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WO2005112979A2 - Procédés de prévention et de traitement de la mucosite alimentaire - Google Patents

Procédés de prévention et de traitement de la mucosite alimentaire Download PDF

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Publication number
WO2005112979A2
WO2005112979A2 PCT/US2005/010545 US2005010545W WO2005112979A2 WO 2005112979 A2 WO2005112979 A2 WO 2005112979A2 US 2005010545 W US2005010545 W US 2005010545W WO 2005112979 A2 WO2005112979 A2 WO 2005112979A2
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WIPO (PCT)
Prior art keywords
mucositis
protein
day
amino acid
fgf
Prior art date
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PCT/US2005/010545
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WO2005112979A3 (fr
Inventor
Enrique Alvarez
Henri Lichenstein
Namdev K. Pradyumna
Pascal Valax
Zachary Yim
Rajeev A. Chillakuru
William Hahne
Michael E. Jeffers
William J. Larochelle
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CuraGen Corp
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CuraGen Corp
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Priority claimed from US10/842,206 external-priority patent/US20050256039A1/en
Priority claimed from US10/980,459 external-priority patent/US20050256042A1/en
Priority to EP05765938A priority Critical patent/EP1773372A2/fr
Priority to AU2005244737A priority patent/AU2005244737A1/en
Application filed by CuraGen Corp filed Critical CuraGen Corp
Priority to JP2007513139A priority patent/JP2007536381A/ja
Priority to CA002566439A priority patent/CA2566439A1/fr
Priority to EP05851340A priority patent/EP1812460A1/fr
Priority to CA002586213A priority patent/CA2586213A1/fr
Priority to AU2005306898A priority patent/AU2005306898A1/en
Priority to JP2007540036A priority patent/JP2008519032A/ja
Priority to PCT/US2005/039833 priority patent/WO2006055264A1/fr
Publication of WO2005112979A2 publication Critical patent/WO2005112979A2/fr
Anticipated expiration legal-status Critical
Publication of WO2005112979A3 publication Critical patent/WO2005112979A3/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to compositions and methods for preventing and treating alimentary mucositis. More particularly, the present invention relates to compositions comprising FGF- 20, a fragment, a derivative, a variant, a homolog, or an analog thereof, and their uses in preventing and treating alimentary mucositis.
  • Alimentary mucositis refers to a form of mucosal barrier injury to the alimentary tract. Alimentary mucositis may occur at a part or multiple parts of the alimentary tract, from mouth to anus, via, e.g., esophagus, stomach, small intestine, colon, and rectum.
  • Non-limiting examples of alimentary mucositis are oral mucositis, esophagitis, stomatitis, enteritis, and proctitis.
  • Alimentary mucositis are generally caused by one or more insults, most commonly by a chemical(s) or radiation, or a combination thereof. Radiation may be a result of, e.g., radiation therapy, accidental radiation exposure, and radiation exposure from a terrorist attack. See e.g., Moulder, Int. J. Radiat. Biol. 80:3-10 (2004). Chemical insults are commonly from chemotherapy. Oral mucositis commonly occurs as a painful, dose-limiting toxicity of chemotherapy and radiation therapy, especially in cancer patients.
  • the disorder is characterized by, e.g., breakdown of the oral mucosa that results in the formation of ulcerative lesions.
  • the ulcerations that accompany mucositis are frequent portals of entry for indigenous oral bacteria, which often lead to sepsis or bacteremia.
  • Oral mucositis occurs to some degree in more than one-third of patients receiving anti-neoplastic drug therapy.
  • the frequency and severity are significantly greater among patients who are treated with induction therapy for leukemia or with many of the conditioning regimens for bone marrow transplant.
  • moderate to severe oral mucositis is not unusual in more than three-quarters of patients.
  • Moderate to severe mucositis occurs in virtually all patients who receive radiation therapy for tumors of the head and neck and typically begins with cumulative exposures of 15 Gy and then worsens as total doses of 60 Gy or more are reached (See, e.g., Peterson, Curr Opin Oncol 11 (4):261 -266 (1999); Plevova, Oral Oncol. 35(5):453-470 (1999); Knox etal., Drugs Aging 17(4):257-267 (2000); and Sonis etal., J. Clin Oncol 19(8) :2201 -2205 (2001)).
  • Toxicol Pathol 27(1 ):64- 71 (1999) include mucosal protective agents, antibiotics, transforming growth factor (TGF), interleukin-11 (IL-11 ), granulocyte-macrophage colony stimulating factor (GM-CSF), and keratinocyte growth factor (KGF).
  • TGF transforming growth factor
  • IL-11 interleukin-11
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • KGF keratinocyte growth factor
  • Palifermin, Amgen a modified version of naturally occurring human KGF (Palifermin, Amgen) has been approved in the United States for decreasing the incidence and duration of severe oral mucositis in patients with hematologic malignancies receiving myelotoxic therapy requiring hematopoietic stem cell support.
  • esophagitis inflammation of esophagus
  • dysphagia difficulty in swallowing
  • odynophagia painful swallowing
  • substernal in radiation-induced esophagitis
  • retrosternal in chemotherapy-induced esophagitis
  • Esophagitis is commonly managed by topical use of local anesthetics, spasmolytics, analgesic drugs, and treatment of acid reflux.
  • Ranitidine or omeprazole has been recommended for the prophylactic reduction of epigastric pain and heartburn following certain chemotherapies.
  • the severity of the esophagitis requires a temporary interruption of the treatment or modification of the treatment plan. See e.g., Keefe etal., Seminars in Oncology 20:38-47 (2004).
  • Stomatitis inflammation of stomach
  • Stomatitis may be radiation-induced and is characterized by, e.g., dyspepsia and symptomatic gastritis, which are usually temporary.
  • gastric ulcer may occur.
  • histamine-2 receptor antagonists or proton pump inhibitors are commonly used to manage the symptoms associated with stomatitis.
  • Enteritis inflammation of intestines, especially the small intestine
  • enteritis inflammation of intestines, especially the small intestine
  • the main symptoms are nausea, abdominal pain, bloating, and diarrhea.
  • Radiation-induced diarrhea typically occurs during the first two weeks after beginning of radiation therapy.
  • the mechanism of radiation- induced diarrhea involves acute mechanical damage to the epithelial crypt cells of the gastrointestinal tract.
  • Chemotherapeutic agents that commonly associated with diarrhea include, but are not limited to, fluoropyrimidines (e.g., 5-fluorouracil), topisomerase I inhibitors (e.g., irinotecan, topotecan), and other agents (e.g., cisplatin, oxaliplatin, cytarabine). See e.g., Cyprus, Seminars in Oncology Nursing 19:2-5 (2003).
  • Chronic bowel toxicity may also occur after radiation therapy, usually six months to three years after the therapy. Patients often have intermittent constipation and diarrhea, which may cause malnutrition and disturbance of electrolytes. In severe cases, acute intestinal obstruction, fistulas, or bowel perforation may occur. See e.g., Keefe etal., Seminars in Oncology 20:38-47 (2004). Recent studies suggest that p53 and p21 , two transcription factors, may play an important role in chemotherapy-induced enteritis. See, Pritchard etal., Clin. Cancer Res. 6:4389-4395 (2000); Pritchard etal., Cancer Res. 58:5453-5465 (1998); Bilim etal., J. Exp. Clin. Cancer Res.
  • enteritis is mainly managed symptomatically by giving, e.g., conventional antidiarrheal, antiemetic, spasmolytic, and defoaming agents to patients.
  • Somatostatin analogues e.g., octreotide
  • 5-HT antagonists e.g., 5-HT antagonists
  • NK1 antagonists e.g., NK1 antagonists
  • Proctitis inflammation of the rectum
  • Proctitis is common is patients who receive radiation therapy for pelvic tumors.
  • Proctitis may be acute radiation proctitis or chronic radiation proctitis.
  • the main symptoms of acute radiation proctitis are diarrhea, tenesmus (fecal urgency with cramp-like rectal pain), and hematochezia (bloody stools). Many patients also experience symptoms from the upper abdomen even though the small bowel is not included in the radiation field.
  • the main symptoms of chronic radiation proctitis are frequent or clustered bowel movements, anal discharge, rectal pain, urgency, tenesmus, incontinence, and hematochezia. See e.g., Keefe etal., Seminars in Oncology 20:38-47 (2004).
  • Topical anesthetic preparations, loperamide, steroid-containing suppositories may be helpful in amolieorating some symptoms.
  • FGF fibroblast growth factor
  • the fibroblast growth factor (“FGF”) family has more than 20 members, each containing a conserved amino acid core (see, e.g., Powers etal., 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 etal., Int. Rev. Cytol., 185:45-106 (1999)).
  • FGFs transduce signals via high affinity interactions with cell surface tyrosine kinase FGF receptors (FGFRs). These FGF receptors are expressed on most types of cells in tissue culture.
  • FGF receptor-1 which shows the broadest expression pattern of the four known FGF receptors, contains at least seven tyrosine phosphorylation sites. A number of signal transduction molecules are affected by binding with different affinities to these phosphorylation sites. FGFs also bind, albeit with low affinity, to heparin sulfate proteoglycans (HSPGs) present on most cell surfaces and extracellular matrices (ECM). Interactions between FGFs and HSPGs may serve to stabilize FGF/FGFR interactions, and to sequester FGFs and protect them from degradation (Szebenyi and Fallon, Int. Rev. Cytol. 185:45-106. (1999)).
  • HSPGs heparin sulfate proteoglycans
  • GAF Glia-activating factor
  • GAF Glia-activating factor
  • GAF Glia-activating factor
  • the human FGF-9 cDNA encodes a polypeptide of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences found in other family members were also well conserved in the FGF-9 sequence.
  • FGF-9 was found to have no typical signal sequence in its N-terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted efficiently from cDNA-transfected COS cells despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cells. The secreted protein lacked no amino acid residues at the N-terminus with respect to those predicted by the cDNA sequence, except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the FGF-9 gene is highly conserved.
  • FGF-20 a novel human FGF, FGF-20. 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 FGF-16 (64% identity).
  • the present invention provides methods of preventing and/or treating alimentary mucositis comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins.
  • the present invention provides methods of preventing and/or treating alimentary mucositis comprising administering to a subject in need thereof a prophylactically and/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,
  • the present invention provides methods of preventing and/or treating alimentary mucositis comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a protein isolated from a cultured host cell containing an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 , 3, 5, 6, 8, 9, 21 , 23, 25, 27,
  • nucleic acid molecule encoding a 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, or 40; and (c) a nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence of SEQ ID NO: 1 , 3, 5, 6, 8, 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39 or 41 , or a complement of said nucleic acid molecule.
  • the stringent 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 60oC, and at least one wash in 0.2 X SSC, 0.01% BSA.
  • one or more CG53135 proteins are isolated from a cultured eukaryotic cell. In some other specific embodiments, one or more CG53135 proteins are isolated from a cultured prokaryotic cell. In a preferred embodiment, one or more CG53135 proteins are isolated from E. coli.
  • one or more CG53135 proteins isolated from a cultured host cell has a purity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • the present invention provides methods of preventing and/or treating alimentary mucositis comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier, 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 pharmaceutically acceptable carrier used in accordance to the present invention comprises 0.04M sodium acetate, 3% Glycerol (volume/volume), and 0.2M Arginine- HCl at pH 5.3.
  • a pharmaceutically acceptable carrier used in accordance to the present invention comprises 0.1-1 M arginine or a salt thereof, 0.01-0.1 M sodium phosphate monobasic (NaH2P04»H20), and 0.01% -0.1% weight/volume (“w/v”) polysorbate 80 or polysorbate 20.
  • the arginine or a salt thereof used in a pharmaceutically acceptable carrier of the invention is selected from the group consisting of arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride.
  • the arginine or a salt thereof in a pharmaceutically acceptable carrier is of 0.5 M.
  • sodium phosphate monobasic used in a pharmaceutically acceptable carrier in accordance to the present invention is 0.05 M.
  • the polysorbate 80 or polysorbate 20 in a pharmaceutically acceptable carrier used in accordance to the present invention is 0.01% (w/v).
  • compositions of the present invention comprising a pharmaceutically acceptable carrier and one or more CG53135 proteins at a concentration of 0.005-50 mg/ml, 0.5-30 mg/ml, 1 -30 mg/ml, or 1 -10 mg/ml.
  • the compositions of the present invention comprise an isolated protein comprising an amino acid sequence of SEQ ID N0:24.
  • the compositions of the present invention comprise an isolated protein comprising an amino acid sequence of SEQ ID NO:2.
  • the compositions of the present invention comprise two or more CG53135 proteins.
  • compositions of the present invention 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.
  • compositions of the present invention comprise a first protein comprising an amino acid sequence of SEQ ID NO:24, a second protein comprising an amino acid sequence of SEQ ID NO:2, and one or more isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, 30 and 32.
  • compositions of the present invention comprise a first protein comprising an amino acid sequence of SEQ ID NO:24, a second protein comprising an amino acid sequence of SEQ ID NO:2, 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.
  • the compositions of the invention are lyophilized.
  • alimentary mucositis that can be prevented or treated include, but is not limited to, oral mucositis, enteritis, esophagitis, stomatitis, and proctitis.
  • Such alimentary mucositis may be caused by a chemical insult, a biological insult, radiation, or a combination thereof.
  • the effective amount to prevent and/or treat alimentary mucositis is between 0.001 - 3 mg/kg, 0.01 - 1 mg/kg, or 0.01-0.5 mg/kg (protein concentration measured by UV assay).
  • the effective amount to prevent and/or treat alimentary mucositis is about 0.03 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, or about 3 mg/kg ((protein concentration measured by UV assay).
  • a composition comprising one or more CG53135 proteins is administered to a subject in need thereof as a single dose 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 (protein concentration measured by UV assay).
  • a composition comprising one or more CG53135 proteins is administered to a subject in need thereof as a multiple dosing with each unit dosage of 0.001-0.5 mg/kg, 0.01-0.2 mg/kg, 0.03 mg/kg, 0.1 mg/kg, or 0.2 mg/kg.
  • a composition comprising one or more CG53135 proteins is administered to a subject in need thereof parenterally (e.g., intravenous administration or subcutaneous administration). In some embodiments, a composition comprising one or more CG53135 proteins is administered to a subject in need thereof by rectal administration, transdermal administration, or transmucosal administration (e.g., nasal administration).
  • 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), or its fragments, derivatives, variants, homologs, or analogs.
  • a CG53135 protein retains at least some biological activity of FGF-20 (SEQ ID NO: 2).
  • 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.
  • CG53135-01 SEQ ID NOs:1 and 2 represents the first identified FGF-20 (see U.S. Patent Application No.
  • CG53135-05 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 NO:2 (the aspartic acid at position 206 is changed to asparagine, " 206 D ⁇ 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 1 shows a summary of some of the CG53135 family members.
  • 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.
  • the invention includes nucleic acid molecules that can hybridize to FGF-20 under stringent hybridization conditions. Table 1. Summary of some of the CG53135 family members
  • the term "effective amount” refers to the amount of a therapy (e.g., a composition comprising a CG53135 protein) which is sufficient to reduce and/or ameliorate the severity and/or duration of alimentary mucositis or one or more symptoms thereof, prevent the advancement of alimentary mucositis, cause regression of alimentary mucositis, prevent the recurrence, development, or onset of one or more symptoms associated with alimentary mucositis, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
  • a therapy e.g., a composition comprising a CG53135 protein
  • 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.
  • 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.
  • 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.
  • 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 SSC, 0.01% BSA.
  • stringent hybridization conditions are hybridization at 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.1XSSC, 0.2% SDS at about 68 °C.
  • 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.
  • 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.
  • 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.
  • 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").
  • the protein agent When the protein agent is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein agent preparation.
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein agent preparation.
  • the protein agent 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.
  • protein agents disclosed herein are isolated.
  • nucleic acid molecules refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule.
  • 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.
  • nucleic acid molecules are isolated.
  • the terms “prevent,” “preventing,” and “prevention” refer to the prevention of the recurrence, onset, or development of alimentary mucositis 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.
  • a therapy e.g., a composition comprising a CG53135 protein
  • 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 alimentary mucositis or one or more symptoms thereof, or to enhance or improve the prophylactic effect(s) of another therapy.
  • 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 subject is a mammal, preferably a human, who has been exposed to or is going to be exposed to an insult that may induce alimentary mucositis (such as radiation, chemotherapy, chemical warfare agents, or biological insults).
  • the subject is a farm animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat) that has been exposed to or is going to be exposed to a similar insult.
  • the term "subject” is used interchangeably with “patient” in the present invention.
  • the terms “treat,” “treatment,” and “treating” refer to the reduction of the progression, severity, and/or duration of alimentary mucositis 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).
  • therapies e.g., a composition comprising a CG53135 protein.
  • therapeutically effective amount refers to the amount of a therapy
  • composition comprising a CG53135 protein
  • a composition comprising a CG53135 protein
  • FIG. 1. RP-HPLC analysis of CG53135-05 E. coli purified product (by Process 1 and 2, respectively, see Section 6.18.1 and 6.18.2 for description of the processes).
  • FIG. 2. Tryptic map of CG53135-05 E. coli purified product (by Process 1 and 2, respectively).
  • FIG. 3. 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).
  • FIG. 4. CG53135 stimulates Growth of NIH 3T3 Fibroblasts. Duplicate wells of serum starved
  • NIH 3T3 cells were treated for 1 day with purified CG53135-01 (1 ug) 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).
  • FIG. 5. CG53135 induces DNA synthesis in 786-0 Kidney Epithelial cells. Serum starved 786-0 cells were left untreated or treated with partially purified CG53135-01 (from 5 ng/uL 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).
  • SE standard error
  • FIG. 7 Effect of CG53135 E. coli purified product in the treatment of radiation-induced mucositis.
  • the total number of days in which animals in each group exhibited a mucositis score > 3 was summed and expressed as a percentage of the total number of days scored.
  • Statistical significance of observed differences with the respective vehicle control was calculated using chi- square analysis.
  • FIG. 7. 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.
  • FIG. 8 shows the cell positions in the crypt.
  • FIG. 9 shows the crypt survival curve comparing prophylactic administration of CG53135-05 E. coli purified product treatment to PBS control group following different radiation dosages.
  • FIG. 10 shows the effect of prophylactic administration of CG53135-05 E. coli purified product on mice intestinal crypt survival after radiation insult.
  • FIG. 11 (A) and (B) show the mean daily mocositis 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).
  • FIG. 12 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).
  • FIG. 13 shows percent weight gain in animals with mucositis treated with CG53135-05 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.
  • FIG. 14 shows Weight change represented as Area Under the Curve (AUC) gain in animals with mucositis treated with CG53135-05 E. coli purified product.
  • AUC Area Under the Curve
  • 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. 15 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.
  • AUC Area Under the Curve
  • 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. 16 shows duration of severe mucositis following treatment with CG53135-05 E. coli purified product. Number of days with mucositis scores of 3 or higher.
  • FIG. 17 (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. 17 (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. 18 shows 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. 19 shows analysis of diarrhea score for each day of observation.
  • the present invention provides methods for the prevention and/or treatment of alimentary mucositis.
  • the present invention provides Fibroblast Growth Factor (FGF) 20, its variants, derivatives, homologs, and analogs (collectively referred to as "CG53135") that can be used in the treatment and/or prevention of alimentary mucositis.
  • FGF Fibroblast Growth Factor
  • CG53135 has stimulatory effects on the proliferation of epithelial cells and mesenchymal cells, as well as stimulatory effects on stem cells, such as but are not limited to, intestinal stem cells and hematopoietic stem cells, and therefore is effective in preventing and/or treating alimentary mucositis.
  • compositions 5.1 CG53135 The present invention provides for compositions comprising CG53135 for prevention and/or treatment of alimentary mucositis.
  • 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), or its fragments, derivatives, variants, homologs, or analogs.
  • 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.
  • a CG53135 is CG53135-12 (SEQ ID NOs:21 and 22), which is a single nucleotide polymorphism ("SNP") of FGF-20 (i.e., 206 D ⁇ N).
  • SNP single nucleotide polymorphism
  • CG53135 refers to a nucleic acid molecule encoding a FGF-20 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.
  • the invention provides a fragment of a CG53135 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.
  • CG53135 protein fragment includes, but is not limited to, residues 2-211 , 3-211 , 9-211 , 12-211 , 15-211 , 24-211 , 52-211 , 54-211 , 55-211 , 63-196, 63-211 , or 63-194 of FGF-20 (SEQ ID NO:2).
  • the nucleic acid encodes a protein fragment that includes residues 2-211 , 3-211 , 9-211 , 12-211 , 15-211 , 24-211 , 52-211 , 54-211 , 55-211 , 63-196, 63-211 , or 63-194 of SEQ ID NO:2.
  • the invention also encompasses derivatives and analogs of FGF-20.
  • 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.
  • 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.
  • 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.
  • a wild-type FGF-20 nucleic acid sequence is codon optimized to the nucleic acid sequence of SEQ ID NO:8 (CG53135-05).
  • 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.
  • 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.
  • 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.
  • a derivative or an analog of FGF-20 can also be made according to the methods described in, e.g., PCT Publication No. WO 2004/018499 A2, and US Publication Nos.
  • 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.
  • 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.
  • restriction endonuclease(s) the sequence of a derivative or analog of FGF-20.
  • 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. etal., 1978, J. Biol. Chem 253:6551), use of TAB.RTM. linkers (Pharmacia), etc.
  • Manipulations of the FGF-20 sequence may also be made at the protein level.
  • 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.
  • analogs and derivatives of FGF-20 can be chemically synthesized.
  • 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.
  • 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, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids.
  • 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.
  • the non-FGF-20 amino acid sequence is fused at the amino-terminus of an FGF-20 or a fragment thereof.
  • 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.
  • a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • 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.
  • 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. Natl. 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.
  • chimeric genes comprising an essential portion of FGF-20 molecule fused to a heterologous (non-FGF-20) protein-encoding sequence may be constructed.
  • 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.
  • chimeric construction can be used to target FGF-20 to a specific site.
  • 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), ⁇ -galactosidase, a maltose binding protein (MalE), a cellulose binding protein (CenA) or a mannose protein, etc.
  • a CG53135 protein is carbamylated.
  • 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.
  • 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.
  • CG53135 refers to CG53135-01 (SEQ ID NOs:1 and 2), CG53135-02
  • 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.
  • a CG53135 is carbamylated, for example, a carbamylated CG53135-13 protein or a carbamylated CG53135-05 protein.
  • any techniques known in the art can be used in purifying a CG53135 protein, including but 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).
  • separation by precipitation 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
  • separation in solution e.g., gel
  • CG53135 proteins employed in a composition of the invention can be in the range of 80 to 100 percent of purity, or at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of purity.
  • one or more CG53135 proteins employed in a composition of the invention has a purity of at least 99%.
  • CG53135 is purified to apparent homogeneity, as assayed, e.g., by sodium dodecyl sulfate polyacrylamide gel electrophoresis. 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 refers to a nucleic acid sequence encoding a CG53135 protein operably associated with one or more regulatory regions that 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 that are 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.
  • RNA polymerase a promoter which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.,
  • 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
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metaliothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K
  • bacterial cells such as Escherichia coli and eukaryotic cells are used for the expression of a recombinant CG53135 molecule.
  • 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 etal., 1986, Gene 45:101 ; and Cockett etal., 1990, Bio/Technology 8:2).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the CG53135 molecule being expressed.
  • vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited to, the E. coli expression vector pCR2.1 TOPO (Invitrogen); plN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101- 3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509) and the like.
  • pFLAG Sigma
  • pMAL NEB
  • pET Novagen
  • GST glutathione 5-transferase
  • 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.
  • 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.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • 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).
  • an AcNPV promoter e.g., the polyhedrin promoter
  • a number of viral-based expression systems may be utilized.
  • 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 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).
  • 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.
  • eukaryotic host cells that 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.
  • mammalian host cells include, but are not limited to, PC12, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030 and HsS78Bst cells.
  • Expression in a bacterial or yeast system can be used if post-translational modifications are found to be non- essential for a desired activity of CG53135.
  • E. coli is used to express a CG53135 sequence.
  • stable expression in cells is preferred.
  • Cell lines that stably express CG53135 may be engineered by using a vector that contains a selectable marker.
  • 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.
  • 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.
  • 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.
  • CG531335 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.
  • 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.
  • N- ⁇ -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- ⁇ -deprotected amino acid to an ⁇ -carboxyl group of an N- ⁇ -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- ⁇ -protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.
  • Any methods known in the art can be used to determine the identity of a purified CG53135 protein in a composition used in accordance to the instant invention. Such methods include, but are not limited to, Western Blot, sequencing (e.g., 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, and SDS-PAGE.
  • sequencing e.g., Edman sequencing
  • liquid chromatography e.g., HPLC, RP-HPLC with both UV and electrospray mass spectrometric detection
  • mass spectrometry e.g., total amino acid analysis, peptide mapping, and SDS-PAGE.
  • the secondary, tertiary and/or quaternary structure of a CG53135 protein can analyzed by any methods known in the art, e.g., 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
  • the purity of a CG53135 protein in a composition used in accordance to the instant invention can be analyzed by any 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”), and Western Blot (e.g., host cell protein Western Blot).
  • SDS-PAGE sodium dodecyl sulphate polyacrylamide gel
  • a CG53135 protein in a composition used in accordance to the instant invention is at least 97%, at least 98%, or at least 99% pure by densitometry. In another preferred embodiment, a CG53135 protein in a composition used in accordance to the instant invention is more than 97%, more than 98%, or more than 99% pure by densitometry.
  • the biological activities and/or potency of CG53135 used in accordance with the present invention can be determined by any methods known in the art. For example, compositions for use in therapy in accordance to the methods of the present invention can be tested in suitable cell lines for one or more activities that FGF-20 possesses (e.g., cellular proliferation stimulatory activity).
  • compositions for use in a therapy in accordance to the methods of the present invention can also be tested in suitable animal model systems prior to testing in humans.
  • animal model systems include, but are not limited to, mucositis models in rats, mice, hamsters, chicken, cows, monkeys, rabbits, etc.
  • mice oral mucositis model Xu et al., Radlother Oncol 1 :369-374 (1984); hamster oral mucositis model, Sonis, In: Teicher B (ed) Tumor models in cancer research, Humana Press, Totowa, New Jersey (2002); rat gastrointestinal mucositis model, Gibson et al., J Gastroenterol Hepato 18:1095-1100 (2003); mouse intestinal stem cells, Potten et al., Gut 36(6):864-873 (1995).
  • an index can be developed that combines observational examination of the animals as well as their survival status.
  • Non-limiting examples are given in Section 6.5, infra.
  • Any staging/scoring system for human patients known in the art may also be used, for example, World Health Organization (WHO) oral mucositis (OM) scoring system an/or the Oral Mucositis Assessment Scale (OMAS) may be used to evaluate the effectiveness of the compositions of the invention in preventing and/or treating oral mucositis.
  • WHO World Health Organization
  • OM oral mucositis scoring system
  • OFMAS Oral Mucositis Assessment Scale
  • any assays known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utilities of the combinatorial therapies disclosed herein for prevention and/or treatment of alimentary mucositis.
  • CG53135 The effectiveness of CG53135 on preventing and/or treating alimentary mucositis can be monitored by any methods known to one skilled in the art, including but not limited to, clinical evaluation, and measuring the level of CG53135 biomarkers in a biosample.
  • CG53135 biomarkers include, but are not limited to, CXCL1 , IL-6, and IL-8. Any adverse effects during the use of CG53135 alone or in combination with another therapy (e.g., another therapeutic or prophylactic agent) are preferably also monitored.
  • Examples of adverse effects of chemotherapy during a cancer treatment include, but are not limited to, gastrointestinal toxicity such as, but not limited to, early and late forming diarrhea and flatulence; nausea; vomiting; anorexia; leukopenia; anemia; neutropenia; asthenia; abdominal cramping; fever; pain; loss of body weight; dehydration; alopecia; dyspnea; insomnia; dizziness, mucositis, xerostomia, and kidney failure, as well as constipation, nerve and muscle effects, temporary or permanent damage to kidneys and bladder, flu-like symptoms, fluid retention, and temporary or permanent infertility.
  • Adverse effects from radiation therapy include, but are not limited to, fatigue, dry mouth, and loss of appetite.
  • adverse effects include gastrointestinal toxicity such as, but not limited to, early and late forming diarrhea and flatulence; nausea; vomiting; anorexia; leukopenia; anemia; neutropenia; asthenia; abdominal cramping; fever; pain; loss of body weight; dehydration; alopecia; dyspnea; insomnia; dizziness, mucositis, xerostomia, and kidney failure.
  • Adverse effects from biological therapies/immunotherapies include, but are not limited to, rashes or swellings at the site of administration, flu-like symptoms such as fever, chills and fatigue, digestive tract problems and allergic reactions.
  • Adverse effects from hormonal therapies include but are not limited to nausea, fertility problems, depression, loss of appetite, eye problems, headache, and weight fluctuation. Additional undesired effects typically experienced by patients are numerous and known in the art. Many are described in the Physicians' Desk Reference (58th ed., 2004).
  • the present invention provides methods of preventing and/or treating alimentary mucositis comprising administering to a subject in need thereof an effective amount of a composition comprising one or more isolated CG53135 proteins.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • an insult e.g., a chemical insult, a radiation insult, a biological insult, or a combination thereof
  • 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.
  • an insult e.g., a chemical insult, a radiation insult, a biological insult, or a combination thereof
  • 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.
  • the present invention provides a method of preventing and/or treating alimentary mucositis comprising cyclically administering a composition comprising one or more CG53135 proteins.
  • 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.
  • 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.
  • 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.
  • a composition comprising one or more isolated 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.
  • 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.
  • 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), interleukin-11 (IL-11), granulocyte-macrophage colony stimulating factor (GM-CSF), keratinocyte growth factor (KGF), L-glutamine, Amifostene, and
  • 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.
  • 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.
  • 5.5 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 LD 50 (the dose lethal to 50% of the population) and the ED 5 o (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 LD 5 o/ED 5 o.
  • Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred.
  • 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 ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • 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 IC 50 (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.
  • 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).
  • 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 0.01-1 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.
  • 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.
  • each patient 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.
  • a small amount e.g., 1/100 to 1/10 of the prescribed dose
  • the injection site is examined one and two hours after the test. If no reaction is detected, then the full dose is administered.
  • 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 PHARMACEUTICAL COMPOSITIONS The compositions used in accordance to the present invention can be administered to a subject at a prophylactically or therapeutically effective amount to prevent and/or treat alimentary mucositis.
  • Various delivery systems are known and can be used to administer
  • Such delivery systems include, but are not limited to, encapsulation in liposomes, microparticles, microcapsules, 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, transmucosal, rectal, and oral routes.
  • compositions used in accordance to the methods 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., eye mucosa, oral mucosa, vaginal mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local.
  • 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.
  • dual chambered syringes e.g., Vetter Lyo-Ject dual-chambered syringe by Vetter Pharmar-Fertist
  • Vetter Lyo-Ject dual-chambered syringe by Vetter Pharmar-Fertist
  • Such systems are desirable for lyophilized formulations, and are especially useful in an emergency setting.
  • administration can be by direct injection at the site (or former site) of rapidly proliferating tissues that are most sensitive to an insult, such as radiation, chemotherapy, or chemical/biological warfare agent.
  • the nucleic acid can be administered in vivo to promote expression of their encoded proteins (e.g., CG53135 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 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.
  • their encoded proteins e.g., CG53135 proteins
  • a nucleic acid of the invention 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.
  • 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.
  • the pharmaceutical compositions are formulated to be suitable for the route of administration to a subject.
  • the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • carrier refers to a diluent, adjuvant, bulking agent (e.g.,arginine in various salt forms, 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.
  • oils e.g., oils of petroleum, animal, vegetable or synthetic origins, such as peanut oil, soybean oil, mineral oil, sesame oil and the like
  • 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.
  • 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.
  • 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.
  • compositions for intravenous administration are solutions in sterile isotonic or hypertonic aqueous buffer.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compositions comprising CG53135 may be complexed to lipids, in particular to cationic lipids.
  • 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.
  • 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.
  • a pressurized volatile e.g., a gaseous propellant, such as Freon or hydrofluorocarbons
  • 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.
  • 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., dichlorodifluoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, other hydrofluorocarbons, carbon dioxide or other suitable gas).
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, other hydrofluorocarbons, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Microcapsules 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.
  • 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).
  • 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
  • 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).
  • 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
  • preservatives e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid
  • compositions of the invention are orally administered in conjunction with one or more penetration enhancers, e.g., alcohols, surfactants and chelators.
  • penetration enhancers include, but are not limited to, fatty acids and esters or salts thereof, bile acids and salts thereof.
  • combinations of penetration enhancers are used, e.g., alcohols, fatty acids/salts in combination with bile acids/salts.
  • sodium salt of iauric acid, capric acid is used in combination with UDCA.
  • 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, polyalkylacrylat.es, 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.
  • PLGA poly(DL-lactic-co-glycolic acid
  • PEG polyethyieneglycol
  • 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.
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • 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.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry iyophilized powder or water free concentrate in a sealed container, such as a vial, ampoule or sachette, indicating the quantity of active agent.
  • a sealed container such as a vial, ampoule or sachette, indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion container containing sterile pharmaceutical grade water or saline.
  • 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.
  • compositions comprising CG53135 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.
  • 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.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, as an emulsion in an acceptable oil
  • sparingly soluble derivatives for example, as a sparingly soluble salt.
  • the ingredients of the compositions used in accordance to the methods of the invention are derived from a subject that is the same species origin or species reactivity as recipient of such compositions.
  • a formulation used in accordance to the methods of the invention comprises 0.02 M - 0.2 M acetate, 0.5-5% glycerol, 0.2-0.5 M arginine-HCI, and one ore more CG53135 proteins, preferably 0.5-5 mg/ml (UV).
  • a formulation used in accordance to the methods of the invention comprises 0.04M sodium acetate, 3% glycerol (volume/volume), 0.2 M arginine-HCI at pH 5.3, and one or more isolated CG53135 proteins, preferably 0.8 mg/ml (UV).
  • a formulation used in accordance to the methods of the invention comprises 0.01-1 M of a stabilizer, such as arginine in various salt forms, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, 0.01-0.1 M sodium phosphate monobasic (NaH P0 4 -H 2 0), 0.01 %-0.1% weight volume (“w/v”) polysorbate 80 or polysorbate 20, and one or more CG53135 proteins, preferably 0.005-50 mg/ml (UV).
  • a stabilizer such as arginine in various salt forms, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose
  • 0.01 %-0.1% weight volume (“w/v”) polysorbate 80 or polysorbate 20 0.01 %-0.1% weight volume
  • CG53135 proteins preferably 0.005-50 mg/ml
  • a formulation used in accordance to the methods of the invention comprises 30mM sodium citrate, pH 6.1 , 2mM EDTA, 200mM sorbitol, 50mM KCI, 20% glycerol, and one or more isolated CG53135 proteins.
  • kits for carrying out the therapeutic regimens of the invention comprise in one or more containers prophylactically or therapeutically effective amounts of the composition of the invention (e.g., a composition comprising one or more CG53135 proteins) in pharmaceutically acceptable form.
  • 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.
  • 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.
  • a kit of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the formulation, and/or a packaged alcohol pad.
  • kits comprising a plurality of containers each comprising a pharmaceutical formulation or composition comprising a dose of the composition of the invention (e.g., a composition comprising one or more CG53135 proteins) sufficient for a single administration.
  • the packaging material and container are designed to protect the stability of the product during storage and shipment.
  • compositions of the invention are stored in containers with biocompatible detergents, including but not limited to, lecithin, taurocholic acid, and cholesterol; or with other proteins, including but not limited to, gamma globulins and serum albumins.
  • EXAMPLE 1 IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN FGF-20 NUCLEIC ACID SEQUENCES This example demonstrated how some of the single nucleotide polymorphisms (SNPs) of FGF-20 were identified.
  • 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.
  • 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.
  • 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.
  • 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.
  • CG53135-01 (the full-length CG53135 gene) was cloned as a Bgl ll-Xho I fragment into the Bam Hl-Xho I sites in mammalian expression vector, pcDNA3.1 V5His (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.
  • 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 ll-Xho I fragment into the Bam Hl- Xho I sites of mammalian expression vector pCEP4/Sec (CuraGen Corporation).
  • the resultant construct, plgK-FGF-20 (construct 1b) has a heterologous immunoglobulin kappa (IgK) signal sequence that could aid in secretion of CG53135-01.
  • CG53135-01 was detected in the conditioned medium using an anti-V5 antibody.
  • a Bgl ll-Xho I fragment encoding the full- length CG53135-01 gene was cloned into the Bam Hl-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.
  • CG53135-01 protein was detected in the soluble fraction of the cells.
  • CG53135-05 a codon-optimized, full-length FGF- 20 gene
  • CG53135-02 a codon-optimized deletion construct of FGF-20, with the N-terminal amino acids 2-54 removed
  • CAATG Nde I restriction site
  • CG53135-0 The full-length, codon-optimized version of CG53135 is referred to as CG53135-05.
  • oligonucleotide primers were designed to amplify the deleted CG53135 gene from pCRScript-CG53135.
  • 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.
  • Nde l-Hind III fragment was isolated from pCR2.1-53135del and subcloned into Nde l-Hind Ill-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.
  • the final purified protein product includes some truncated form of FGF-20 (e.g., 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)) in addition to the full length FGF-20, and a protein consisting of amino acids 3-211 (CG53135-13, SEQ ID NO:24) of FGF-20 constitutes the majority of the final purified protein product. 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.
  • FGF-20 e.g., 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)
  • CG53135-05 E. coli purified product is used herein to refer to a purified protein product from E. coli expressing a CG53135-05 construct.
  • 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).
  • RP-HPLC Assay Peak Identification Purified drug substance (by both Process 1 and Process 2, respectively, as described in Section 6.18 below) 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 ⁇ m, 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. 1 ).
  • Table 5 Edman sequencing data for the first 20 amino acids of CG53135-05 E. coli purified product for Process 1 and 2, respectively.
  • 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 ⁇ L of 6 N HCl, 0.2% phenol containing 2 nmol norleucine as an internal standard. Samples were dried in a Speed Vac Concentrator and dissolved in 100 ⁇ L 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 6 below. Note that Cys and trp are destroyed during acid hydrolysis of the protein. Asn and gin 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.
  • 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 ⁇ m; 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.
  • Bioassay 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.
  • BrdU bromodeoxyuridine
  • 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% C0 2 /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% C0 2 /air.
  • BrdU (10 mM final concentration) was added and incubated with the cells for 2 hours at 37°C in 10% C0 2 /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 7).
  • the Pl 200 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 Pl 200 of 0.7 - 11 ng/mL (Table 7).
  • EXAMPLE 4 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.
  • 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% C0 2 /air (to subconf luence at 5% C0 2 for dedifferentiated chondrocytes and NHOst). Cells were then starved in respective basal growth medium for 24-72 hours.
  • coli or pCEP4/Sec or pCEP4/Sec-FGF 20X enriched conditioned medium was added (10 ⁇ L/100 ⁇ L of culture) for 18 hours.
  • BrdU (10 ⁇ M 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.
  • 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.
  • NHOst primary human osteoblasts
  • HSMC human aorta smooth muscle cells
  • NIH 3T3 cells were cultured with 1 ⁇ g CG53135-01 or control for 48 hours and then counted (FIG. 4).
  • CG53135 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. 5).
  • 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.
  • CG53135 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.
  • HUVEC human umbilical vein endothelial cells
  • BAEC bovine aortic endothelial cells
  • HMVEC-d human endothelial, dermal capillary.
  • HUVEC human umbilical vein endothelial cells
  • BAEC bovine aortic endothelial cells
  • HMVEC-d human endothelial, dermal capillary.
  • 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.
  • 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 CG53135-05 protein products, and the results are summarized in the Conclusion section below.
  • 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.
  • CG53135- 02 also induces proliferation of mesenchymal and epithelial cells.
  • CG53135-02 induces proliferation of endothelial cells.
  • EXAMPLE 5 ACTIVITY OF CG53135 IN HAMSTER MODEL OF ACUTE RADIATION- INDUCED ORAL MUCOSITIS (N-152 STUDY) CG53135 protein was evaluated for activity in a hamster model of radiation-induced oral mucositis, and its activity compared with KGF-2, another FGF family member.
  • KGF-2 also referred to as FGF-10, is active in models of wound healing and inflammatory bowel disease (Miceli etal. J.
  • Molecules that show activity in the acute radiation model may be further evaluated in the more complex models of fractionated radiation, chemotherapy, or concomitant therapy.
  • an acute radiation dose of approximately 40 Gy on Day 0 is administered in order to induce severe mucositis. This dose results in predictable ulcerative oral mucositis that typically peaks around Day 16-18.
  • CG53135-05 protein used in this study was purified as Batch Dev 08-02.
  • the recombinant human DNA protein, CG53135-05 was expressed using Escherichia coli BLR (DE3) cells (Novagen, Darmstadt, Germany). These cells were transformed with full-length, codon-optimized CG53135-05 using pET24a vector (Novagen).
  • a GMP manufacturing cell bank (MCB) of these cells was produced.
  • CG53135-05 was purified from clarified cell lysate by 2 cycles of ion exchange chromatography and ammonium sulfate precipitation. The final precipitate was washed with purified water and suspended in formulation buffer as follows: 30 mM citrate (pH 6.0), 2 mM EDTA, 200 mM sorbitol, 50 mM KCI, 20% glycerin.
  • Animals were acutely radiated with a single dose of radiation (40 Gy/dose) on the left buccal mucosa on Day 0. Animals were treated once daily with vehicle or CG53135-05 E coli purified product intraperitoneally (IP) or topically following acute radiation. Animals in Groups 1 to 5 received IP injection of test materials once per day. For Groups 6 to 8, test material was applied topically to the cheek pouch three times per day. The following dosing schedules were used: Day 3 to Day 15 (Groups 2, 3 and 7), and Day -5 (i.e., five days prior to radiation) to Day -2 (i.e., two days prior to radiation), then Day 3 to Day 15 (Groups 1 , 4, 5, 6 and 8). Doses of CG53135-05 E.
  • coli purified product were 300 ⁇ g/day (Groups 2, 4, 7 and 8) and 600 ⁇ g/day (Group 3).
  • the KGF-2 dose was 300 ⁇ g/day (Group 5).
  • Mucositis was evaluated on alternate days beginning on Day 6 and continued until the conclusion of the experiment on Day 28 (i.e., Days 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 & 28).
  • Clinically relevant oral mucositis e.g., mucositis score of ⁇ 3 developed -14 days after radiation.
  • Each hamster was weighed daily for the period of the study (i.e., Day -5 to Day 28) and its survival was recorded in order to assess possible differences in animal weight among treatment groups as an indication for mucositis severity or possible toxicity resulting from the treatments.
  • Mucositis was scored visually by comparison to a validated photographic scale, ranging from 0 (normal) to 5 (for severe ulceration). The clinical scale is described in Table 9.
  • 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.
  • a photograph was taken of each animal's mucosa using a standardized technique.
  • all film was developed and the photographs randomly numbered for blinded scoring.
  • 2 independent, trained observers graded the photographs in blinded fashion using the above-described scale.
  • the actual blinded score was be based upon the average of the score assigned by the 2 blinded, independent evaluators. Only the scores from blinded photographic evaluation was statistically analyzed and reported in the results.
  • coli purified product resulted in a significant reduction in the severity of mucositis compared with untreated control animals (p ⁇ 0.001 by Chi-square analysis) and significantly reduced mean daily mucositis scores for 3 of 12 scoring days compared with the vehicle control group.
  • administration of 1200 ⁇ g CG53135-05 E coli purified product significantly reduced the severity of mucositis relative to the vehicle control group (p ⁇ 0.001 by Chi-square analysis) and significantly reduced mean daily mucositis scores for 5 scoring days. No significant difference in body weight was observed in any of the treatment regimens when compared with the controls.
  • 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).
  • CG53135 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.
  • EXAMPLE 7 EFFECT OF CG53135-05 ADMINISTRATION ON HAMSTER EPITHELIAL PROLIFERATION IN V7VO (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).
  • 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.
  • EXAMPLE 8 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).
  • mice were sacrificed at various times after a single 12 mg/kg (IP) 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 14).
  • mice 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 14).
  • 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 1Gy 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 14. Study Design
  • 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.
  • Table 15 The comparisons shown in Table 15 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 14), stem cell radiosensitivity was assessed.
  • 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 15).
  • EXAMPLE 9 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 ClonoquantTM 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.
  • Escherichia coli BLR (DE3) cells (Novagen, Madison, Wl) 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.
  • MMCB manufacturing master cell bank
  • 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 18. 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.
  • 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 19. Table 19. Description of Mucositis Score Values
  • 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.
  • a photograph was taken of each animal's mucosa using a standardized technique.
  • 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.
  • 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.
  • AUC area under the curve
  • 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 coli purified product at 24 mg/kg or 48 mg/kg on day 1 to a low of 2.63 in the group treated with CG53135- 05 E. coli purified product at 12 mg/kg on day 1.
  • 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 20 and Figure 11. Further, Figure 12 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.
  • Table 21 Mucositis scores as performed by using the Mann-Whitney Rank-Sum test
  • Weight Change the mean daily percentage weight change for each group is shown in Figure 13.
  • 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.
  • EXAMPLE 11 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
  • 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 6.10.
  • 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 on only one day after reaching a score of 2 did not show significance when compared with either control group.
  • CG53135-05 drug substance in the formulation as described in Section 6.18.1 , referred as "CG53135-05 drug substance" in this example
  • administered intravenously in human patients with advanced (Stage 4) cancer single rising- dose tolerance
  • 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.
  • a value of the OMAS system is obtained by summing the erythema and ulceration/pseudomembrane scores.
  • Adverse events 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.
  • 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).
  • conditioning regimens including melphalan (Mel 200), cyclophosphamide, carmustine and etoposide (CBV), carboplatin and thiotepa (CT), and busulfan/cyclophosphamide (targeted BuCy).
  • the median day of neutrophil engraftment (as determined by ANC>500/uL) occurred on day 13 after stem cell infusion.
  • EXAMPLE 13 MULTIPLE DOSES OF CG53135 FOR PREVENTING OR TREATING MUCOSITIS.
  • Preclinical studies using a validated hamster oral mucositis ("OM") model have supported the exploration of the effects of CG53135 in treating patients with active OM.
  • the results of the study further demonstrated that multiple-dosing regimen was more efficacious in reducing the duration and severity of OM (Study N-318 in Example 6.11).
  • a third phase I clinical trial can be conducted to assess the safety of 3 consecutive daily doses of CG53135-05 drug substance at 0.03, 0.1 , or 0.2 mg/kg (UV) on patients with active OM from CT with or without TBI as conditioning for AHSCT or on leukemia patients receiving chemotherapy.
  • OM scores, pharmacokinetics, and pharmacodynamics (PD) will be assessed.
  • the proposed single doses of 0.03, 0.1 , or 0.2 mg/kg (UV) have been administered on cancer patients in the two phase I clinical trials conducted to assess safety and tolerability of single dose CG53135-05 drug substance.
  • CG53135-05 drug substance was well tolerated with the proposed trial doses among the patients that have completed the trial.
  • the third phase I trial proposes daily administration of pharmacologic doses of CG53135-05 drug substance for 3 consecutive days. Given the limited duration of therapy of three days and the potential benefit of the information derived from the study (positive safety data will justify further detailed exploration of these safety issues), the risk- benefit profile is considered to be favorable. Blood samples will be collected to monitor changes in hematology and clinical chemistry and to measure levels of CG53135-05 drug substance and antibody to CG53135. Urine will be collected for urinalysis parameters. Electrocardiogram (ECG) as well as physical and eye examinations will also be performed for assessment of safety. Blood samples will be collected to assess biomarkers of drug activity. In the context of this study, biomarkers are defined as characteristics that are objectively measured and evaluated as indicators of pharmacologic responses to treatment with CG53135-05 drug substance. Biomarkers that may be monitored include cytokines.
  • WHO Health Organization
  • OM scoring system will be used to measure efficacy.
  • Patients receiving an autologous stem cell transplant following myelo-ablative chemotherapy and/or Total Body Irradiation (TBI) will be screened to enroll patients.
  • Patients will receive autologous stem cells twenty-four hours after completion of the chemotherapy with or without total body irradiation (TBI) regimen.
  • Patients will receive a CG53135-05 drug substance at least twenty-four hours after completion of the stem cell infusion.
  • Patients will be monitored post-drug administration until discharged or the first day of neutrophil engraftment (ANC > 500/ ⁇ l).
  • ANC neutrophil engraftment
  • Blood samples will be collected to measure levels of the CG53135, antibody to CG53135, and to monitor changes in hematology and clinical chemistry.
  • Urine will be collected for urinalysis parameters.
  • Electrocardiogram (ECG) and physical examinations will also be performed for assessment of safety. Blood samples will be collected to assess biomarkers of drug activity.
  • the primary end point of efficacy will be demonstrated as the duration and severity of oral mucositis.
  • the secondary end points will include days with fever, total parenteral nutrition, number of infections, use of intravenous narcotic analgesics, time to engraftment, time to discharge, incidence and duration of diarrhea, as well as mortality / progression-free disease at 90 days.
  • EXAMPLE 15 TREATMENT OF ORAL MUCOSITIS DERIVED FROM CHEMOTHERAPY OR COMBINATION OF CHEMOTHERAPY AND RADIATION THERAPY
  • a phase II clinical trial may be conducted to evaluate the efficacy of a CG53135-05 drug substance in treating oral mucositis from cancer patients that receive chemotherapy, radiation therapy, or a combination of chemotherapy and radiation therapy. This will be a double blind placebo controlled study with a single dose or multiple doses of a CG53135-05 drug substance intravenously administered.
  • the World Health Organization (WHO) OM scoring system will be used to measure efficacy of the treatment. Patients will be monitored daily for the onset of oral mucositis.
  • WHO World Health Organization
  • Treatment with a CG53135-05 drug substance will be initiated when patients develop oral mucositis with a WHO score of 1 or 2.
  • a CG53135-05 drug substance will be administered as a single dose or multiple doses.
  • Patients will be monitored post-drug administration until discharged or the first day of neutrophil engraftment (ANC>500/ ⁇ l) in bone marrow transplant patients. Patients will return for follow-up 2 weeks after discharge, 30 days and 90 days following the infusion of the drug.
  • Blood samples will be collected to measure levels of CG53135, antibody to CG53135, and to monitor changes in hematology and clinical chemistry.
  • Urine will be collected for urinalysis parameters.
  • Electrocardiogram (ECG) and physical examinations will also be performed for assessment of safety.
  • Blood samples will be collected to assess biomarkers of drug activity.
  • the primary end point of efficacy will be demonstrated as incidence of severe oral mucositis (WHO OM Grade 3 or 4) or as the duration and severity of oral mucositis.
  • the secondary end points will include days with fever, total parenteral nutrition, number of infections, use of intravenous narcotic analgesics, time to engraftment, time to discharge, incidence and duration of diarrhea, as well as mortality/progression-free disease at 90 days. 6.16 EXAMPLE 16: CG53135 REDUCES THE INCIDENCE.
  • 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.
  • 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:
  • 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. (FIG. 18A and 18B) 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. 19)
  • the primary aim of this study was to investigate whether CG53135 reduces CPT-11 -induced Gl mucositis in an in vivo animal model. The secondary aim was to test varying schedules of administration of CG53135.
  • 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 mortality. Animals were euthanized 168 hours following diarrhea induction. At euthanasia, tissues were harvested for histopathological evaluation of the gastrointestinal tract.
  • CG53135 pre-treated animals (16 mg/kg) demonstrated an improvement in gastrointestinal mucositis as measured by a reduction in the incidence of diarrhea. A reduction in overall mortality was also noted in this group. This has important implications for the use of CG53135 in Gl mucositis in humans, and should be further studied.
  • EXAMPLE 18 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.
  • 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
  • Fermentation and Primary Recovery Recombinant CG53135-05 was expressed using Escherichia coli 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.
  • MMCB Manufacturing Master Cell Bank
  • 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.
  • IPTG isopropyl-beta-D-thiogalactoside
  • 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.
  • the SP Sepharose FF pool was filtered through a 0.2 ⁇ m PES filter and frozen at -80°C.
  • 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 NaCl, 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.
  • Phenyl loading buffer 100 mM sodium citrate, 500 mM L-arginine, 750 mM NaCl, 5 mM EDTA, pH 6.0.
  • 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 ⁇ m PES filter and frozen at -80°C in 1.8 L aliquots.
  • 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 27).
  • 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.
  • PI 200 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 -
  • the new formulation consists 10 mg/mL (UV) of the protein product produced by the process described below ("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.
  • the previous formulation as described in U.S. Application No.
  • the acidic component of the acetate buffer is acetic acid, which sublimes during lyophilization.
  • the loss of acetic acid is at 100% level with the basic component, sodium acetate, being the only buffering agent.
  • This loss of acetic acid to lyophilization increases the pH to > 7.5, which is far from the target pH of 5.3.
  • 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.
  • CG53135-05 was precipitated using the precipitate buffer (50 mM NaPi, 5 mM EDTA, 1 M L-Arginine HCl, 2.5 M (NH 4 ) 2 S0 4 .
  • 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.
  • All formulation contains 0.2 M arginine.
  • An optimal concentration of the sodium phosphate as a buffering salt was observed (Table 32).
  • 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 32 The optimal concentration of sodium phosphate as a buffering salt is 50 mM
  • Table 33 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 7.0. 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%.
  • 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.
  • the improved process steps for the manufacturing of drug substance and drug product are described in Table 34, and each step is explained below. Table 34.
  • MMCB Manufacturing Master Cell Bank
  • MWCB Manufacturing Working Cell Bank
  • 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.
  • 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.
  • 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 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.
  • 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% (W/V).
  • 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.
  • the bed expansion factor is maintained between 2.5 - 3.0 times the packed bed column volume.
  • 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 7.0.
  • the column is further washed with 100 mM sodium phosphate, 5 mM EDTA, 0.9 M Arginine, pH 7.0.
  • the product is eluted with 100 mM sodium phosphate, 5 mM EDTA, 0.2 M Arginine, pH 7.0.
  • CUNO Filtration the PPG eluate is passed through an endotoxin binding CUNO 30ZA 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 7.0. The product is eluted with 50 mM sodium phosphate, 0.5 M Arginine, pH 7.0. 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 ⁇ m filter and subsequently diluted to 10 g/L.
  • Bulk Bottling the retentate from the concentration and diafiltration step is filtered through a 0.22 ⁇ m pore size filter into 2 L single use Teflon bottles. The bottles are frozen at -70°C.
  • Drug Product / Vial 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.
  • the CG53135-05 reference standard was prepared at Diosynth RTP Inc, using a 140L scale manufacturing process that was representative of the bulk drug substance manufacturing process (as described in the General Method of Manufacture).
  • the reference standard was stored as 1 mL aliquots in 2 mL cryovials at -80°C ⁇ 15°C. Purity of the final product was analyzed by SDS-PAGE, RP-HPLC, size exclusion-HPLC, and Western blot. Potency of the drug was measured by growth of NIH 3T3 cells in response to CG53135- 05 E. coli purified product. All data indicated that the final product is suitable for clinical uses.
  • Such alterations and changes may include, for example, different pharmaceutical compositions for the administration of the proteins according to the present invention to a mammal; different amounts of protein in the compositions to be administered; different times and means of administering the proteins according to the present invention; and different materials contained in the administration dose including, for example, combinations of different proteins, or combinations of the proteins according to the present invention together with other biologically active compounds for the same, similar or differing purposes than the desired utility of those proteins specifically disclosed herein.
  • Such changes and alterations also are intended to include modifications in the amino acid sequence of the specific desired proteins described herein in which such changes alter the sequence in a manner as not to change the desired potential of the protein, but as to change solubility of the protein in the pharmaceutical composition to be administered or in the body, absorption of the protein by the body, protection of the protein for either shelf life or within the body until such time as the biological action of the protein is able to bring about the desired effect, and such similar modifications. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims.
  • the invention and the manner and process of making and using it have been thus described in such full, clear, concise and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same.

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Abstract

La présente invention concerne des compositions et des procédés servant à prévenir et à traiter la mucosite alimentaire. Plus particulièrement, la présente invention fournit des procédés servant à prévenir et/ou à traiter la mucosite alimentaire en utilisant des compositions comprenant du FGF-20, un fragment, un dérivé, une variante, un homologue ou un analogue de celui-ci.
PCT/US2005/010545 2004-05-10 2005-03-29 Procédés de prévention et de traitement de la mucosite alimentaire Ceased WO2005112979A2 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
JP2007513139A JP2007536381A (ja) 2004-05-10 2005-03-29 消化管粘膜炎を予防または処置する方法
AU2005244737A AU2005244737A1 (en) 2004-05-10 2005-03-29 Methods of preventing and treating alimentary mucositis
CA002566439A CA2566439A1 (fr) 2004-05-10 2005-03-29 Procedes de prevention et de traitement de la mucosite alimentaire
EP05765938A EP1773372A2 (fr) 2004-05-10 2005-03-29 Procédés de prévention et de traitement de la mucosite alimentaire
PCT/US2005/039833 WO2006055264A1 (fr) 2004-11-03 2005-11-03 Formulations, procedes de production et utilisations du fgf-20
EP05851340A EP1812460A1 (fr) 2004-11-03 2005-11-03 Formulations, procedes de production et utilisations du fgf-20
JP2007540036A JP2008519032A (ja) 2004-11-03 2005-11-03 Ｆｇｆ−２０の調合物、生成方法および使用
CA002586213A CA2586213A1 (fr) 2004-11-03 2005-11-03 Formulations, procedes de production et utilisations du fgf-20
AU2005306898A AU2005306898A1 (en) 2004-11-03 2005-11-03 Formulations, methods of production and uses of FGF-20

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US84217904A 2004-05-10 2004-05-10
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US10/842,206 US20050256039A1 (en) 2003-05-09 2004-05-10 Novel fibroblast growth factors and methods of use thereof
US10/842,179 2004-05-10
US61301304P 2004-09-22 2004-09-22
US60/613,013 2004-09-22
US62355104P 2004-10-28 2004-10-28
US60/623,551 2004-10-28
US10/980,459 US20050256042A1 (en) 2003-05-09 2004-11-03 Methods of preventing and treating alimentary mucositis
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