors was equivalent. EGF also exhibited trophic activity. Cell death (apoptosis) was measured by LDH assay and visually. (FIG.
72) Conclusion:
CG53135 acts similarly to the neurotrophin NGF and to the growth factor EGF to the extent that CG53135 is capable of rescuing PC12 cells from serum starvation-induced cell death. Thus, CG53135 possesses trophic activity. Trophic activity is recognized to have value in the treatment of numerous disorders of the central nervous system. In particular, the ability to protect neuronal cells is important to diseases where neurodegeneration is involved, such as Alzheimer's disease, Parkinson's disease and diseases with catastrophic cell death such as stroke and traumatic brain injury.

6.48. EXAMPLE 48: CG53135 INHIBITS SERUM-WITHDRAWAL INDUCED CASPASE

This experiment was performed to assess the ability of CG53135 to inhibit the activation of pro-apoptotic caspace enzymes, and compare to the action of Nerve Growth Factor (NGF).
Materials and Method:

Materials: PC12 cells, tissue culture plates and medium (DMEM +/- 10% FBS), NGF, CG53135-05 E. coli purified product (by Process 1).

Method: Plate PC12 cells at medium density on poly-lysine coated tissue culture plates in DMEM + 10% FBS. Culture 24 hrs. Administer serum-free media containing NGF
(100 ng/ml) or CG53135 (1000 ng/mi) or no growth factor supplements. Collect cell lysates at various time points (0, 3, 6 and 20 hrs.). Evaluate caspase2, 3, 8, 9 activation by ELISA.

Results:

Serum withdrawal induced caspase activity time-dependently. Both CG53135 and NGF
blocked caspase induction. (FIG. 73) Conclusion:
CG53135 acts similarly to the neurotrophin NGF to the extent that both proteins are able to prevent the activation of apoptosis promoting caspase enzymes upon apoptotic stimuli. Caspases have been implicated in a number of diseases of the CNS involving neuronal death, including Alzheimer's disease, Parkinson's disease, stroke and traumatic brain injury.
Therefore, CG53135 has value in the treatment of these diseases.

6.49. EXAMPLE 49: CG53135 INDUCES NEURITE OUTGROWTH BY NEURONAL PC12 CELLS
This experiment was performed to assess the neuritogenic qualities of CG53135-05 and compare to the action of Nerve Growth Factor (NGF) Materials and Method:

Materials: PC12 cells, tissue culture plates and medium (DMEM +/- 10% FBS), NGF, CG53135-05 E. coli purified product (by Process 1).

Method: Plate PC12 cells at low density on poly-lysine coated tissue culture plates in DMEM
+ 10% FBS. Culture 24 hrs. Administer serum-free media containing NGF or CG53135 at 100 ng/ml or no growth factor supplements. Photograph at 72 hrs to visualize neurite outgrowth.

Results:

CG53135 induced neurite outgrowth in a dose-dependent fashion. The maximal extent of neurite outgrowth was achieved at 1000 ng/ml. The maximal extent of neurite outgrowth induced by both growth factors was equivalent. EGF did not induce neurite outgrowth.
(FIG. 74) Conclusion:
CG53135 acts similarly to the neurotrophin NGF to the extent that CG53135 is capable of inducing similar neurite outgrowth. The capability of NGF to induce neurite outgrowth is an important feature of this growth factor that distinguishes it from other factors such as EGF which do not possess such neurotrophic activity. Thus, CG53135 possesses neurotrophic activity.
Neurotrophic activity is recognized to have value in the treatment of numerous disorders of the central nervous system. In particular, the ability to induce neurite outgrowth is important to diseases where neurodegeneration is involved, such as Alzheimer's disease, Parkinson's disease and diseases with pathological structural changes or neural architecture are involved such as stroke and traumatic brain injury.

6.50. EXAMPLE 50: CG53135 ACTIVATES MAP KINASE IN NEURONAL PC12 CELLS
This experiment was performed to assess the MAPK activating action of CG53135 and compare to the action of Nerve Growth Factor (NGF), Epidermal Growth Factor (EGF) and Basic FGF (bFGF).

Materials and Method:

Materials: PC12 cells, tissue culture plates and medium (DMEM +/- 10% FBS), NGF, EGF, bFGF, CG53135-05 E. coli purified product (by Process 1), EGF, MAPKK inhibitor Method: Plate PC12 cells at medium density on poly-lysine coated tissue culture plates in DMEM + 10% FBS. Culture 24 hours. Administer serum-free media containing NGF
(100 ng/ml), EGF (100 ng/ml) or CG53135 (100 ng/ml) or no growth factor supplements. Pre-treat separate cultures with PD98059 before treating with CG53135 or NGF. Lyse cells 10 min post treatment, perform western blot with anti-phospho MAPK antibody to assess MAPK
activation. Also, evaluate time course of MAPK activation by CG53135 and bFGF in human cortical neuronal cell line HCNIA
at 0, 10 min, 1 hour and 3 hours.

Results:

CG53135 induced robust MAPK activation in a MAPKK-dependent manner. CG53135 exhibits gradual and sustained MAPK activation timecourse, superior to bFGF.
(FIG. 75) Conclusion:

CG53135 acts similarly to the neurotrophin NGF in the induction of MAPK, a key intracellular signaling molecule involved in cell survival and neuronal differentiation. The activation of this trophic pathway, which also is involved in processes underlying learning and memory, is recognized to have value in the treatment of numerous disorders of the central nervous system. In particular, the ability to protect neuronal cells is important to diseases where neurodegeneration is involved, such as Alzheimer's disease, Parkinson's disease and diseases with catastrophic cell death such as stroke and traumatic brain injury. The ability to stimulate intracellular pathways involved in learning and memory also is likely to have relevance to disorders involving memory dysfunction, such as Alzheimer's and age-related memory loss.

7. EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

DEMANDE OU BREVET VOLUMINEUX

LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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VOLUME

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Claims (73)

1. A formulation comprising about 0.1-1 M arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, about 0.01-0.1 M sodium phosphate monobasic (NaH2PO4-H2O), about 0.01 %-0.1% weight/volume ("w/v") polysorbate 80 or polysorbate 20, and an isolated fibroblast growth factor.

2. A formulation comprising about 0.01-1 M arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, about 0.01-0.1 M sodium phosphate monobasic (NaH2PO4-H2O), about 0.01 %-0.1 % weight/volume ("w/v") polysorbate 80 or polysorbate 20, and an isolated protein selected from the group consisting of:

(a) ~a protein comprising an amino acid sequence of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40;

(b) ~a protein with one or more amino acid substitutions to the protein of (a), wherein said substitutions are no more than 15% of the amino acid sequence of SEQ ID
NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and wherein said protein with one or more amino acid substitutions retains cell proliferation stimulatory activity; and (c) ~a fragment of the protein of (a) or (b), which fragment retains cell proliferation stimulatory activity.

3. The formulation of claim 1 or 2, wherein said arginine in a salt form is selected from the group consisting of arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride.

4. The formulation of claim I or 2, wherein said arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium or sucrose is of 0.01-0.7 M.

5. The formulation of claim 1 or 2 comprising an arginine in a salt form at a concentration of 0.5 M.

6. The formulation of claim I or 2, wherein said sodium phosphate monobasic is 0.05 M.

7. The formulation of claim I or 2, wherein said polysorbate 80 or polysorbate 20 is 0.01 %
(w/v).

8. The formulation of claim 1 or 2 comprising polysorbate 80.

9. The formulation of claim 1 or 2 comprising polysorbate 20.

10. The formulation of claim I or 2, wherein said protein is at a concentration of 0.5-30 mg/ml.

11. The formulation of claim 1 or 2, wherein said protein is at a concentration of 10 mg/ml.

12. The formulation of claim 2, wherein said protein comprises an amino acid sequence of SEQ
ID NO:24.

13. The formulation of claim 2, wherein said protein comprises an amino acid sequence of SEQ
ID NO:2.

14. The formulation of claim 1 or 2 comprising two or more proteins.

15. The formulation of claim 14, wherein said proteins comprise a first protein comprising an amino acid sequence of SEQ ID NO:24, and a second protein comprising an amino acid sequence of SEQ ID NO:2.

16. The formulation of claim 1 or 2 is lyophilized or spray dried.

17. The formulation of claim 2, wherein said isolated protein is of about 0.005 mg/ml to about 50 mg/ml.

18. A formulation comprising about 10 mg/ml of an isolated protein comprising an amino acid sequence of SEQ ID NO:24, 0.5 M arginine sulfate, 0.05 M sodium phosphate monobasic, and 0.01 % (w/v) polysorbate 80.

19. A formulation comprising about 10 mg/ml of an isolated protein comprising an amino acid sequence of SEQ ID NO:2, 0.5 M arginine sulfate, 0.05 M sodium phosphate monobasic, and 0.01%
(w/v) polysorbate 80.

20. A formulation comprising 0.5 M arginine sulfate, 0.05 M sodium phosphate monobasic, 0.01 % (w/v) polysorbate 80, and about 10 mg/ml of a mixture of isolated proteins, wherein said proteins comprise a first protein comprising an amino acid sequence of SEQ ID
NO:24, and a second protein comprising an amino acid sequence of SEQ ID NO:2.

21. The formulation of claim 20 further comprising an isolated protein, wherein said protein comprises an amino acid sequence selected from the group consisting of SEQ ID
NOs:26, 28, 30 and 32.

22. The formulation of claim 21, wherein one or more of the isolated protein is carbamylated.

23. The formulation of claim 20 further comprising a third protein comprising an amino acid sequence of SEQ ID NO:28, a fourth protein comprising an amino acid sequence of SEQ ID NO:30, and a fifth protein comprising an amino acid sequence of SEQ ID NO:32.

24. The formulation of any of claims 2, 18-23, wherein said isolated protein is at least 98% pure.

25. A method of increasing solubility of a fibroblast growth factor ("FGF") in an aqueous solution comprising adding arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, or a combination thereof to said solution to a final concentration of 0.01 - 1 M.

26. The method of claim 25, wherein said fibroblast growth factor is an isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID
NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

27. The method of claim 25 or 26, wherein said arginine in a salt form is selected from the group consisting of arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride.

28. The method of claim 25 or 26, wherein said final concentration of arginine in a salt form is 0.01 - 0.7 M.

29. The method of claim 25 or 26, wherein said final concentration of arginine in a salt form is 0.5 M.

30. The method of claim 25 further comprising adding acetate, succinate, tartrate, or a combination thereof to said solution.

31. A method of increasing solubility of a FGF in a solution comprising adding acetate, succinate, tartrate or a combination thereof to said solution.

32. The method of claim 31, wherein said acetate, succinate, tartrate, or a combination thereof has a final concentration of 0.01-0.2 M in said solution.

33. A method of producing an isolated protein comprising the steps of:

(1) ~fermenting an E. coli cell containing a vector comprising SEQ ID NO:8;
(2) ~chilling the fermented culture to 10-15°C;

(3) ~diluting the chilled culture with a lysis buffer comprising 50-100 mM
sodium phosphate, 60 mM ethylene diamine tetraacetic acid, 7.5 mM DTT, and 3.5-5 M urea;

(4) ~lysing the cells in the diluted culture;

(5) ~loading the resultant cell lysate onto a pre-equilibrated cation exchange column, and flushing the column with a buffer comprising 50-100 mM sodium phosphate, 40 mM

EDTA, 10 mM sodium sulfate, and 3-5 M urea;

(6) ~washing the flushed column with a buffer comprising 50-100 mM sodium phosphate, 5 mM EDTA, 10-25 mM sodium sulfate, and 2.22 mM dextrose;

(7) ~washing the column again with an elution buffer comprising 50-100 mM
sodium phosphate, 5 mM EDTA, 150-250 mM sodium sulfate, and 0.5-1 M L-arginine;

(8) ~loading the resultant eluate onto a hydrophobic interaction chromatography column pre-equilibrated with 50-100 mM sodium phosphate, 150-250 mM sodium sulfate, 5 mM
EDTA, and 1 M arginine;

(9) ~washing the resulting column with a solution comprising 100-250 mM sodium phosphate, 5 mM EDTA, and 0.8-1 M arginine; and (10) ~washing the column again with a solution comprising 50-100 mM sodium phosphate, 5 mM EDTA, and 0.1-0.3 M arginine to elute the protein.

34. The method of claim 33 further comprising the steps of:
(11) ~concentrating the resultant eluate;

(12) filtering the retentate obtained together with a solution comprising 50 mM sodium phosphate, 0.5 M arginine;

(13) concentrating the filtered retentate; and (14) filtering the concentrated retentate.

35. The method of claim 33, wherein said fermenting in step (1) comprises the steps of:

(a) culturing E. coli cells containing a vector comprising SEQ ID NO:8 to exponential growth phase with 2.5 to 4.5 OD600 units in a chemically defined seed medium;
(b) inoculating cells of step (a) to a seed medium and culturing the cells to an exponential growth phase with 3.0 to 5.0 OD600 units;

(c) transferring the cells of step (b) to a chemically defined batch medium;

(d) culturing the cells of step (c) to 25-35 units OD600, and adding additional chemically defined medium with a feeding rate of 0.7 g/kg broth/minute;

(e) culturing the cells of step (d) to 135 to 165 units OD600; and (f) culturing the cells of step (e) for about four hours.

36. The method of claim 33, wherein said step (3) further comprising adding polyethyleneimine to the diluted cell culture.

37. The method of claim 33, wherein said step (10) further comprising the steps of:
(a) passing the eluate through a charged endotoxin binding filter; and (b) flushing the filter of step (a) first with water, and then with a buffer comprising 50-100 mM sodium phosphate, 5 mM EDTA, 0.1-0.3 M arginine to elute the protein.

38. The method of claim 33, wherein said lysing in step (4) comprises passing through a high pressure homgenizer.

39. The method of claim 33, wherein said step (10) further comprising the steps of:

(a) loading the eluate onto a pre-equilibrated hydrophobic interaction chromatography column, wherein said column is equilibrated with 50-100 mM sodium phosphate, 100 mM ammonium sulfate, 800-1000 mM sodium chloride, 0.5-1 M arginine; and (b) washing the column of step (a) with 50-100 mM sodium phosphate, 0.5-1 M
arginine to produce an eluate.

40. The method of claim 33, wherein said step (10) further comprising the step of adding 1%
polysorbate 80 or polysobate 20 to the eluate of step (10) to a final concentration of 0.01 %(w/v).

41. The method of claim 33, wherein said step (10) further comprising the steps of:

(a) passing the eluate through a charged endotoxin binding filter;

(b) flushing the filter of step (a) first with water, and then with a buffer comprising 50-100 mM sodium phosphate, 5 mM EDTA, 0.1-0.3 M arginine to produce a filtrate;

(c) loading the filtrate of step (b) onto a pre-equilibrated hydrophobic interaction chromatography column, wherein said column is equilibrated with 50-100 mM
sodium phosphate, 10-100 mM ammonium sulfate, 800-1000 mM sodium chloride, 0.5-1 M arginine;

(d) washing the column of step (c) with 50-100 mM sodium phosphate, 0.5-1 M
arginine to produce an eluate; and (e) adding 1% polysorbate 80 or polysorbate 20 to the eluate of step (d) to a final concentration of 0.01% (w/v).

42. The method of claim 34 further comprising a step of lyophilizing or spray drying the filtered solution of step (14).

43. One or more isolated protein produced by the method of any of claims 32-42.

44. The isolated protein of claim 43 is at least 98% pure.

45. A pharmaceutical composition comprising the isolated protein of claim 43, and a pharmaceutically acceptable carrier.

46. A pharmaceutical composition comprising the isolated protein of claim 44, and a pharmaceutically acceptable carrier.

47. A formulation comprising about 0.01-1 M arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium or sucrose, or a combination thereof, about 0.01-0.1 M
sodium phosphate monobasic (NaH2PO4.cndot.H2O), about 0.01% -0.1 % weight/volume ("w/v") polysorbate 80 or polysorbate 20, and the isolated protein of claim 42.

48. The formulation of claim 2 or 47 for prevention or treatment of alimentary mucositis.

49. The formulation of claim 2 or 47 for prevention or treatment of inflammatory bowel disease ("IBD").

50. The formulation of claim 2 or 47 for prevention or treatment of osteoarthritis.

51. The formulation of claim 2 or 47 for prevention or treatment of a disorder associated with radiation exposure or a symptom thereof.

52. The formulation of claim 2 or 47 for prevention or treatment of a disorder of central nerve system.

53. The formulation of claim 2 or 47 for prevention or treatment of a cardiovascular disease.

54. A method of treating or preventing alimentary mucositis comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

55. The method of claim 54, wherein the effective amount is between 0.001-3 mg/kg.

56. The method of claim 54, wherein the administering is a single dose administered at a dosage of 0.001-1 mg/kg, 0.01-0.5 mg/kg, 0.01-0.2 mg/kg, 0.03 mg/kg, 0.1 mg/kg, or 0.2 mg/kg.

57. The method of claim 54, wherein the administering is a multiple dose administered at a dosage with each unit dosage of 0.001-1 mg/kg, 0.01-0.5 mg/kg, 0.01-0.2 mg/kg, 0.03 mg/kg, 0.1 mg/kg, or 0.2 mg/kg.

58. The method of claim 54, wherein the alimentary mucositis is oral mucositis, enteritis, esophagitis, stomatitis, or proctitis.

59. The method of claim 54, wherein the alimentary mucositis is caused by a chemical insult, a biological insult, radiation, or a combination thereof.

60. A method of treating or preventing inflammatory bowel disease comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

61. The method of claim 60, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

62. A method of treating or preventing irritable bowel syndrome comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

63. A method of stimulating proliferation, differentiation, or migration of epithelial cells or mesenchymal cells comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

64. A method of treating or preventing arthritis or cartilage degeneration comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

65. A method of stimulating cartilage regeneration or repair comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

66. A method of treating or preventing stroke or a neurodegenerative disease comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

67. A method of treating or preventing cardiovascular disease comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

68. A method of treating or preventing a disorder caused by an insult affecting rapidly proliferating tissue or one or more symptoms thereof comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

69. The method of claim 68, wherein the insult is radiation exposure, exposure to a chemical agent or a microorganism, or a combination thereof.

70. A method of upregulating oxygen scavenging pathways comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

71. A method of stimulating secretion of an endogenous cytokine or an endogenous chemokine from a cell of a subject, wherein the method comprises administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

72. A method of stimulating hematopoietic stem cell proliferation comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.

73. A method of optimizing hematopoietic stem cell engraftment comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the formulation of any of claims 1-24.