EP1626696A2 - Nouveaux facteurs de croissance des fibroblastes et procedes pour les utiliser - Google Patents

Nouveaux facteurs de croissance des fibroblastes et procedes pour les utiliser

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Publication number
EP1626696A2
EP1626696A2 EP04751821A EP04751821A EP1626696A2 EP 1626696 A2 EP1626696 A2 EP 1626696A2 EP 04751821 A EP04751821 A EP 04751821A EP 04751821 A EP04751821 A EP 04751821A EP 1626696 A2 EP1626696 A2 EP 1626696A2
Authority
EP
European Patent Office
Prior art keywords
fgf
polypeptide
cartilage
seq
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04751821A
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German (de)
English (en)
Inventor
Jeffrey Peterson
Paul Sciore
Peter Mezes
Henri Lichtenstein
Michael Jeffers
William Larochelle
Marie Ruiz-Martinez
Galina Chernaya
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CuraGen Corp
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CuraGen Corp
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Filing date
Publication date
Application filed by CuraGen Corp filed Critical CuraGen Corp
Publication of EP1626696A2 publication Critical patent/EP1626696A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to compositions and methods of treatment of various conditions, including but are not limited to, stroke, wound healing, and joint diseases (e.g., osteoarthritis and rheumatoid arthritis). More particularly, the present invention relates to compositions comprising a member of the fibroblast growth factor family, FGF-CX (also known as CG53135-05 or FGF-20), its related polypeptides, nucleic acids encoding such polypeptides, and their uses for treating a condition, such as but is not limited to, stroke, would healing, and joint diseases (e.g., osteoarthritis and rheumatoid arthritis).
  • FGF-CX also known as CG53135-05 or FGF-20
  • FGF-1 basic FGF
  • FGF-2 basic FGF
  • FGFR-1 FGF receptor-1
  • FGFs and their receptors in brains of perinatal and adult mice has been examined. Messenger RNA of all FGF genes, with the exception of FGF-4, is detected in these tissues.
  • FGF-3, FGF-6, FGF-7 and FGF-8 genes demonstrate higher expression in the late embryonic stages than in postnatal stages, suggesting that these members are involved in the late stages of brain development.
  • FGF-1 and FGF-5 increased after birth.
  • FGF-6 expression in perinatal mice has been reported to be restricted to the central nervous system and skeletal muscles, with intense signals in the developing cerebrum in embryos but in cerebellum in 5 -day-old neonates.
  • FGF-receptor (FGFR)-4 a cognate receptor for FGF-6, demonstrates similar spatiotemporal expression, suggesting that FGF-6 and FGFR-4 play significant roles in the maturation of nervous system as a Ugand-receptor system.
  • these results strongly suggest that the various FGFs and their receptors are involved in the regulation of a variety of developmental processes of brain, such as proliferation and migration of neuronal progenitor cells, neuronal and glial differentiation, neurite extensions, and synapse formation. See, e.g., Ozawa et ah, Brain Res. Mol. Brain Res. 199641(l-2):279-88.
  • FGF polypeptide family include the FGF receptor tyrosine kinase (FGFRTK) family and the FGF receptor heparan sulfate proteoglycan (FGFRHS) family. These members interact to regulate active and specific FGFR signal transduction complexes. These regulatory activities are diversified throughout a broad range of organs and tissues, and in both normal and tumor tissues, in mammals. Regulated alternative messenger RNA (mRNA) splicing and combination of variant subdomains give rise to diversity of FGFRTK monomers.
  • mRNA alternative messenger RNA
  • Divalent cations cooperate with the FGFRHS to conformationally restrict FGFRTK trans-phosphorylation, which causes depression of kinase activity and facilitates appropriate activation of the FGFR complex by FGF.
  • FGFRTK trans-phosphorylation
  • different point mutations in the FGFRTK commonly cause craniofacial and skeletal abnormalities of graded severity by graded increases in FGF-independent activity of total FGFR complexes.
  • Other processes in which FGF family exerts important effects are liver growth and function, and prostate tumor progression.
  • GAF Glia-activating factor
  • GAF is a heparin- binding growth factor that was purified from the culture supernatant of a human glioma cell line. See, Miyamoto etah, 1993, Mol. Cell Biol. 13(7): 4251-4259. GAF shows a spectrum of activity slightly different from those of other known growth factors, and is designated as FGF-9.
  • 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. [007] FGFs have been shown to induce neuronal sprouting. See Proc. Natl. Acad.
  • Osteoarthritis is a degenerative joint disease and a frequent cause of joint pain that affects a large and growing population. OA is estimated to be the most common cause of disability in adults. The disease typically manifests itself in the 2nd to 3rd decades, with most people over forty years exhibiting some pathologic change in weight bearing joints, although the change may be asymptomatic. A systematic review of incidence and prevalence of OA of the knee in people older than 55 years in the United Kingdom reported an incidence of 25 percent per year, a prevalence of disability of 10 percent, and severe disability in about two to three percent.
  • OA results from a complex interplay of multiple factors, including joint integrity, genetics, local inflammation, mechanical forces, and cellular and biochemical processes. Characteristic features of the disease are degradation of articular cartilage, hypertrophy of bone at the margins, and changes in the synovial membrane, typically accompanied by pain and stiffness of the joint. For the majority of patients, OA is linked to one or more factors, such as aging, occupation, trauma, and repetitive and small insults over time. The pathophysiologic process of OA is almost always progressive.
  • CG53135-05 and their variants belong to the FGF family that regulates proliferation (see U.S. Application Serial No. 10/174,394, which is incorporated herein by its entirety).
  • the identification of a polymorphism (CG53135-12) in the gene encoding an FGF20-like protein, CG53135-01, in humans, and a method for identifying individuals who are carriers of the genetic risk-altering factor for OA have been described in U.S. Application Serial No. 10/702,126 (“the '126 application”), which is incorporated herein by its entirety.
  • the '126 application describes a DNA-based diagnostic test for identifying individuals with increased risk for OA and resultant musculoskeletal complications.
  • Non-pharmaceutical interventions include behaviour modification, weight loss, exercise, walking aids, avoidance of aggravating activities, as well as joint irrigation, and arthroscopic and surgical interventions.
  • Current pharmaceutical interventions include nonsteroidal antiinflammatory drugs, intraarticular corticosteroids, and colchicine.
  • FGF- 18 has been shown to repair damaged cartilage in a rat meniscal tear model for OA (See Paper #0199, 50th Annual Meeting of the Orthopaedic Research Society, San Francisco CA, 2004).
  • the present invention provides methods of preventing or treating a disease
  • FGF-CX polypeptide which has homology to Fibroblast Growth Factor (FGF) protein.
  • FGF Fibroblast Growth Factor
  • present invention also encompasses FGF-CX polynucleotide sequences and the FGF-CX polypeptides encoded by these nucleic acid sequences, and fragments, homologs, analogs, and derivatives thereof.
  • the diseases to be prevented or treated include, but are not limited to, joint diseases (non-limiting examples being arthritis, osteoarthritis, joint pathology, ligament and tendon injuries, and meniscal injuries), ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, and neurodegenerative diseases (non-hmiting examples being Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease).
  • the invention encompasses an isolated FGF-CX nucleic acid
  • the nucleic acid can include, but not limited to, nucleic acid sequence encoding a polypeptide at least 85% identical to a polypeptide comprising the amino acid sequence of Table A (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36).
  • the invention also encompasses the polypeptides resulting from the proteolytic cleavage, of CG53135-05 (SEQ ID NO: 2) that includes SEQ ID Nos: 37, 38, 39, 40.
  • the nucleic acid can be, but is not limited to, a genomic DNA fragment, and a cDNA molecule.
  • the present invention also encompasses a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
  • the present invention further encompasses host cells transformed with a recombinant expression vector comprising any of the nucleic acid molecules described above.
  • the invention provides a pharmaceutical composition that comprises a FGF-CX nucleic acid and a pharmaceutically acceptable carrier.
  • the invention provides a substantially purified FGF-CX polypeptide, e.g., any of the FGF-CX polypeptides encoded by a FGF-CX nucleic acid, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also provides a pharmaceutical composition that comprises a FGF-CX polypeptide and a pharmaceutically acceptable carrier.
  • the invention provides an antibody that binds specifically to a FGF-CX polypeptide.
  • the antibody can be, but is not limited to, a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also provides a pharmaceutical composition including FGF-CX antibody and a pharmaceutically acceptable carrier.
  • the present invention also emcompasses isolated antibodies that bind to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.
  • kits comprising antibodies that bind to a polypeptide encoded by any of the nucleic acid molecules described above and a negative control antibody.
  • the invention encompasses a method for producing a FGF-CX polypeptide.
  • the method includes providing a cell containing a FGF-CX nucleic acid, e.g., a vector that includes a FGF-CX nucleic acid, and culturing the cell under conditions sufficient to express the FGF-CX polypeptide encoded by the nucleic acid.
  • the expressed FGF-CX polypeptide is then recovered from the cell.
  • the cell produces little or no endogenous FGF-CX polypeptide.
  • the cell can be, e.g., a prokaryotic cell or eukaryotic cell.
  • the present invention provides a method of inducing an immune response in a subject against a polypeptide encoded by any of the nucleic acid molecules disclosed above by administering to the mammal an amount of the polypeptide sufficient to induce the immune response.
  • the present invention also provides methods of identifying a compound that binds to FGF-CX polypeptide by contacting the FGF-CX polypeptide with a compound and determining whether the compound binds to the FGF-CX polypeptide.
  • the invention provides a prophylactic treatment with FGF-CX polypeptide wherein an injury that predisposes the subject to osteoarthritis has occurred but the cartilage is intact.
  • the invention also provides a therapeutic treatment with FGF-CX polypeptide wherein intrinsic or extrinsic factors (e.g. genetic predisposition or meniscal injury, respectively) has led to osteoarthritic changes and cartilage damage.
  • intrinsic or extrinsic factors e.g. genetic predisposition or meniscal injury, respectively
  • Figure 1 shows Liquid Chromatography and Mass Spectrometry analysis of
  • CG53135-05 was injected onto the phenyl-hexyl column in an aqueous mobile phase containing 95% water, 5% acetonitrile, and 0.1% triftuoroacetic acid. The protein was then eluted by using a non-linear gradient with an organic mobile phase containing 95% acetonitrile, 5% water, and 0.085% triftuoroacetic acid. Each of the 4 peaks was characterized using LC/ESI/MS, MALDI-TOF MS, and N-terminal amino acid sequencing.
  • Figure 2A and 2B depict Peptide Map of CG53135-05.
  • the upper tracing in each panel represents that of CG53135-05 and the lower tracing in each panel represents an identical sample treated similarly but without CG53135-05.
  • Figure 2A Detection at 214 nm to monitor CG53135 peptides.
  • Figure 2B Detection at 295 nm to monitor tryptophan- containing peptides.
  • Figure 3 shows Receptor Binding Specificity of CG53135.
  • Figure 4 shows the results of Forelimb Placing Test.
  • the mean and standard error of the score for groups receiving vehicle are represented over time.
  • Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA.
  • Figure 5 shows the results of Hindlimb Placing Test.
  • the mean and standard error of the score for groups receiving vehicle are represented over time.
  • Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA.
  • Figure 6 shows the results of Body Swing Test.
  • the mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 ⁇ g/injection CG53135-05 (square), and 2.5 ⁇ g/injection CG53135-05 (triangles) are represented over time.
  • a score range of -50% swings to the right indicates no impairment, whereas 0% swings to the right swing indicates maximal impairment.
  • Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA.
  • Figure 7 shows the results of Cylinder Test. The mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 ⁇ g injection CG53135-05 (square), and 2.5 ⁇ g/injection CG53135-05 (triangles) are represented over time.
  • Figure 8 shows the results of Body Weight. The mean and standard errors of the weights for groups receiving vehicle (diamonds), 1.0 ⁇ g/injection CG53135-05 (square), and 2.5 ⁇ g/injection CG53135-05 (triangles) is represented over time.
  • Figure 9 shows the effect of CG53135-05 on Pro-MMP production in
  • Figure 10 shows the effect of CG53135-05 on Pro-MMP production in
  • Figure 11 shows the effect of CG53135-05 on T P production in SW1353 cells.
  • Figure 12 shows the effect of intra-articular injection of CG53135-05 in the
  • Figure 13 shows results of intra-articular injection of CG53135-05 in the
  • Figure 15 shows results of intra-articular injection CG53135-05 in the
  • Figure 16 shows results of intra-articular injection of CG53135-05 in the
  • Figure 17 shows results of intra-articular injection of CG53135-05 in
  • the present invention provides methods of preventing or treating a joint disease (e.g., osteoarthritis, other related joint pathologies, such as but are not limited to, ligament and tendon injuries within the ligament and tendon itself, or within their respective insertion sites, meniscal tears, other joint disorders where matrix deposition occurs, joint disorders where remodeling and repair are required, and cartilage and joint pathology occurred as a result of an inflammatory disease (e.g., rheumatoid arthritis)) in a subject comprising administering to the subject a composition comprising an FGF-CX polypeptide.
  • a joint disease e.g., osteoarthritis, other related joint pathologies, such as but are not limited to, ligament and tendon injuries within the ligament and tendon itself, or within their respective insertion sites, meniscal tears, other joint disorders where matrix deposition occurs, joint disorders where remodeling and repair are required, and cartilage and joint pathology occurred as a result of an inflammatory disease (e.g., rheumatoid arthritis))
  • the present invention also encompasses methods of using FGF-CX to improve functional recovery following middle cerebral artery (MCA) occlusion in rats.
  • MCA middle cerebral artery
  • the present invention evaluates the efficacy and safety of FGF-CX in a model that assesses these parameters.
  • administering FGF-CX will be beneficial in the treatment of pathological conditions including, but are not limited to, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, and neurodegenerative diseases (such as Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease).
  • FGF-CX polypeptides FGF-CX polypeptides, nucleic acids encoding the polypeptides, and methods of making such polypeptides are described in U.S. Application Serial Nos. 09/494,585 and
  • FGF-CX is used interchangeably with the term “CG53135,” “CG53135-05,” and "FGF-20.”
  • FGF-CX nucleic acids include isolated nucleic acids that encode FGF-CX polypeptide or a portion thereof, FGF-CX polypeptides, vectors containing these nucleic acids, host cells transformed with the FGF-CX nucleic acids, anti- FGF-CX antibodies, and pharmaceutical compositions.
  • methods of making FGF-CX polypeptides as well as methods of screening, diagnosing, treating conditions using these compounds, and methods of screening compounds that modulate FGF- CX polypeptide activity.
  • Table A provides a summary of the FGF-CX nucleic acids and their encoded polypeptides.
  • the terms “subject” and “subjects” refer to an animal, preferably a mammal, including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey, such as a cynomolgous monkey, chimpanzee, and a human), and more preferably a human.
  • a non-primate e.g., a cow, pig, horse, cat, dog, rat, and mouse
  • a primate e.g., a monkey, such as a cynomolgous monkey, chimpanzee, and a human
  • the subject is a mammal, preferably a human, who is suffering from a joint disease (e.g., osteoarthritis, other osteoarthritis-related disorders), ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases (non-limiting examples being Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease).
  • the subject is a mammal, preferably a human, who are at risk for a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases.
  • the subject is a mammal, preferably a human, who is suffering from a joint disease, but who is not suffering from stroke or a neurodegenerative disease.
  • the term "subject" is used interchangeably with "patient” in the present invention.
  • the term "therapeuticaHy effective amount” refers to the amount of a therapy (e.g., FGF-CX polypeptide), which is sufficient to reduce the severity of a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases), reduce the duration of a disease, prevent the advancement of a disease, cause regression of a disease, ameliorate one or more symptoms associated with a disease, or enhance or improve the therapeutic effect(s) of another therapy.
  • a therapy e.g., FGF-CX polypeptide
  • compositions comprising FGF-CX can also be administered in combination with one or more other therapies to prevent, treat, or ameliorate a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases) or one or more symptoms thereof.
  • a disease e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases
  • a disease e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases
  • a disease e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases
  • FGF-CX polypeptide and/or another therapy are administered in a sub-optimal amount, e.g., an amount that does not manifest detectable therapeutic benefits when administered alone, as determined by methods known in the art.
  • a sub-optimal amount e.g., an amount that does not manifest detectable therapeutic benefits when administered alone, as determined by methods known in the art.
  • co-administration of FGF-CX polypeptide and another therapy results in an overall improvement in effectiveness of treatment.
  • FGF-CX polypeptide and one or more other therapies are administered within the same patient visit.
  • FGF-CX polypeptide is administered prior to the administration of one or more other therapies.
  • the FGF-CX polypeptide is administered subsequent to the administration of one or more other therapies.
  • FGF-CX polypeptide and one or more other therapies are cyclically administered to a subject. Cycling therapy involves the administration of FGF-CX polypeptide for a period of time, followed by the administration of one or more other therapies for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment.
  • Toxicity and therapeutic efficacy of a composition of the invention e.g. , FGF-
  • CX polypeptide can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED 50 (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 of LD 5 0/ED 50 .
  • Compositions that exhibit large therapeutic indices are preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such composition 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 for use in humans.
  • the dosage of complexes lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed, the route of administration utilized, the severity of the disease, age and weight of the subject, and other factors normally considered by a medical professional (e.g., a physician).
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell cultures. 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. [INSERT PRFERRED DOSAGE RANGES HERE]
  • Various delivery systems are known and can be used to administer a composition of the invention.
  • 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, rectal, and oral routes.
  • compositions of the invention may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, virginal mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, virginal mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • compositions of the invention may be desirable to administer locally to the area in need of treatment. This may be achieved by, for example, local infusion during surgery, or topical apphcation, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant (said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers).
  • administration can be by direct injection at the site (or former site) of rapidly proliferating tissues which are most sensitive to an insult such radiation, chemotherapy, or chemical warfare agent.
  • the nucleic acid can be administered in vivo to promote expression of their encoded proteins (e.g., FGF-CX polypeptide), 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 retro viral 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.
  • a nucleic acid of the invention can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • compositions of the invention include 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 one or more compositions (e.g., FGF-CX polypeptide) of the invention, 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. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the prophylactic or therapeutic agent 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, binders, tablet disintegrating 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, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, 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 of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, liquid syrups, tablets, capsules, gel capsules, soft gels, pills, powders, enemas, sustained-release formulations and the like.
  • the compositions of the present invention 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, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions of the present invention may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature, these formulations vary in the components and the consistency of the final product.
  • the preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • a pharmaceutical composition of the invention 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 aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • compositions of the invention are to be administered topically, the compositions can be formulated in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • Preferred topical formulations include those in which the polypeptides of the invention are in admixture with a topical delivery agent, such as but not limited to, lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include, but are not limited to, neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoyiphosphatidyl choline DMPC, distearolyphosphatidyl choline), negative (e.g. dimyristoyiphosphatidyl glycerol DMPG), and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
  • the polypeptides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes.
  • the polypeptides may be complexed to lipids, in particular to cationic lipids.
  • Preferred fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a Cl-10 alkyl ester (e.g.
  • isopropylmyristate IPM monoglyceride, diglyceride, or pharmaceutically acceptable salt thereof.
  • 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 in a squeeze bottle.
  • a pressurized volatile e.g., a gaseous propellant, such as freon
  • 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.
  • the composition can be formulated in an aerosol form, spray, mist or in the form of drops.
  • prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions 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. 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).
  • the preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or, therapeutic agent(s).
  • compositions of the invention are orally administered in conjunction with one or more penetration enhancers, e.g., surfactants and chelators.
  • Preferred surfactants 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., fatty acids/salts in combination with bile acids/salts.
  • sodium salt of lauric acid, capric acid is used in combination with UDCA.
  • Further penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
  • Compositions of the invention may be delivered orally in granular form including, but is not limited to, sprayed dried particles, or complexed to form micro or nanoparticles.
  • Complexing agents that can be used for complexing with the peptide of the invention include, but are not limited to, poly-amino acids, polyimines, polyacrylates, polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates, cationized gelatins, albumins, acrylates, polyethyleneglycols (PEG), polyalkylcyanoacrylates, 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.
  • PEG polyethyleneglycol
  • the method of the invention may comprise pulmonary administration, e.g. , by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent.
  • the method of the invention may comprise administration of a composition 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.
  • 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 lignocaine 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 lyophilized powder or water free concentrate in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity of active agent.
  • compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include, but are not limited to, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • Non-limiting examples of pharmaceutically acceptable salts are acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium acetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glucaptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphateldiphosphate, polygalacturonate, salicylate, ste
  • compositions 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.
  • the compositions may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
  • ingredients of the compositions of the invention e.g., a, a, a, a, a, a, a, a, a, a, a, a, a, a,
  • FGF-CX are derived from a subject that is the same species origin or species reactivity as recipient of such compositions.
  • 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., FGF-CX polypeptide) in pharmaceutically acceptable form.
  • the composition in a vial of a kit of the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
  • 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 pharmaceutically acceptable solution e.g., saline, dextrose solution, etc.
  • kits of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the formulation, and or a packaged alcohol pad. Instructions are optionally included for administration of the formulations of the invention by a clinician or by the patient.
  • kits comprising a plurality of containers each comprising a pharmaceutical formulation or composition comprising a dose of the composition of the invention (e.g., FGF-CX polypeptide) sufficient for a single administration.
  • a pharmaceutical formulation or composition comprising a dose of the composition of the invention (e.g., FGF-CX polypeptide) sufficient for a single administration.
  • 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.
  • the products of the invention include instructions for use or other informational material that advise the physician, technician, or patient on how to appropriately prevent or treat the disease or disorder in question.
  • FGF-CXld 250059596 SEQ ID NO: 8 185 aa MW at 20762.3kD Protein Sequence
  • FGF-CXlj CG53135-02 iSEQ ID NO: 20 158 aa MW at l8254.6kD Protein Sequence iMAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGM NDKGELYGSEKLT SEC I FR ⁇ QFEENWY TYS SNI YKHGDTGRRYFVALNKDGTPRDGARSKRHQKFTH FLPRPVDPERVP ⁇ LYKDLLMYT
  • FGF-CX11, CG53135-06 ISEQ ID NO: 24 179 aa !MW at 20146.7kD Protein Sequence
  • FGF-CXlm CG53135-07 SEQ JD NO: 25 54 bp DNA Sequence ORF Start: ATG at 1
  • FGF -CXIa MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL FGF -CXlb MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL FGF -CXlc FGF -CXld FGF -CXle FGF -CXlf -TRSILRRRQLYCRTGFHLQILPDGSVQGT FGF -CXlg FGF -CXlh FGF -CXli FGF -CXlj MAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGT FGF -CXl MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAA ⁇ RSARGGPGAAQLAHL FGF -CX11 MAPLAEVGGFLGGLEGLGQPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSV
  • FGF-CXld (SEQ ID NO 8)
  • FGF-CXle (SEQ ID NO 10)
  • FGF-CXlh (SEQ ID NO 16)
  • FGF-CXli (SEQ ID NO 18)
  • PSG a new signal peptide prediction method
  • N-region length 6; pos.chg 0; neg.chg 1 H-region: length 8; peak value 0.00 PSG score: -4.40
  • GvH von Heijne's method for signal seq. recognition
  • GvH score (threshold: -2.1): -5.49 possible cleavage site: between 16 and 17
  • membrane topology type lb (cytoplasmic tail 94 to 211)
  • Gavel prediction of cleavage sites for mitochondrial preseq cleavage site motif not found
  • NUCDISC discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 12.3%
  • KDEL ER retention motif in the C-terminus: none
  • SKL peroxisomal targeting signal in the C-terminus: none
  • VAC possible vacuolar targeting motif
  • Actinin-type actin-binding motif type 1 : none type 2 : none
  • NMYR N-myristoylation pattern : none
  • Prenylation motif none memYQRL: transport motif from cell surface to Golgi: none
  • NNCN Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 89
  • COIL Lupas's algorithm to detect coiled-coil regions total : 0 residues
  • CG53135-05 resulted in variants that have high activity in the proliferation assays.
  • these variants detailed in this section are expected to have same utility as that of CG53135- 05.
  • fractions were collected using an automated fraction-collector (Agilent 1100) and the fractions characterized by liquid chromatography electrospray ionization ion trap mass spectrometry (LC/ESI/MS), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and N-terminal amino acid sequencing.
  • LC/ESI/MS liquid chromatography electrospray ionization ion trap mass spectrometry
  • MALDI-TOF-MS matrix-assisted laser desorption ionization time of flight mass spectrometry
  • N-terminal amino acid sequencing N-terminal amino acid sequencing.
  • Peak 0 is not a protein, is not product-related, and is not a residual process impurity (i.e., it is not DNA, endotoxin, kanamycin, or IPTG).
  • the CG53135-05-related species (Peaks 1, 2, 3, and 4) eluted in mobile phase of 36% acetonitrile, 63% water, and 0.1% trifluoroacetic acid. Similar fractions were then pooled and the samples concentrated using an Amicon 10,000 dalton cut-off filter (Millipore, Bedford, MA) in a 4°C centrifuge.
  • the samples were diluted 16-fold in 200 mM arginine, 40 mM sodium acetate, and 3% glycerol and then concentrated to a volume of approximately 500 ⁇ l.
  • the concentration of the pooled fractions was determined using amino acid analysis.
  • the XP DECA was set to acquire a full MS scan between 400 and 1400 m/z followed by full MS/MS scans between 400 and 2000 m/z of the top 3 ions from the preceding MS scan.
  • Data were processed using TurboSequest (Thermofinngan).
  • Database searching and protein identification was performed using MASCOT (Matrix Sciences, Manchester, UK).
  • MASCOT reports a probability-based MOWSE score and percent coverage for the identified protein based on molecular mass of the peptides, MS/MS sequence information, mass accuracy, and number of peptides detected.
  • Table 2 contains the peak number for the CG53135-05-related species, confidence score provide by MASCOT, and percent coverage obtained from MS/MS spectra.
  • NA . data not available, not a protein, not product-related
  • the molecular weight determined by MALDI-TOF and N-terminus sequencing can identify the 4 species. For peak 1, 4 different species were detected via N- terminal sequencing, 2 of which were also detected by MALDI-TOF. These results are also in agreement with the coverage obtained using LC/MS.
  • the polypeptide sequences of each species derived by N-terminal sequencing are given in Table 4.
  • CG53135-05 were hydrolyzed for 16 h at 115°C in 100 mL of 6 N HC1, 0.2% phenol containing 2 nmol norleucine as an internal standard. Samples were dried in a Speed Vac Concentrator and dissolved in 100 mL sample buffer containing 2 nmol homoserine as an internal standard. The amino acids in each sample were separated on a Beckman Model 7300 ion-exchange instrument. The amino acid composition of CG53135-05 was consistent with the theoretical amino acid composition.
  • the chromatogram in Figure 2A is consistent with the 20 peptides expected from the digestion of CG53135-05 with trypsin, and the chromatogram in Figure 2B reveals a single peak as expected for the single tryptophan residue in CG53135-05.
  • 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% CO 2 /air and then starved in Dulbecco's modified Eagle's medium for 24 - 72 h.
  • CG53135-05-related species were added and incubated for 18 h at 37°C in 10% CO 2 /air.
  • BrdU (10 mM final concentration) was added and incubated with the cells for 2 h 37°C in 10% CO /air. Incorporation of BrdU was measured by enzyme-linked immunosorbent assay according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN).
  • FGF family members transduce signals intracellularly via high affinity interactions with cell surface immunoglobulin (Ig) domain-containing tyrosine kinase FGF receptors (FGFRs).
  • Ig immunoglobulin domain-containing tyrosine kinase FGF receptors
  • Four distinct human genes encode FGFRs (Powers et ah, Endocr Relat Cancer 2000, 7:165-97; Klint and Claesson-Welsh, Front Biosci 1999, 4:D165-77; Xu et ah, Cell Tissue Res 1999, 296:33-43).
  • a related fifth human sequence lacking a kinase domain has recently been identified and named FGFR-5 (Kim et ah, Biochim Biophys Acta 2001, 1518:152-6).
  • 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 serve to stabilize FGF/FGFR interactions and to sequester FGF and protect it from degradation (Powers et al, Endocr Relat Cancer 2000, 7:165-97; Szebenyi and Fallon, Lit Rev Cytol 1999,185:45-106).
  • HSPGs heparin sulfate proteoglycans
  • FGF receptor-1 (FGFR-1), which shows the broadest expression pattern of the four 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.
  • FGFR-1, FGFR-2 and FGFR-3 each recognize FGF-1, FGF-2, FGF-4 and
  • FGF-8 In addition, FGFR-1 & FGFR-2 bind FGF-3, FGF-5, FGF-6, FGF-10 and FGF-17 (Powers et ah, Endocr Relat Cancer 2000, 7:165-97). Binding of various FGF ligands varies with each receptor splice form, thus allowing a wide repertoire of FGF-mediated signaling events through a limited number of receptor coding genes. Tissue-specific alternate splicing permits cells expressing a single FGFR gene to significantly diversify their biological response by generating distinct receptor isoforms that may exhibit different ligand specificity and function.
  • FGFR-4 binds FGF-1, FGF-2, FGF-4, FGF-6, FGF-8 and FGF-9 but not FGF- 3, FGF-5 or FGF-7.
  • FGF-7, or keratinocyte growth factor- 1 (KGF-1) is only recognized by FGFR-2, whereas FGF-9 binds to FGFR-2, FGFR-3 and FGFR-4.
  • Receptor specificity of FGFs-11 to -19 is not well understood (Powers et ah, Endocr Relat Cancer 2000, 7:165-97; Ornitz et al, J Biol Chem 1996, 271:15292-7).
  • FGFR-1, FGFR-2 and FGFR-3 are widely expressed, suggesting an important functional role in tissue homeostasis. Protein expression patterns for tissue-specific isoforms have not yet been determined.
  • FGFR-4 has a more limited expression pattern being notably absent from lung, oviduct, placenta, testis, prostate, thyroid, parathyroid, and sympathetic ganglia, tissues where all three other receptors are predominantly expressed (Hughes, J Histochem Cytochem 1997, 45:1005-19).
  • the nickel column was washed with PBS/0.5M NaCl + 1M L-arginine and bound proteins were eluted with a linear gradient of imidazole (0-0.5 M). Fractions containing CG53135 (100-150 mM imidazole) were pooled and dialyzed against 1 X 10 6 volumes of PBS pH 8.0 containing 1M L-arginine. The protein sample was stored at -80°C.
  • recombinant CG53135-01, aFGF, or PDGF-BB final concentrations of 10, 5 and 3 ng/mL, respectively
  • soluble receptors final concentrations of 0.2, 1 and 5 ug/mL
  • Factor concentrations represent the amount of hgand needed to generate a half maximal BrdU response in NIH 3T3 cells.
  • Soluble FGFRs were Fc chimeras of the following receptor forms (FGFRl ⁇ (IIIc); FGFR2 ⁇ (IIIb); FGFR2 ⁇ (IIIb); FGFR2QC(IIIC); FGFR3 ⁇ (IIIc); FGFR4) and were obtained from R&D Systems (Minneapolis, MN).
  • the BrdU assay was performed according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN) using a 4 h BrdU incorporation time.
  • Example 4 Treatment of Stroke [00103] Thirty male Sprague Dawley rats were allocated to treatment groups as indicated in the study design Table 8 below.
  • MCA Middle cerebral artery
  • Rats were given CG53135-05 or vehicle [40mM acetate, 200 mM mannitol (pH 5.3)] by percutaneous injection into the cisterna magna, once at 1 day, (approximately 24 hours) and once at 3 days, (approximately 72 hours) after MCA. Animals were given test article (2 dose groups) or vehicle treatment according to the study design. Clinical Observations/Signs
  • Body Weight Animals were weighed on Days 1, 3, 7, 14 and 21.
  • Forelimb Placing Test Assessment Score The forelimb placing test measures sensorimotor function in each forelimb as the animal places the limb on a table top in response to visual, tactile, and proprioceptive stimuh.
  • Hie forelimb placing test consists of the following evaluations and scoring, where the combined total score for the forelimb placing test reflects a range from 0 (no impairment) to 10 (maximal impairment): visual placing (forward, sideways):, 0 - 4 tactile placing (dorsal,lateral): 0 - 4 proprioceptive placing: 0 - 2 Total score for all forelimb tests: 0 - 10
  • Hindlimb Placing Test Assessment Score Similarly, the hindlimb placing test measures sensorimotor function of the hindlimb as the animal places it on a tabletop in response to tactile and proprioceptive stimuli.
  • the hindhmb placing test consists of the following evaluations and scoring, where the combined total score for the hindlimb placing test reflects a range from 0 (no impairment) to 6 (maximal impairment): tactile placing (dorsal,lateral): 0 - 4 proprioceptive placing: 0 - 2
  • Body Swing Test the body swing test was carried out on all animals on Day
  • the cylinder test measures spontaneous motor activity of the forelimbs. Animals are placed in a narrow glass cylinder (16.5 x 25 cm) and videotaped for 5 min on the day before stroke surgery and at weekly intervals thereafter. Videotapes are then scored independently by one experienced observer and up to 50 spontaneous movements will be counted ( ⁇ 5 min per rat per day). Spontaneous movements include those made by each forelimb to initiate rearing, to land on or to move laterally along the wall of the cylinder, or to land on the floor after rearing.
  • the area of cerebral infarcts on each of seven slices (+4.7, +2.7, +0.7, -1.3, -3.3, -5.3, and -7.3 mm compared with Bregma) was determined using a computer interface imaging system using the indirect method (area of the intact contralateral hemisphere - area of the intact ipsilateral hemisphere) to correct for brain shrinkage during processing. Jnfarct volume was then expressed as a percentage of the intact contralateral hemispheric volume. Volumes of the infarction in the cortex and striatum were also determined separately using these same methods. H&E stained section was examined for histological changes such as hemorrhage, abscess or tumor formation.
  • Forelimb Placing Test on days -1, 1, 3, 7, 14, and 21 relative to MCA occlusion, animals were examined by using a limb placing test to assess sensorimotor function in the forelimb in response to visual, tactile and proprioceptive stimuli (Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. & Finklestein, S. P. (1997) Proc. Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res.
  • Visual placing (scored 0-4), tactile placing (scored 0-4), and proprioceptive placing (scored 0-2) were summed to generate a range of potential total scores from 0 to 12, with 12 representing maximal impairment (Figure 4).
  • Hindlimb Placing on days -1, 1, 3, 7, 14, and 21 relative to MCA occlusion, animals were examined by using a limb placing test to assess sensorimotor function in the hindlimb in response to tactile and proprioceptive stimuli [Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. & Finklestein, S. P. (1997) Proc. Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res. 573, 44-60]. Tactile placing (scored 0-4), and proprioceptive placing (scored 0-2) were summed to generate a range of potential total scores from 0 to 6, with 6 representing maximal impairment (Figure 5).
  • Body Swing Test On days -1, 1, 3, 7, 14, and 21 relative to MCA occlusion, animals were examined by using a body swing test to assess side preference as the animal is held approximately one inch above the surface of the table, and swings to the right or the left side.
  • a body swing test to assess side preference as the animal is held approximately one inch above the surface of the table, and swings to the right or the left side.
  • Cylinder Test On days -1, 1, 3, 7, 14, and 21 relative to MCA occlusion, animals were examined by cylinder test to assess spontaneous motor activity of the forelimbs (Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. & Finklestein, S. P. (1997) Proc. Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res.
  • CG53135-05 administration will be useful in the treatment of pathological conditions including ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning and neurodegenerative diseases (such as Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease).
  • pathological conditions including ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning and neurodegenerative diseases (such as Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease).
  • MMPs matrix metalloproteinases
  • OA Osteoarthritis
  • RA Rheumatoid Arthritis
  • MMP matrix metalloproteinase
  • SW1353 chondrosarcoma cell line ATCC HTB-94. This cell line is a well-established chondrocytic cellular model for matrix metalloproteinases (MMP) production.
  • SW1353 cells were plated in a 24-well plate at 1 xlO 5 cells/ml (1 ml) in DMEM medium- 10 % FBS. Following overnight incubation, the medium was replaced with DMEM + 0.2 % Lactabulmin serum.
  • CG53135-05 was added to the wells at doses ranging from 10 to 5000 ng/ml, in the absence or presence of JL-1 beta (0.1 to 1 ng/ml, R&D systems Minneapolis, MN), TNF-alpha (10 ng/ml, R&D systems) or vehicle control to a final volume of 0.5 ml.
  • TL-1 beta and TNF-alpha are both potent stimulators of MMP activity. All treatments were done in triplicate wells. After 24 h, the supernatants were collected and Pro- MMP-1, and -13, as well as TJMP-1 (tissue inhibitor of matrix metalloproteinase), a natural inhibitor of MMP activity, was measured by ELIS A (R&D systems). The measurements were normalized to the number of cells by an MTS assay.
  • CG53135-05 significantly decreased MMP-13 production in the presence of either IL-1 beta or TNF-alpha as demonstrated in Figure 14 and Figurel5 respectively.
  • JL-1 beta and TNF-alpha are both potent stimulators of MMP activity.
  • MMP-13 affinity for type It collagen, the main collagen that is degraded in OA is ten times higher that of MMP- 1. Since MMP-13 expression increases in OA and RA, the decrease of MMP-13 observed with addition of CG53135-05 indicates that the protein can be used as an OA and RA therapeutic ( Figure 10).
  • CG53135-05 up-regulated the production of TJMP-1, a natural inhibitor of MMP activity (Figure 11).
  • CG53135-05 This enhancement of TJMP-1 production by CG53135-05 is beneficial in reducing the matrix breakdown by MMP-1 and -13 observed in OA and RA.
  • CG53135-05 had no effect on MMP-3 production constitutively or after JL-1 induction (data not shown.).
  • CG53135-05 FGF-20 showed increase in basal expression of MMP-1 in SW1353 cells (data not shown).
  • the rats were injected intra-articularly three times per week for 2 weeks with vehicle solution (8mM acetate, 40 mM arginine, and 0.6% glycerol (pH 5.3) in approximately 1% hyaluronic acid), 10 ⁇ g CG 53135-05 or 100 ⁇ g CG 53135-05.
  • vehicle solution 8mM acetate, 40 mM arginine, and 0.6% glycerol (pH 5.3) in approximately 1% hyaluronic acid
  • the rats were divided into three treatment groups with three animals in each group: two groups received CG53135 and one received only the vehicle control.
  • the rats were anesthetized with Isoflurane and injected through the patellar tendon into the area of the cruciate attachments of both knees.
  • CG53135 was injected at doses of 0.1 mg/ml (0.01 mg/joint) or 1.0 mg/ml (0.1 mg/joint).
  • Controls were injected with the vehicle solution as described above. Injections were done Monday, Wednesday and Friday for 2 weeks. The animals were terminated on day 15 at which time they were injected ip with BRDU (100 mg kg) in order to pulse label proliferating cells.
  • Morphologic Pathology Vehicle injected rats had minimal to mild synovial hyperplasia, inflammation and fibroplasia with none to minimal matrix deposition in fibrotic synovium.
  • Articular cartilage had no proteoglycan loss or fibrillation.
  • the central area of the joint where the cruciates attach and in which the intra-articular injections are made had none to minimal fibroplasia and cartilage/bone damage. No marginal zone chondrogenesis was present.
  • Knees injected with 10 ⁇ g CG 53135-05 had mild to moderate synovial hyperplasia, inflammation and fibroplasia with minimal to moderate matrix deposition in fibrotic synovium.
  • Articular cartilage had no proteoglycan loss or fibrillation.
  • the central area of the joint where the cmciates attach and in which the intra-articular injections are made had none to minimal fibroplasia and cartilage/bone damage.
  • One knee had minimal marginal zone chondrogenesis.
  • Knees injected with 100 ⁇ g CG 53135-05 had moderate to marked synovial hyperplasia, inflammation and fibroplasia with moderate matrix deposition in fibrotic synovium.
  • Articular cartilage had none to minimal proteoglycan loss or fibrillation.
  • the central area of the joint where the cruciates attach and in which the intra-articular injections are made had minimal to marked fibroplasia and cartilage/bone damage. All knees had mild to moderate marginal zone chondrogenesis.
  • One animal had chondrogenesis in areas associated with articular cartilage.
  • Example 7 Intra-articular injection of CG53135-05 in Meniscal Tear model of Rat Osteoarthritis: Prophylactic and Therapeutic Dosing
  • Example 6 utilized CG53135-05 administration into the joints of normal rats to identify effects on relevant cell populations by histomorphometric analysis.
  • CG53135-05 induced significant marginal zone chondrogenesis similar to that seen with other growth factors such as TGF-beta, suggesting an effect on pluripotent stem cells within the marginal zone.
  • TGF-beta growth factors
  • the potentially adverse effects of observed synovial fibroplasia and bone resorption could have been due to either FGF-20 activity or endotoxin levels within the non-clinical grade hyaluronic acid used to formulate the protein.
  • Prophylactic Dosing intra-articular dosing (CG53135-05) of the right knee joint was initiated on the day of surgery and is continued for 2 weeks post-surgery with intra- articular injections given Thursday, Saturday, and Monday (day 0, 2, 4, 7, 9, and 11) with rats under Isoflurane anesthesia.
  • Indomethacin a nonsteroidal anti-inflammatory drug, was dosed (lmg/kg/day) daily by the oral route starting on the day of surgery to reduce any potential inflammation due to the injection.
  • Body weights were recorded on days 0, 7 and 14. After animal termination on day 14 post-surgery, both knees were collected for histopathologic evaluation. The study design is shown in Table 10.
  • intra-articular dosing CG53135-05
  • intra-articular injections given Friday, Sunday, and Tuesday (day 22, 25, 27, 29, 32, and 34) with rats under Isoflurane anesthesia.
  • Indomethacin is dosed daily by the oral route starting on the day of surgery.
  • Body weights are recorded on days 0, 7, 14, 21, 28, and 35.
  • both knees are collected for histopathologic evaluation.
  • Table 11 The study design is shown in Table 11.
  • Results of prophylactic dosing study include the standards followed for this model. Multiple sections (3 levels) of right knee were analyzed microscopically and scored according to the following methods. In scoring the 3 sections, the worst case scenario for the 2 halves on each of the 3 slides representing 3 levels was determined for cartilage degeneration and osteophyte formation. This value for each parameter for each slide was then averaged to determine overall subjective cartilage degeneration scores for tibia and femur and osteophyte scores for tibia.
  • Cartilage degeneration was scored none to severe (numerical values 0-5) for depth and area (surface divided into thirds) using the following criteria:
  • l minimum degeneration, chondrocyte and proteoglycan loss with or without fibrillation involving the superficial zone
  • Total Tibial Cartilage Degeneration Width ⁇ m a micrometer measurement of total extent of tibial plateau affected by any severity of degeneration (Total Tibial Cartilage Degeneration Width ⁇ m) extended from the origination of the osteophyte or marginal zone if no osteophyte was present with adjacent cartilage degeneration (outside 1/3) across the surface to the point where tangential layer and underlying cartilage appeared histologically normal.
  • a micrometer depth of any type of lesion (cell/proteoglycan loss, change in metachromasia, but may have good retention of collagenous matrix and no fibrillation) expressed as a ratio of depth of changed area vs. depth to tidemark was included and taken over 4 equally spaced points on the tibial surface. These measurements were taken (1st) matrix adjacent to osteophyte (2nd) 1/4 of the distance across the tibial plateau (3rd) 1/2 of the distance across the tibial plateau (4th) 3/4 of the distance across the tibial plateau. This measurement was the most critical analysis of any type of microscopic change present. The depth to tidemark measurement (denominator) also gives an indication of cartilage thickness across the tibial plateau and therefore allows comparisons across groups when trying to determine if hypertrophy or hyperplasia has occurred.
  • Results of the prophylactic dosing study indicate that intra-articular injection of 100 ⁇ g of CG53135-05 in knee joints of rats with medial meniscal tear results in chondroprotective effects as a result of both inhibition of cartilage degeneration and stimulation of cartilage repair. Some joints had layering of proliferated new cartilage over existing normal appearing or damaged cartilage. This observation is particularly exciting as it demonstrates the potential for resurfacing to occur.
  • CG53135-05 in osteoarthritis include:
  • Results of the therapeutic dosing study The data described demonstrated the potential chondroproliferative activities of CG53135-05 administered intra-articularly. However, protein injected joints had markedly increased inflammation, fibroplasia and connective tissue resorptive process.
  • Indomethacin treatment was not effective in reducing the inflammatory changes and it had no beneficial effects on inhibiting the resorptive processes occurring in bone.
  • following modification to the therapeutic dosing study can be attempted: Increasing the dosing interval to once or twice weekly and/or increasing the study duration to allow time for the prohferative tissue to fill the large cartilage defects induced by this disease process.
  • Another possibility is to investigate the effects of CG53135-05 in a larger species such as the dog as dogs have less of a tendency to proliferate connective tissue and resorb bone in response to various stimuli than rodents.
  • Such alterations and changes may include, but not limited to, different compositions for the administration of the polypeptides according to the present invention to a subject; different amounts of the polypeptide; different times and means of administration; different materials contained in the administration dose including, for example, combinations of different peptides, or combinations of peptides with different biologically active compounds.
  • Such changes and alterations also are intended to include modifications in the amino acid sequence of the specific polypeptides described herein in which such changes alter the sequence in a manner as not to change the functionality of the polypeptide, but as to change solubility of the peptide in the composition to be administered to a subject, absorption of the peptide by the body, protection of the polypeptide for either shelf life or within the body until such time as the biological action of the peptide 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 of the present invention.

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Abstract

La présente invention concerne des compositions et des procédés pour traiter diverses pathologies, notamment un accident vasculaire cérébral, une cicatrisation et des maladies articulaires (par ex. l'ostéoarthrose et la polyarthrite rhumatoïde). Cette invention concerne plus particulièrement des compositions comprenant un membre de la famille des facteurs de croissance des fibroblastes, FGF-CX (également connu sous la dénomination CG53135-05 ou FGF-20), ses polypeptides associés, des acides nucléiques codant de tels polypeptides, ainsi que leurs utilisations pour traiter une pathologie, notamment un accident vasculaire cérébral, une cicatrisation et des maladies articulaires (par ex. l'ostéoarthrose et la polyarthrite rhumatoïde).
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