JP2007516223A - Novel fibroblast growth factor and method of use thereof - Google Patents

Abstract

The present invention relates to compositions and methods for the treatment of a variety of conditions including, but not limited to, stroke, wound healing and joint disease (eg, osteoarthritis and rheumatoid arthritis). More particularly, the present invention relates to a member of the fibroblast growth factor family, FGF-CX (also known as CG53135-05 or FGF-20), related polypeptides, and nucleic acids encoding such polypeptides. Compositions comprising and their use for treating conditions such as, but not limited to, stroke, wound healing and joint diseases (eg, osteoarthritis and rheumatoid arthritis).

Description

Detailed Description of the Invention

  The present invention claims the benefit of US Provisional Application No. 60 / 469,353, dated May 9, 2003, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD The present invention relates to compositions and methods for the treatment of a variety of conditions including, but not limited to, stroke, wound healing and joint diseases (eg, osteoarthritis and rheumatoid arthritis). More particularly, the present invention relates to a member of the fibroblast growth factor family, FGF-CX (also known as CG53135-05 or FGF-20), related polypeptides, and nucleic acids encoding such polypeptides. Compositions comprising and their use for treating conditions such as, but not limited to, stroke, wound healing and joint diseases (eg, but not limited to osteoarthritis and rheumatoid arthritis).

BACKGROUND ART The FGF protein family, which includes acidic FGF (FGF-1) and basic FGF (FGF-2) as its prototypical members, binds to four related receptor tyrosine kinases. These FGF receptors are expressed in most types of cells in tissue culture. It has been reported that dimerization of FGF receptor monomers at the time of ligand binding is one prerequisite for activation of the kinase domain leading to receptor transphosphorylation. FGF receptor-1 (FGFR-1), which exhibits the widest expression pattern among the four FGF receptors, contains at least seven tyrosine phosphorylation sites. Numerous signal transduction molecules are affected by binding to these phosphorylation sites with different affinities.

  Expression of FGF and its receptor in the brain of perinatal and adult mice has been investigated. Messenger RNAs of all FGF genes except FGF-4 were detected in these tissues, and FGF-3, FGF-6, FGF-7 and FGF-8 genes were further expressed in late embryogenesis rather than late birth. High expression suggests that these members are involved in later stages of brain development. In contrast, FGF-1 and FGF-5 expression increased after birth. In particular, FGF-6 expression in perinatal mice is restricted to the central nervous system and skeletal muscle and is accompanied by a strong signal in the developing cerebrum in the embryo except for the cerebellum of the newborn 5th day Has been reported. The cognate receptor for FGF-6, FGF-receptor (FGFR) -4, shows similar spatiotemporal expression, and FGF-6R and FGFR-4 play a significant role in the maturation of the nervous system as a ligand-receptor system. Suggests to fulfill. According to Ozawa et al., These results indicate that various FGFs and their receptors regulate various developmental processes in the brain such as proliferation and migration of neuronal progenitor cells, neuronal and glial differentiation, neurite dilation and synaptogenesis. It strongly suggests that it is involved in See, for example, Ozawa et al., Brain Studies, Molecular Brain Studies (1996), 41 (1-2): 279-88.

  Other members of the FGF polypeptide family include the FGF receptor tyrosine kinase (FGFRTK) family and the FGF receptor heparan sulfate prothioglycan (FGFRHS) family. These members interact to regulate an active and specific FGFR signal transduction complex. These regulatory activities diversify throughout a wide range of organs and tissues in the mammalian body and in both normal and tumor tissues. Regulated alternative messenger RNA (mRNA) splicing and combination of mutant subdomains generates a wide variety of FGFRTK monomers. A divalent cation cooperates with FGFRHS to conformally limit the FGFRTK trans-phosphorylation reaction, which causes a loss of kinase activity and facilitates proper activation of FGFR by FGF. For example, it has been found that different point mutations within FGFRTK generally cause graded severe cephalic and skeletal abnormalities due to a gradual increase in FGF-independent activity of the entire FGFR complex. Other processes in which the FGF family has significant effects include liver growth and function, and prostate tumor progression.

  Another FGF family member, the glial activator (GAF), is a heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. See Miyamoto et al., 1993, Mol. Cell. Biol. 13 (7): 4251-4259. GAF exhibits a spectrum of activity that is slightly different from that of other known growth factors and is referred to as FGF-9. Human FGF-9 cDNA encodes a 208 amino acid polypeptide. Sequence similarity with other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences found within other family members were well conserved within the FGF-9 sequence. FGF-9 was found to have no standard signal sequence at its N-terminus, similar to that in acidic and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in the conventional manner. However, FGF-9 was found to be efficiently secreted from COS cells transfected with cDNA despite the lack of a standard signal sequence. It could only be detected in the cell culture medium. The secreted protein lacked any amino acid residues at the N-terminus compared to that predicted by the cDNA sequence, except for the starting methionine. Rat FGF-9 cDNA was also cloned, and structural analysis indicated that the FGF-9 gene was highly conserved.

  FGF has been shown to induce neuronal sprouting. See Proc. Natl. Acad. Sci. USA 1997, 94 (15): 8179-84. Kawamata et al., Journal of Cerebral Blood Flow and Metabolism, 16: 542-547, 1996, has a wide variety of basic FGF, a polypeptide of 154 amino acids in length of 18 kDa. It was proposed to support the survival and growth of brain neurons. US Patent Application No. 2002 / 0151496A1 suggests that FGF-20 is a neurotrophic factor and stimulates the survival of cells derived from neurons.

2.1. Inflammation: Osteoarthritis and Rheumatoid Arthritis Osteoarthritis (“OA”) is a degenerative joint disease that often causes arthralgia affecting a wide and growing population. OA is estimated to be the most common cause of disability in adults. The disease typically appears in people in their 20s and 30s, and although most people over the 40s show some pathological changes in weight bearing joints, the changes can be asymptomatic. A systematic review of the incidence and prevalence of knee OA for people over the age of 55 in the UK has a 25 percent incidence, 10 percent prevalence of disability and about 2-3 percent per year Reported severe disability. The National Health and Nutrition Examination Survey (Center for Health Statistics, Centers for Disease Control and Prevention) found that the prevalence of this disease was less than 0.1 percent between 25 and 34 years, compared with 55 years Over 80% were found to be over 80%. OA is the result of a complex interaction of a number of factors including joint integrity, genetic features, local inflammation, mechanical forces and cellular and biochemical processes. Characteristic features of the disease are articular cartilage degradation, peripheral bone hypertrophy and synovial changes that typically accompany joint pain and stiffening. For most patients, OA is linked to one or more factors such as aging, occupation, trauma, and repetitive minor damage over time. The pathophysiological process of OA is almost always progressive.

  CG53135-05 and variants thereof belong to the FGF family that regulates growth (see US patent application Ser. No. 10 / 174,394, which is incorporated by reference herein in its entirety). A method for identifying an individual that is a carrier of a genetic risk modifier for OA and the identification of a polymorphism in the gene encoding FGF20-like protein, CG53135-01 in the human body (CG53135-12) It has been described in US patent application Ser. No. 10 / 702,126 (“the '126 application”), which is hereby incorporated by reference. The '126 application describes a DNA-based diagnostic test to identify individuals with an increased risk of OA and consequent musculoskeletal complications.

  There are multiple well-proven treatment modalities for OA, ranging from non-pharmaceutical to pharmaceutical intervention. Non-pharmaceutical interventions include behavior modification, weight loss, exercise, gait assistance, avoidance of exacerbating activities and joint cleaning and arthroscopic surgery and surgery. Current pharmaceutical interventions include non-steroidal anti-inflammatory drugs, corticosteroids by intra-articular injection, and colchicine. Furthermore, FGF-18 has been shown to repair damaged cartilage in a rat meniscal tear model for OA (Article 0199, 50th Annual Meeting of the Orthopedic Association). See Orthopaedic Research Society, San Francisco CA, 2004)).

  However, because existing therapies have not succeeded in reducing the incidence or severity of disease, satisfactory treatment of OA is also an unmet medical need. Thus, treatments that can successfully treat osteoarthritis are potentially damaging to the health care system while potentially saving millions of dollars in the costs associated with open surgery, disability and supplementary assistance services. It has the advantageous effect of reducing the rate.

  Citation or discussion herein is not to be considered as an admission that it is prior art to the present invention.

DISCLOSURE OF THE INVENTION The present invention relates to a disease (eg, stroke, joint disease) comprising administering to a subject in need thereof a FGF-CX polypeptide having homology to a fibroblast growth factor (FGF) protein. And a method for preventing or treating trauma). The invention also encompasses FGF-CX polynucleotide sequences and FGF-CX polypeptides encoded by these nucleic acid sequences and fragments, homologues, analogs and derivatives thereof.

  According to the present invention, diseases to be prevented or treated include joint diseases (non-limiting examples are arthritis, osteoarthritis, joint lesions, ligament and tendon damage and meniscal damage), ischemic stroke , Including hemorrhagic stroke, trauma, spinal cord injury, heavy metal or toxin poisoning, and neurodegenerative diseases (non-limiting examples are Alzheimer, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease) However, it is not limited to these.

  In one aspect, the invention provides an isolated FGF-CX nucleic acid encoding a FGF-CX polypeptide (SEQ ID NOs: 1, 3, 5, 7, 9, 11, as shown in Table A). 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35) or a fragment, homologue, analogue or derivative thereof. The nucleic acid includes the amino acid sequence shown in Table A (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36). Nucleic acid sequences that encode a polypeptide that has at least 85% identity to the comprising polypeptide can be included, but are not limited to. The invention also encompasses a polypeptide resulting from protein cleavage of CG5313-05 (SEQ ID NO: 2) comprising SEQ ID NO: 37, 38, 39, 40. The nucleic acid can be, but is not limited to, genomic DNA fragments and cDNA molecules.

  The invention also encompasses vectors containing one or more of the nucleic acids described herein, and cells containing the vectors or nucleic acids described herein.

  The invention further encompasses a host cell transformed with a recombinant expression vector comprising any of the nucleic acid molecules described above.

  In one embodiment, the invention provides a pharmaceutical composition comprising FGF-CX nucleic acid and a pharmaceutically acceptable carrier. In another embodiment, the invention provides a substantially purified FGF-CX polypeptide, such as any of the FGF-CX polypeptides encoded by FGF-CX nucleic acids and fragments, homologues, analogs thereof. And derivatives. The invention also provides a pharmaceutical composition comprising an FGF-CX polypeptide and a pharmaceutically acceptable carrier.

  In another embodiment, the invention provides an antibody that specifically binds to an FGF-CX polypeptide. The antibody can be, but is not limited to, a monoclonal or polyclonal antibody or a fragment, homologue, analog and derivative thereof. The invention also provides a pharmaceutical composition comprising an FGF-CX antibody and a pharmaceutically acceptable carrier. The invention also encompasses an isolated antibody that binds to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.

  The invention further provides a kit comprising an antibody that binds to a polypeptide encoded by any of the nucleic acid molecules described above and a negative control antibody.

  The invention includes a method for producing an FGF-CX polypeptide. The method provides cells containing FGF-CX nucleic acid, eg, a vector containing FGF-CX nucleic acid, and cultures the cells under conditions sufficient to express the FGF-CX polypeptide encoded by the nucleic acid. Including doing. The expressed FGF-CX polypeptide is then recovered from the cells. Preferably, the cell produces little or no endogenous FGF-CX polypeptide. The cell can be, for example, a prokaryotic cell or a eukaryotic cell.

  The present invention elicits an immune response in a subject against a polypeptide encoded by any of the nucleic acid molecules disclosed above by administering to the mammal a sufficient amount of the polypeptide to elicit an immune response. Provide a way to do it.

  The invention also identifies a compound that binds to an FGF-CX polypeptide by contacting the FGF-CX polypeptide with one compound and determining whether the compound binds to the FGF-CX polypeptide. A method is also provided.

  The invention provides prophylactic treatment with an FGF-CX polypeptide that renders the cartilage intact even if an injury occurs that predisposes the subject to osteoarthritis.

  The invention also provides therapeutic treatment with FGF-CX polypeptides where endogenous or extrinsic factors (eg, genetic predisposition or meniscal injury, respectively) cause osteoarthritic changes and cartilage damage. Also provide.

  The present invention relates to articular disorders in a subject's body (eg, osteoarthritis, other related joint lesions, such as, without limitation, ligament and tendon injury within the ligament and tendon itself or within the respective insertion site, meniscus Methods for preventing or treating plate tears, other joint disorders that result in matrix deposition, joint disorders that require remodeling and repair, and cartilage and joint lesions (eg, rheumatoid arthritis) resulting from inflammatory diseases) Wherein a method comprising administering to the subject a composition comprising an FGF-CX polypeptide is provided.

  The invention also encompasses methods of using FGF-CX to improve functional recovery after middle cerebral artery (MCA) occlusion in the rat body. Since strokes can result in disruption of motor and motor nerves, sensory segregation, visual function, language, memory, or other intellectual abilities, the present invention provides a model for evaluating these parameters. Is evaluating the efficacy and safety of FGF-CX. According to the present invention, administration of FGF-CX can be achieved by ischemic stroke, hemorrhagic stroke, trauma, spinal cord injury, heavy metal or toxin poisoning, and neurodegenerative diseases (eg, Alzheimer, Parkinson's disease, amyotrophic lateral sclerosis, Huntington). Disease)), which is advantageous in the treatment of pathological conditions including but not limited to.

  FGF-CX polypeptides, nucleic acids encoding the polypeptides, and methods of making such polypeptides, both in US patent application Ser. No. 09 / 494,585, which are incorporated herein by reference in their entirety. 10 / 174,394. FGF-CX is used interchangeably with the words “CG53185”, “CG53135-05” and “FGF-20”.

  Included in the invention are FGF-CX nucleic acids, isolated nucleic acids encoding FGF-CX polypeptides or portions thereof, FGF-CX polypeptides, vectors containing these nucleic acids, FGF-CX Host cells transformed with nucleic acids, anti-FGF-CX antibodies and pharmaceutical compositions. Also disclosed are methods of making FGF-CX polypeptides, methods of screening, diagnosing and treating symptoms using these compounds, and methods of screening for compounds that modulate FGF-CX polypeptide activity. . Table A provides a summary of FGF-CX nucleic acids and their encoded polypeptides.

  As used herein, the term “subject” refers to animals, preferably non-primates (eg, cows, pigs, horses, cats, dogs, rats and mice) and primates (eg, monkeys such as cynomolgus monkeys, chimpanzees and humans). Means mammals, more preferably humans. In some embodiments, the subject has a joint disease (eg, osteoarthritis, other osteoarthritis related disorders), ischemic stroke, hemorrhagic stroke, trauma, spinal cord injury, heavy metal or toxin poisoning, and neurodegenerative disease ( Non-limiting examples are mammals, preferably humans, suffering from Alzheimer, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease). In another embodiment, the subject is a mammal, preferably a human, at risk for joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord injury, heavy metal or toxin poisoning, or neurodegenerative disease. In one embodiment, the subject is a mammal, preferably a human, who suffers from a joint disease but does not suffer from a stroke or neurodegenerative disease. The term “subject” is used interchangeably with “patient” in the present invention.

  As used herein, the term “therapeutically effective amount” refers to a disease (eg, joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord injury, heavy metal or toxin poisoning, or neurodegenerative disease). Reduce severity, reduce disease duration, prevent disease progression, cause disease regression, improve one or more symptoms associated with the disease, or enhance the therapeutic effect of another therapy Or the amount of therapy (eg, FGF-CX polypeptide) sufficient to improve.

  A composition comprising FGF-CX prevents, treats or treats a disease (eg, joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord injury, heavy metal or toxin poisoning, or neurodegenerative disease) or one or more symptoms thereof It can also be administered in combination with one or more other therapies to improve. In a preferred embodiment, the composition comprising FGF-CX exhibits a disease or one or more symptoms thereof, such as joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord injury, heavy metal or toxin poisoning, or neurodegenerative disease. It is administered in combination with one or more other therapies known to be used in preventing, treating or ameliorating.

  In one embodiment, during the combination therapy, the FGF-CX polypeptide and / or another therapy is a therapeutically detectable when administered alone, eg, as determined by methods known in the art. Administered in sub-optimal amounts, such as those that do not show the benefits of. In such methods, co-administration of the FGF-CX polypeptide and another therapy results in an overall improvement in the effectiveness of the treatment.

  In one embodiment, the FGF-CX polypeptide and one or more other therapies are administered at the same visit of the patient. In another embodiment, the FGF-CX polypeptide is administered prior to administration of one or more other therapies. In yet another aspect, the FGF-CX polypeptide is administered after administration of one or more other therapies. In one particular embodiment, the FGF-CX polypeptide and one or more other therapies are administered periodically to one subject. Cyclic therapy involves administration of a period of FGF-CX polypeptide followed by administration of one or more other therapies for a period of time and this sequential administration. Cycle therapy can reduce the development of resistance to one or more therapies, avoid or reduce the side effects associated with one of the therapies, and / or improve the efficacy of the treatment.

The toxic and therapeutic efficacy (eg, FGF-CX polypeptide) of the composition of the invention is therapeutically effective, eg, LD ₅₀ (dose lethal for 50% of the population) and ED ₅₀ (50% of the population) Can be determined by standard pharmaceutical procedures in cell culture or laboratory animals to determine doses. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED ₅₀ . Compositions that exhibit large therapeutic indices are preferred. A delivery system that targets such a composition to the site of infected tissue to minimize potential damage to uninfected cells and reduce side effects, although compositions that exhibit toxic side effects may also be used Consideration should be given to designing

In one embodiment, data obtained from cell culture assays and animal studies can be used in formulating a dosage range for use in humans. The dosage of the complex is preferably within a range of circulating concentrations including the ED ₅₀ with little or no toxicity. The dosage will be in this range depending on the dosage form utilized, the route of administration utilized, the severity of the disease, the age and weight of the subject and other factors normally taken into consideration by medical professionals (eg physicians). Can vary within. For any composition used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes an IC50 (ie, the concentration of test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in the human body. Levels in plasma can be measured, for example, by high performance liquid chromatography. [Insert preferred dosage range here].

  Appropriate and recommended dosages, formulations and routes for treatment methods such as chemotherapeutic agents, radiotherapy and biological / immunotherapeutic agents such as cytokines are known in the art and are described in the physician tabletop reference ( Physician's Desk Reference (58th edition, 2004).

  Various delivery systems are known and can be used to administer the compositions of the invention. Such delivery systems include liposomes, microparticles, encapsulation in microcapsules, expression by recombinant cells, receptor-mediated endocytosis, nucleic acids of the invention as part of a retrovirus or other vector, etc. However, it is not limited to these. Introduction methods include intradermal, intramuscular, intraperitoneal, intrathecal, intraventricular, epidural, intravenous, subcutaneous, intranasal, intratumoral, transdermal, rectal and oral administration. It is not limited. The compositions of the invention can be administered by any convenient route, such as infusion or bolus injection, absorption through epithelial or dermal mucosal layers (eg, oral mucosa, vaginal mucosa, rectum and intestinal mucosa, etc.) In combination with other biologically active agents. Administration can be systemic or local.

  In some embodiments, it may be desirable to administer the pharmaceutical composition of the invention locally to the area in need of treatment. This can be done, for example, by topical infusion during surgery or by topical application in combination with, for example, a post-surgical wound dressing, or by injection, or by catheters, suppositories or implants (this implant is a sialastic membrane) Or made of a porous, non-porous or gelatin-like material containing membranes or fibers. In one embodiment, administration may be by direct injection at the site of fast growing tissue (or the original site) that is most sensitive to injury such as radiation, chemotherapy or chemical weapons.

  In some embodiments where the composition of the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid is in a form such that the nucleic acid is constructed as part of a suitable nucleic acid expression vector and is intracellular. For example by using a retroviral vector or by direct injection or by using a microparticle bombard (eg gene gun) or by coating with a lipid or cell surface receptor or transfection agent, or in the nucleus etc. Can be administered in vivo to promote expression of its encoded protein (eg, FGF-CX polypeptide) by administering it in linkage to a homeobox-like peptide known to enter . Alternatively, the nucleic acids of the invention can be introduced into cells and incorporated into host cell DNA for expression by homologous recombination.

  The compositions of the invention include bulk pharmaceutical compositions useful in the manufacture of pharmaceutical compositions that can be used for the preparation of unit dosage forms. In a preferred embodiment, the composition of the invention is a pharmaceutical composition. Such compositions comprise a prophylactically or therapeutically effective amount of one or more compositions of the invention (eg, FGF-CX polypeptide) and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is formulated to be appropriate for the route of administration to the subject.

  In one embodiment, the term “pharmaceutically acceptable” is approved by a federal or state government agency for use in animals, and more particularly in the human body, or the United States Pharmacopeia or others. Is listed in the generally recognized pharmacopoeia. The term “carrier” refers to a diluent, ashband (eg, Freundash band (complete and incomplete), excipient or vehicle) administered with a prophylactic or therapeutic agent, such as water or Sterile liquids such as oils (eg petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil etc.) or diluents, fragrances, solubilizers, lubricants, suspending agents, binders, It can be a solid carrier such as a tablet disintegrating agent or one or more substances that can also act as an encapsulating material Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectables.Suitable pharmaceutical excipients include starch, glucose, lactose Sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol or combinations thereof. Without being limited, the composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired.

  The compositions of the present invention include a number of possible dosage forms including, but not limited to, liquid syrups, tablets, capsules, gel capsules, soft gels, pills, powders, enemas, sustained release formulations and the like. Can be formulated in either form. The compositions of the present invention can 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 can also contain stabilizers. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like.

  In some embodiments, the composition of the present invention may be formulated and used as a foam. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. Although fundamentally similar in nature, these formulations differ in terms of final product components and consistency. The preparation of such compositions and formulations is generally known to those of skill in the pharmaceutical and formulation arts and is applicable to formulating the compositions of the present invention.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. In certain embodiments, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, intratumoral or topical administration to humans. . Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a local anesthetic and solubilizer such as lidocaine to ease pain at the site of injection.

  For topical application of the composition of the invention, the composition may 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. In preferred topical formulations, the polypeptides of the invention are mixed with topical delivery agents such as, but not limited to, lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. The prescription in state is included. Preferred lipids and liposomes include neutral (eg, dioleoylphosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (eg, dimyristoyl phosphatidylglycerol DMPG), and cations (eg, dioleoyl). Tetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA), but are not limited to these. The polypeptides of the invention may be encapsulated in liposomes or may form complexes to liposomes, particularly cationic liposomes. Alternatively, the polypeptide can be complexed to a lipid, particularly a cationic lipid. Preferred fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linolenic acid, linolenic acid, dicouple, tricouple, monoolein, dilaurin, Glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C1-10 alkyl ester (eg, isopropyl myristate IPM), monoglyceride, diglyceride, or a pharmaceutically acceptable salt thereof Including, but not limited to. For non-sprayed topical dosage forms, viscous to semi-solid or solid forms typically contain one or more excipients or carriers that are compatible with topical application and preferably have a dynamic viscosity higher than water. Used. Other suitable topical dosage forms include active ingredients, preferably in combination with a solid or liquid inert carrier, in a mixture with pressurized volatiles (eg, gaseous high pressure gases such as freon) or squeeze bottles A sprayable aerosol preparation packaged therein is included. If desired, humectants or wetting agents can also be added to the pharmaceutical composition and dosage form. Examples of such additive components are well known in the art.

  Where the method of the invention includes intranasal administration of one composition, the composition can be formulated in the form of an aerosol, spray, mist or droplets. In particular, a prophylactic or therapeutic agent for use according to the present invention uses a suitable high pressure gas (eg dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas) It can be properly delivered in the form of an aerosol spray from a pressurized pack or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver the calculated amount. Capsules and cartridges (eg comprised of gelatin) can be formulated for use in an inhaler or inhaler containing a powder mixture of the compound and a suitable powder, such as lactose or starch.

  When the method of the invention comprises oral administration, the composition is in the form of a powder, granules, microparticles, nanoparticles, suspension or solution in an aqueous or non-aqueous medium, capsule, gel capsule, sachet, tablet or minitablet. It can be prescribed. Thickeners, fragrances, diluents, emulsifiers, dispersion aids or binders may be desirable. Tablets or capsules may contain a binder (eg, pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropylmethylcellulose); a filler (eg, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); a lubricant (eg, stearin By conventional means with pharmaceutically acceptable excipients such as tablet splitting substances (eg potato starch or sodium starch glycolate); or wetting agents (eg sodium lauryl phosphate); It can be prepared. The tablets can be coated by methods well known in the art. Liquid preparations for oral administration include suspensions (eg, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (eg, lecithin, or acacia); non-aqueous vehicles (eg, almond oil, oily esters) , Ethyl alcohol, or rectified vegetable oils); and pharmaceutically acceptable additives such as preservatives (eg, methyl or propyl-p-hydroxybenzoate or sorbic acid). The preparation may likewise contain buffer salts, fragrances, colorants and sweeteners as appropriate. Preparations for oral administration can be suitably formulated for sustained, controlled or sustained release of a prophylactic or therapeutic agent.

  In one embodiment, the compositions of the invention are administered orally in combination with one or more penetration enhancers, such as surfactants and chelating agents. Preferred surfactants include, but are not limited to, fatty acids and esters or salts thereof, bile acids and salts thereof. In some embodiments, a combination of penetration enhancers is used, eg, fatty acids / salts in combination with bile acids / salts. In one particular embodiment, lauric acid, the sodium salt of capric acid is used in combination with UDCA. Additional penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The composition of the invention may be administered orally in granular form, including but not limited to sprayed dry particles, or complexed to form microparticles or nanoparticles. You can also. Complexing agents that can be used to complex with the peptides of the invention (eg, FGF-CX polypeptides) include poly-amino acids, polyimines, polyacrylates, polyalkyl acrylates, polyoxyethanes. ), Polyalkyl cyanoacrylate, cationized gelatin, albumin, acrylate, polyethylene glycol (PEG), polyalkyl cyanoacrylate, DEAE-derivatized polyimine, porlan, cellulose, and starch. is not. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P (TDAE), polyaminostyrene ( For example, p-amino), poly (methyl cyanoacrylate), poly (ethyl cyanoacrylate), poly (butyl cyanoacrylate), poly (isobutyl cyanoacrylate), poly (isohexyl cyanoacrylate), DEAE- Methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly (DL-lactic acid), poly (D, L-lactic acid-co- Recall acid (PLGA), alginate, and polyethylene glycol (PEG) including but not limited to these.

  The method of the invention may include pulmonary administration of the composition formulated with an aerosolizing agent, for example using an inhaler or nebulizer.

  The methods of the invention can include administration of a composition formulated for parenteral administration by infusion (eg, bolus infusion or continuous infusion). Injectable formulations may take the form of unit dosage forms (eg, ampoules or multiple dose containers) with added preservatives. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form to comprise a suitable vehicle (eg, sterile, pyrogen-free water) prior to use.

  In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a local anesthetic and solubilizer such as lignocaine to ease pain at the site of the injection. In general, the ingredients are supplied in a unit dosage form or separately as a lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

  The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino acids such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, sodium, potassium, ammonium, calcium, ferric hydroxide And those formed with free carboxyl groups such as, but not limited to, those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Non-limiting examples of pharmaceutically acceptable salts include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium acetate, cansylate, carbonate, chloride Citrate, dihydrochloride, edetate, edicylate, estrate, esylate, fumarate, glucaptate, gluconate, glutamate, glucorylarsanylate, hexylresorcinate, hydrabamine, bromide Hydronate, Hydrochloride, Hydroxynaphthoate, Iodide, Isethionate, Lactate, Lactobionate, Malate, Maleate, Mandelate Mesylate, Methyl bromide, Methyl nitride, Sulfide Methyl, mucato, napsic acid, nitrate, pamoate (embonate), pantothenate, phosphaterdiphosphate, polygalacturonic acid Salt, salicylate, stearate, basic acetate, succinate, sulfate, tannate, tartrate, theocrate, triethiodide, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum , Calcium, lithium, magnesium, potassium, sodium and zinc.

  In addition to the formulations described above, the composition can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition may be formulated with a suitable polymer or hydrophobic material (eg, as an emulsion in an acceptable oil) or ion exchange resin, or as a slightly less soluble derivative such as, for example, a slightly less soluble salt. it can. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.

  In one embodiment, the components of the inventive composition (eg, FGF-CX) are derived from a subject that is of the same species origin or species reactivity as the recipient of such composition.

  The invention also provides a kit for carrying out the treatment regimen of the invention. Such kits contain a prophylactically or therapeutically effective amount of a composition of the invention (eg, FGF-CX polypeptide) in one or more containers in a pharmaceutically acceptable form. The composition contained in the vial of the kit of the invention may be in the form of a pharmaceutically acceptable solution in combination with, for example, sterile saline, dextrose solution or buffer solution or other pharmaceutically acceptable sterile fluid. Can take. Alternatively, the composition can be lyophilized or dried. In this case, the kit optionally further comprises a pharmaceutically acceptable solution (eg, saline, dextrose solution, etc.) that is preferably sterile to reconstitute the composition to form a solution for injection purposes. Is contained in the container.

  In another embodiment, the inventive kit further comprises a needle or syringe and / or a packaged alcohol pad, preferably packaged in sterile form, for injecting the formulation. Instructions for administration of the inventive formulation by a clinician or patient are optionally included.

  In some embodiments, the invention comprises a kit comprising a plurality of containers each containing a pharmaceutical formulation or composition comprising a dose of a composition of the invention (eg, FGF-CX polypeptide) sufficient for a single administration. I will provide a.

  As is the case with all pharmaceutical products, packaging materials and containers are designed to protect the stability of the product during storage and shipment. In one embodiment, the composition of the invention comprises a biocompatible detergent comprising (but not limited to) lecithin, taurocholic acid and cholesterol; or gamma globulin and serum albulin (provided that It is stored in a container with other proteins (but not limited to these). In addition, the product of the invention includes instructions for use or other informational material that advises a physician, technician or patient on how to properly prevent or treat the disease or disorder in question. .

  The invention is further illustrated using the following examples.

5.1. Example 1: Sequence Analysis Details of sequence relatedness and domain analysis for each FGF-CX are presented in Table 1A. The FGF-CX1 clone was analyzed and the nucleotide and encoded polypeptide sequences are shown in Table 1A.

  A clustered double (ClustalW) comparison of the protein sequences described above produces the following sequence alignment shown in Table 1B.

  Further analysis of the FGF-CX1a protein produced the following properties shown in Table 1C.

  A search for the FGF-CX1a protein against the Geneseq database, a proprietary database containing sequences published in patents and patent publications, generated a plurality of homologous proteins shown in Table 1D.

  In a BLAST search of the public sequence database, the FGF-CX1a protein was found to have homology to the proteins shown in the BLASTP data in Table 1E.

  PFam analysis predicts that the FGF-CX1a protein contains the domains shown in Table 1F.

5.2. Example 2: Liquid chromatography, mass spectrometry and N-terminal sequencing of the product CG53135-05 by proteolysis of CG53135-05 (FGF-20) resulted in variants with high activity in proliferation assays. . Thus, these variants detailed in this section are expected to have the same utility as CG53135-05.

Liquid chromatography (LC) and mass spectrometry (MS) of CG53135-05
Purified CG53135-05 was purified from a phenyl-hexyl column (Luna 5 mm) using a standard HPLC system (Agilent 1100, Agilent) in a mobile phase containing acetonitrile, water and trifluoroacetic acid. , 250 mm x 3 mm, Phenomenex). The resulting analysis shows a detectable level of micro-heterogeneity showing one large peak (# 3) and three small peaks (# 1, 2, 4 and 5) associated with CG53135-05 (FIG. 1). Revealed sex. To characterize these species, fractions were collected using an automated fraction collector (Agilent 1100), liquid chromatography electrospray ionization ion trap mass spectroscopy (LC / ESI / MS), matrix assisted Fractions were characterized using laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and N-terminal amino acid sequencing.

  Peak 0 is not a protein, is not product related, and is not a residual process impurity (ie, not DNA, endotoxin, kanamycin or IPTG). CG53135-05 related species (peaks 1, 2, 3 and 4) eluted a mobile phase of 36% acetonitrile, 63% water and 0.1% trifluoroacetic acid. Similar fractions were then pooled and the sample concentrated using an Amicon 10,000 Dalton cutoff filter (Millipore, Bedford, Mass.) In a 4 ° C. centrifuge. . 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 μl. The concentration of pooled fractions was determined using amino acid analysis.

  All four CG53135-05 related species (peaks 1, 2, 3 and 4) were digested proteolytically using trypsin and the peptides were analyzed using liquid chromatography with mass spectrometric detection. XP DECA nanospray / ion trap instrument (ThermoFinnigan), San Jose, CA, interfaced with the ultimate nanoflow chromatography system (LC Packings, Amsterdam, The Netherlands). )) Was used for mass spectrometry. Data was collected via Xcalibur software (thermofin gun) using automated MS-MS / MS switching. Within the Instrument Method file, all MS scans between 400 and 1400 m / z followed by all MS / MS between 400 and 2000 m / z of the top three ions from the preceding MS scan XPDECA was set to acquire a scan. Data was processed using TurboSequence (thermofingan). Database search and protein identification was performed using MASCOT (Matrix Sciences, Manchester, UK). MASCOT reports a probability-based MOWSE score and coverage percentage for the identified proteins based on the molecular mass of the peptide, MS / MS sequence information, mass accuracy and the number of peptides detected. Table 2 contains peak numbers for CG53135-05 related species, confidence scores provided by MASCOT, and percentage coverage obtained from MS / MS spectra.

ＮＡ＝ データ入手不可

NA = Data not available

  To determine the identity of CG53135-05 related species, the collected fractions were analyzed by MALDI-TOF and N-terminal sequencing. The N-terminal amino acid of purified CG53135-05 was qualitatively determined. CG53135-05 protein was resolved by SDS-PAGE and transferred to a polyvinylidene fluoride membrane by electrophoresis. A major band of approximately 23 kDa stained with Coomassie from the membrane was excised and analyzed by an automated Edman sequencer (Procise, Applied Biosystems, Foster City, Calif.). . Table 3 presents the molecular weights obtained for each species and the variants determined by N-terminal sequencing (where N = full length CG53135).

ＮＡ＝データ入手不可。タンパク質でなく製品関連でもない。

NA = data not available. It is neither protein nor product related.

  The molecular weight determined by MALDI-TOF and N-terminal sequencing can identify four species. For peak 1, four different species were detected via N-terminal sequencing, two of which were also detected by MALDI-TOF. These results are also consistent with the coverage obtained using LC / MS. The polypeptide sequences for each species derived by N-terminal sequencing are shown in Table 4.

多重アラインメント

Multiple alignment

Total amino acid analysis The amino acid composition of CG53135-05 was determined. A sample of CG53135-05 was hydrolyzed for 16 hours at 115 ° C. in 0.2% phenol, 100 mL 6N HCl containing 2 nmol norleucine as an internal standard. The sample was dried in a high speed Vac concentrator and dissolved in 100 mL of sample buffer containing 2 nmol homoserine as an internal standard. The amino acids in each sample were separated on a Beckman 7300 type ion exchange instrument. The amino acid composition of CG53135-05 was consistent with the theoretical amino acid composition.

  An experimental amino acid composition was used to derive the extinction coefficient used in concentration estimation via UV absorbance (protein estimation using the Bradford method). The extinction coefficient at λmax is 0.97 mL / mg · cm.

^Ａ 処方中の余剰のａｒｇのため未判定。
^Ｂ 分析中に酸加水分解においてｃｙｓが破壊されることから未判定。
^Ｃ 分析中に酸加水分解においてｔｒｐが破壊されることから未判定。
^Ｄ 酸加水分解中ａｓｎはａｓｐに、ｇｌｎはｇｌｕ酸に転換されることになる。従って、ａｓｘはａｓｎとａｓｐの和を表わし、一方ｇｌｘはｇｌｎとｇｌｕの和を表わす。

^A Not determined due to surplus arg in prescription.
Undetermined because cys is destroyed in acid hydrolysis during ^B analysis.
Not determined because trp is destroyed in acid hydrolysis during ^C analysis.
^{During D} acid hydrolysis, asn will be converted to asp and gln to gluco acid. Thus, asx represents the sum of asn and asp, while glx represents the sum of gln and glu.

Peptide mapping purified CG53135-05 (25 mg) was denatured, reduced in urea and dithiothreitol at 50 ° C. and alkylated with iodoacetate. After reducing the urea concentration, the samples were treated with trypsin at 20 ° C. for 40 hours. The resulting peptide fragments were separated by RP-HPLC (by using a C-18 column with an acetonitrile gradient in trifluoroacetate) to give peptide maps (FIGS. 2A and 2B). The chromatogram in FIG. 2A is consistent with the 20 peptides expected from digestion of CG53135-05 with trypsin, and the chromatogram in FIG. 2B shows a single tryptophan residue within CG53135-05. Reveals a single peak as expected for.

The biological activity of CG53135-05 related species collected from the four peaks identified by biological assays LC and MS was compared to serum-deficient cultured NIH3T3 mouse embryonic fibers with various doses of isolated CG53135-05 related species. Measured by measurement of bromodeoxyuridine (BrdU) incorporation during blast treatment and DNA synthesis. For this assay, cells were cultured in Dulbecco's modified Eagle 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 depleted in Dulbecco's modified Eagle medium for 24-72 hours. CG53135-05 related species were added and incubated at 37 ° C. for 18 hours in 10% CO ₂ / air. BrdU (10 mM final concentration) was added and allowed to incubate with the cells for 2 hours at 37 ° C. in 10% CO ₂ / air. BrdU incorporation was measured by enzyme immunosorbent method according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN).

Peak 4 was not included in this assay because only insufficient material was collected (Peak 4 is less than 3% of the total peak area for CG53135-05). Material collected from CG53135-05 and all three remaining fractions (ie peaks 1, 2 and 3) induced DNA synthesis in NIH3T3 mouse fibroblasts in a dose-dependent manner (Table 7). PI ₂₀₀ was defined as the concentration of protein that resulted in BrdU incorporation at twice the background. CG53135-05 and CG53135-05 related species recovered from all three measurable peaks demonstrated similar biological activity with a PI ₂₀₀ of 0.7-11 ng / mL (Table 9).

5.3. Example 3: Receptor binding specificity of CG53135 (Study L-116.01)
FGF family members transduce signals inside the cell through high affinity interactions with the cell surface immunoglobulin (Ig) domain containing tyrosine kinase FGF receptor (FGFR). Four distinct human genes encode FGFR (Powers et al., Endocr Relat Cancer 2000, 7: 165-97; Klint and Claesson-Welsh, Life Frontier of Science (1999), 4: D165-77; Xu et al., Cell Tissue Res 1999, 296: 33-43). A related fifth human sequence lacking the kinase domain was recently identified and named FGFR-5 (Kim et al., Biochim Biophys Acta 2001, 1518: 152). -6). Each of these receptors can bind several different members of this family (Kim et al., Biochemistry and Biophysics 2001, 1518: 152-6; Ornitz et al., Biochemistry Journal (J Biol Chem)). 1996, 271: 15292-7). FGF also binds to heparin sulfate proteoglycans (HSPG) that are present on most cell surfaces and extracellular matrix (ECM), albeit with low affinity. The interaction between FGF and HSPG stabilizes the FGF / FGFR interaction and serves to sequester FGF and protect it from segregation (Powers et al., Endocrine-Related Cancer 2000, 7: 165-97; Seveni (Szebenyi) and Fallon, International Cytology Review (Int Rev Cytol 1999, 185: 45-106). It has been reported that dimerization of the FGF receptor monomer at the time of ligand binding is a necessary condition for activation of the kinase domain that leads to receptor transphosphorylation. FGF receptor-1 (FGFR-1), which shows the maximum expression pattern of four FGF receptors, contains at least seven tyrosine phosphorylation sites. Numerous signal transduction molecules are affected by binding to these phosphorylation sites with different affinities.

  FGFR-1, FGFR-2 and FGFR-3 recognize FGF-1, FGF-2, FGF-4 and FGF-8, respectively. In addition, FGFR-1 and FGFR-2 bind to FGF-3, FGF-5, FGF-6, FGF-10 and FGF-17 (Powers et al., Endocrine related cancer 2000, 7: 165-97). The 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. By tissue-specific alternating splicing, cells expressing a single FGFR gene can significantly diversify their biological responses by generating completely different receptor isoforms that can exhibit different ligand specificities and functions. It becomes possible. FGFR-4 binds to 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 recognized only by FGFR-2, while FGF-9 binds to FGFR-2, FGFR-3 and FGFR-4. The receptor specificity of FGF11-FGF19 is not well understood (Powers et al., Endocrine-Related Cancer 2000, 7: 165-97; Ornitz et al., Biochemistry Journal 1996, 271: 15292-7).

  Immunohistological studies in normal human adult tissues from major organ systems (J Hughes, Histochemistry Cytochem 1997, 45: 1005-19) are FGFR-1, FGFR- 2 and FGFR-3 have been widely expressed, suggesting an important functional role in tissue homeostasis. The protein expression pattern for tissue specific isoforms has not yet been determined. FGFR-4 is more limited than it is significantly absent in the tissues where all other three receptors are predominantly expressed, lung, oviduct, placenta, testis, prostate, thyroid, parathyroid and sympathetic ganglia. (Hughes, Histochemistry and Cytochemistry Journal 1997, 45: 1005-19).

  To determine the receptor binding specificity of CG53135, we investigated the effect of soluble FGFR on the induction of DNA synthesis in NIH3T3 cells by recombinant CG53135-01 produced in E. coli.

Materials and Methods Purification of proteins from Escherichia coli: For production of E. coli, plasmid pETMY-hFGF20X was transformed into E. coli expression host BL21 (Novagen, Madison, Wis.). (Madison, WI)), induction of protein CG53135 expression was performed according to the manufacturer's instructions. Bacteria of pETMYhFGF20X / BL21 E. coli were grown in LB medium at 37 ° C. Bacteriophage lambda (CE6) was added at an OD of 0.6 until the final multiplicity of infection was 5. The infected medium was further incubated at 27 ° C. for 3 hours. Following induction, whole cells were harvested and proteins were analyzed by Western blot using anti-HisGly antibody (Invitrogen. Low speed centrifugation (5000 rpm in GS-3 rotator for 15 min at 4 ° C.). Cells were harvested, suspended in phosphate buffered saline (PBS) containing 0.5 M NaCl and 1 M arginine and disrupted by passing twice through a microfluidizer. The soluble protein fraction (supernatant) was clarified by separation and filtration through a 0.2 micron low protein binding membrane, after which the protein sample was washed with a metal chelation column (pre-filled with nickel sulfate). The nickel column was washed with PBS / 0.5M NaCl + 1M L-arginine and bound. Protein were pooled fractions containing the .CG53135 eluted with a linear gradient of imidazole (0-0.5 M) (100-150 mM imidazole), PBSpH8 of 1 × 10 ⁶ volumes containing L- arginine 1M Dialyzed against 0. Protein samples were stored at -80 ° C.

  Receptor specificity: NIH3T3 cells were cultured in 96-well plates to approximately 100% confluence, washed, supplemented with DMEM (Life Technologies) without supplementation, and incubated for 24 hours. Recombinant CG53135-01 or control protein was then added to the cells for 18 hours, the control proteins used were aFGF (positive control) and platelet derived growth factor-BB (PDGFBB) (negative control). To analyze the effect of soluble FGFR on CG53135 activity, recombinant CG53135-01, aFGF, or PDGF-BB (final concentrations of 10, 5 and 3 ng / mL, respectively) were dissolved in soluble receptors (0.2, 1 And 5 μg / mL final concentration) and into serum-deficient NIH3T3 cells Incubated for 30 minutes at 37 ° C. before addition of the factor concentration represents the amount of ligand required to generate a half-value BrdU response in NIH3T3 cells.The soluble FGFR is expressed in the following receptor form (FGFR1β (IIIc FGFR2β (IIIb); FGFR2α (IIIb); FGFR2α (IIIc); FGFR3α (IIIc); FGFR4) Fc chimera, obtained from R & D Systems (Minneapolis, Minn.) The BrdU test was performed according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN) using a BrdU uptake time of 4 hours.

Results and Conclusions To determine the receptor binding specificity of CG53135, we investigated the effect of soluble FGFR on the induction of DNA synthesis in NIH3T3 cells by recombinant CG53135-01 produced in E. coli. Soluble receptors for FGFR1β (IIIc), FGFR2β (IIIb), FGFR2α (IIIb), FGFR2α (IIIc), FGFR3α (IIIc) and FGFR4 were utilized. We have found that the soluble form of each of these FGFRs can specifically inhibit the biological activity of CG53135 (FIG. 3).

  Complete or near complete inhibition was obtained with soluble FGFR2α (IIIb), FGFR2β (IIIb), FGFR2α (IIIc) and FGFR3α (IIIc), while partial inhibition was achieved with soluble FGFR1β (IIIc) and FGFR4. None of the soluble receptor reagents interfered with the induction of DNA synthesis by PDGF-BB (FIG. 2), thus demonstrating their specificity. The integrity of each soluble receptor reagent was demonstrated by demonstrating its ability to inhibit the induction of DNA synthesis by aFGF, a factor known to interact with all of the FGFRs under analysis (FIG. 3). .

5.4. Example 4: Treatment of stroke Thirty male Sprague-Dawell rats were assigned to treatment groups as indicated in the study design in Table 8 below.

*投与用量及び体積は、３３０ｇの平均体重に基づくものである。

* Dosage and volume are based on an average body weight of 330 g.

Experimental Procedure Middle Cerebral Artery (MCA) Surgery and Intracisional Injection: Animals were treated for 7 days prior to surgery. Cefazolin sodium (40 mg / kg, ip) was administered the day before and immediately after surgery. At the time of surgery, rats were anesthetized with 2% halothane in a 2: 1 N ₂ O: O ₂ mixture. Body temperature was maintained at 37 ± 0.50 ° C. The proximal right MCA was electrocoagulated from just proximal to the olfactory tract to the inferior cerebral vein and then severed. For instillation in the tank, the animals were again anesthetized as described above and placed in a stereotaxic frame. Rats received CG53135-05 or vehicle [40 mM acetate, 200 mM mannitol (pH 5.3)] once on the first day (about 24 hours) and once on the third day (about 72 hours) from the MCA. It was given by intradermal injection into the cisterna magna. Animals were given test articles (2 dose groups) or vehicle treatment according to the study design.

Clinical observations / animals were observed for signs of seizure (indicated by vigorous movement and tremor around the cage), distress (indicated by loud vocalization) and coma for 1 hour immediately after injection. Animals were also observed daily for mortality and mortality.

  Body weight: Animals were weighed on day 1, day 3, day 7, day 14 and day 21.

  Limb Reposition Test: Limb Reposition Test for all animals on day -1 (preoperative), day 1 (immediately before injection), day 3 and every 7 days thereafter (day 7, day 14, day 21) Eyes)

Forelimb Reposition Test Evaluation Score: The forelimb reposition test measures the sensorimotor function within each forelimb as the animal places the limb on the table top in response to visual, tactile and self-accepting stimuli. The forelimb reposition test consists of the following assessments and scores, where the combined total score for the forelimb reposition test reflects a range from 0 (no dysfunction) to 10 (maximum dysfunction).
Visual repositioning (forward, horizontal): 0-4
Tactile repositioning (back, side): 0-4
Self-accepting repositioning: 0-2
Total score for all forelimb tests: 0-10

Hind limb repositioning test evaluation score: Similarly, the hind limb repositioning test measures the sensorimotor function of the hind limb as the animal places the hind limb on the table top in response to tactile and self-accepting stimuli. The hindlimb reposition test consists of the following evaluations and scores, where the combined total score for the hindlimb reposition test reflects a range from 0 (no dysfunction) to 6 (maximum dysfunction).
Tactile repositioning (back, side): 0-4
Self-accepting repositioning: 0-2
Total score for all hindlimb tests: 0-6

  Body Swing Test: The body swing test was performed for all animals on day -1 (preoperative), day 1 (immediately before injection), day 3 and every 7 days thereafter (day 7, day 14, day 21). Eye).

  The body swing test examines the lateral preference as the animal is held approximately 1 inch above the surface of the table and rocked to the right or left. Count the 30 swings and then calculate a score based on the percentage of swing to the right. (Score range = approximately 50% rightward swing (no functional impairment)-0% rightward swing (maximum functional failure)).

  Cylinder test: A cylinder test was performed on all animals on day -1 (preoperative) and postoperative day 7 (7th, 14th, 21st). The cylinder test measures the locomotor activity of the forelimbs. Animals are placed in a narrow glass cylinder (16.5 x 25 cm) and recorded on videotape for 5 minutes the day before stroke surgery and then at weekly intervals for 5 minutes. The videotape will then be assessed independently by one experienced observer and will count up to 50 locomotor activity (approximately 5 minutes per rat per day). Spontaneous movements include those performed by each forelimb to start rising and land sideways or move sideways along the cylinder wall, or to land on the floor behind the rising.

  Gross examination and histomorphology: On the scheduled end date (day 3), animals were euthanized by intraperitoneal injection of chloral hydrate (500 mg / kg). The brain was examined and removed with the naked eye, postfixed in formalin, dehydrated and embedded in paraffin. A coronal section (5 mm) is cut on a microtome mounted on a glass slide and stained with hematoxylin / eosin (H & E). Cerebral infarction on each of +4.7, +2.7, +0.7, -1.3, -3.3, -5.3 and -7.3 mm compared to 7 slices (Bregma) Were determined using a computer interface imaging system that uses an indirect method to correct for brain contraction during processing (intact contralateral hemisphere area-intact ipsilateral hemisphere area). . The basal volume was then expressed as a percentage of the intact contralateral hemisphere volume. Separately using these same methods, the volume of embolization in the cortex and striatum was also determined. H & E stained sections were examined for histological changes such as bleeding, abscess or tumor formation.

  Statistical analysis: For treatment assignment of each animal, the blinded investigator performed all in-tank injections, behavioral studies and subsequent histological analysis. Data are then expressed as mean ± SEM and will be analyzed by one or two methods (ANOVA) followed by an appropriate paired post-hoc test with correction for multiple comparisons. .

Results Forelimb Reposition Test: Limb rest to assess sensorimotor function in the forelimb in response to visual, tactile and self-accepting stimuli on days 1, 1, 3, 7, 14 and 21 in relation to MCA occlusion The animals were examined by using a repair test (Kawamata T., Dietrich WD, Schallert T., Gotss E., Cocke R.R. Benowitz LI and Finklestein SP (1997) Bulletin of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 94, 8179-8184; De Ryck M. Van Reempts J., De Icheba (Duytschaever) H., Van Deuren (Van Deuren) B. And Kurinke (Clincke) G. (1992) Brain Research (Brain Res.) 573,44-60). Visual repositioning (rating from 0 to 4), tactile repositioning (rating from 0 to 4) and self-accepting setting to obtain a potential total score range of 0-12, with 12 representing maximum dysfunction Corrections (rating from 0 to 2) were summed (Figure 4).

  Hind limb repositioning test: In relation to MCA occlusion, a limb repositioning test was conducted on days 1, 1, 3, 7, 14, and 21 to assess sensorimotor function in the hind limb in response to tactile and self-accepting stimuli. Animals were examined by use (Kawamata T., Dietrich WD, Schallert T., Gotss E., Cocke RR, Benowitz ( Benowitz LI and Finklestein SP (1997) Bulletin of the American Academy of Sciences (Proc. Natl. Acad. Sci. USA) 94, 8179-8184; De Ryck M., Fan. Limpts J., Duitseva (Duitschaver H., Van Deuren B. and Clinke G. (1992) Brain Res. 573, 44-60). Tactile repositioning (rating 0-4) and self-accepting repositioning (rating 0-2) were summed to obtain a potential total score range of 0-6, with 6 representing maximum dysfunction ( FIG. 5).

  Body Swing Test: Lateral as the animal is held approximately 1 inch above the surface of the table and swings to the right or left on days 1, 1, 3, 7, 14, and 21 in relation to MCA occlusion Animals were examined by using body swing test to assess preference (Kawamata T., Dietrich WD, Schallert T., Gotts E., Cook ( Cocke RR, Benowitz LI, and Finklestein SP (1997) Bulletin of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 94, 8179-8184; (De Ryck) M., Fan Limpts (V n Reempts) J., Deyuicheba (Duytschaever) H., Van Deuren (Van Deuren) B. and Kurinke (Clincke), G. (1992) Brain Research (Brain Res.) 573,44-60). Count the 30 swings and calculate a score based on the percentage of swing to the right (FIG. 6).

  Cylinder test: On days 1, 1, 3, 7, 14, and 21 in relation to MCA occlusion, animals were examined by the cylinder test to assess forelimb locomotor activity (Kawamata T. et al. Dietrich WD, Schallert T., Gotts E., Cocke RR, Benowitz LI, and Finklestein SP (1997) Bulletin of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 94, 8179-8184; De Ryck M., Van Reempts J., Duytschaver H., Van deuren (Van Deuren B. and Clinke, G. (1992) Brain Res. 573, 44-60). Briefly, animals are placed in a narrow glass cylinder (16.5 × 25 cm) and recorded on videotape for 5 minutes the day before stroke surgery and then at weekly intervals for 5 minutes. The videotape will then be assessed independently by one experienced observer and will count up to 50 locomotor activity (approximately 5 minutes per rat per day). Spontaneous movements include those performed by each forelimb to start rising and land sideways or move sideways along the cylinder wall, or to land on the floor behind the rising (FIG. 7).

  Body weight: Animals were weighed on days -1, 1, 3, 7, 14, and 21 in relation to MCA occlusion. The results show no significant difference between vehicle and CG53135-05 treatment (Figure 8).

Conclusions Administration of CG53135-05 after MCA occlusion significantly increased recovery in the forelimb (Fig. 4) and hindlimb (Fig. 5) repositioning tests for the contralateral (affected) limb, both at low and high doses. , And suggested that it produces an improvement in the body swing test (FIG. 6). This activity pattern with other therapies in this model has been shown to reflect improvements in cerebral cortex and subcortical (striatum) function, respectively (Dijkhuizen RM, Ren). J, Mandeville JB, Wu O, Ozdag FM, Moskowitz MA, Rosen BR, Finklestein SP. 2001, Bulletin of the National Academy of Sciences 98 (22) ): 12766-71). There were no obvious differences in the voluntary limb use cylinder test (FIG. 7) or animal weight (FIG. 8).

  Therefore, for pathological conditions including ischemic stroke, hemorrhagic stroke, trauma, spinal cord injury, heavy metal or toxin poisoning, and neurodegenerative diseases (eg Alzheimer's, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, etc.) In therapy, administration of CG53135-05 will be useful.

5.5. Example 5: Matrix metalloproteinase production assay Matrix metalloproteinases (MMPs) are a family of related enzymes that degrade extracellular matrix in bone and cartilage. These enzymes act during normal development in tissue differentiation and remodeling. In joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA), increased expression of these enzymes contributes to irreversible matrix degradation. Thus, the effect of CG53135-05 on MMP production was evaluated.

The activity of CG53135 on the production of matrix metalloproteinase (MMP) was evaluated using the SW1353 chondrosarcoma cell line (ATCC HTB-94). This cell line is an established chondrocyte model for matrix metalloproteinases (MMPs). SW1353 cells were plated in 24 well plates at 1 × 10 ⁵ cells / ml (1 ml) in DMEM medium—10% FBS. After overnight incubation, the medium was replaced with DMEM + 0.2% lactalbumin serum. 10 in the absence or presence of IL-1 beta (0.1-1 ng / ml, R & S Systems, Minneapolis, MN), TNF-alpha (10 ng / ml, R & D Systems) or vehicle control to a final volume of 0.5 ml. CG53135-05 was added to the wells at doses in the range of ~ 5000 ng / ml. Both IL-1 beta and TNF-alpha are potent stimulators of MMP activity. All treatments were performed in triplicate wells. After 24 hours, supernatants were collected and pro-MMP-1 and -13 and TIMP-1 (tissue inhibitors of matrix metalloproteinases), which are natural inhibitors of MMP activity, were measured by ELISA (R & D Systems). Measurements were normalized to cell number by MTS assay.

Results CG53135-05 significantly reduced MMP-13 production in the presence of either IL-1 beta or TNF-alpha as demonstrated in FIGS. 14 and 15, respectively. Both IL-1 beta and TNF-alpha are potent stimulators of MMP activity. MMP-13 affinity for type II collagen, the major collagen that is degraded in OA, is 10 times higher than that of MMP-1. Since MMP-13 expression is increased in OA and RA, the decrease in MMP-13 observed with the addition of CG53135-05 indicates that the protein can be used as an OA and RA therapeutic agent. (FIG. 10). Furthermore, CG53135-05 upregulated the production of TIMP-1, which is a natural inhibitor of MMP activity (FIG. 11). This enhancement of TIMP-1 production by CG53135-05 is beneficial in reducing the matrix destruction by MMP-1 and MMP-13 observed in OA and RA. Furthermore, CG53135-05 had no effect on MMP-3 production constitutively or after IL-1 induction (data not shown). Similarly, CG53135-05 (FGF-20) showed increased basal expression of MMP-1 in SW1353 cells (data not shown).

5.6. Example 6: Effect of CG53135-05 on normal rats: Verification of concept on meniscal tear model The effect of CG53135-05 on normal rats is further demonstrated in disease models (eg, meniscal tear model of osteoarthritis in rats). The research was carried out as a verification of the concept to promote the research. The effect of CG53135-05 on synovium and cartilage was assessed by injecting protein into normal male Lewis rats.

Effect of intra-articular injection of CG53135-05 in normal rats Vehicle solution (8 mM acetate, 40 mM arginine, and 0.6% glycerol (pH 5.3) in about 1% hyaluronic acid), 10 μg CG53135 -05 or 100 [mu] g CG53135-05 was injected into the joints of the rats 3 times a week for 2 weeks.

  Study Design: Male Lewis rats weighing 293-325 grams on day 0 were obtained from Harlan Sprague Dawley, Indianapolis, IN and acclimated for 8 days. Rats were divided into 3 treatment groups, each containing 3 animals. Two groups received CG53135 and one group received vehicle control only. Rats were anesthetized with isoflurane and injected through the patella tendon into the cruciate area of both knees. CG53135 was injected at a dose of 0.1 mg / ml (0.01 mg per joint) or 1.0 mg / ml (0.1 mg per joint). Controls were injected with vehicle solution as described above. Injections were made on Monday, Wednesday and Friday for 2 weeks. On day 15, the animals were discarded and at that time BRDU (100 mg / kg) was injected intraperitoneally to pulse label growing cells.

Observation and analysis of lesion markers and relative observation. Rats were observed daily for abnormal swelling or gait changes and weighed weekly.

  Histopathologically preserved and decalcified (5% formic acid) knees are trimmed into two approximately equal longitudinal (ankle) or anterior (knee) halves, treated through graded alcohol and detergent, infiltrated and paraffinized It was embedded in, cut and stained with toluidine blue (knee). Numerous sections (three levels) of the right knee were analyzed microscopically, paying attention to the problem parameters listed below. Each parameter was graded as normal, minimal, mild, moderate, high or severe. Because of the type of modification produced by repeated injections of protein, cartilage was evaluated using descriptive parameters rather than the assessment criteria commonly used in osteoarthritis models. Although animals received BRDU injections prior to disposal, growth changes were readily seen in sections stained with toluidine blue.

result

  Throughout the study, survival stage parameter body weights were similar in both vehicle and protein injected animals (Table 12). The knee injected with 100 μg protein had some evidence of fibrosis clinically during the injection process starting with the third injection.

  Rats receiving morphological pathological vehicle injections have minimal or mild synovial hyperplasia, inflammation, and fibrosis with minimal or minimal matrix deposition in the fibrotic synovium. Was. Articular cartilage did not have any proteoglycan loss or fibrillation. The central area of the joint where the cross was attached and the intraarticular injection was performed had no or minimal fibroproliferation and cartilage / bone damage. There was no marginal cartilage formation.

  A knee that received an injection of 10 μg CG53135-05 had minimal or moderate matrix deposition within the fibrotic synovium, minimal to moderate synovial hyperplasia, inflammation, fibrosis Had. Articular cartilage did not have any proteoglycan loss or fibrillation. The central area of the joint where the cross was attached and the intraarticular injection was performed had no or minimal fibroproliferation and cartilage / bone damage. One knee had minimal cartilage formation in the marginal zone.

  The knee that received an injection of 100 μg CG53135-05 had moderate to high synovial hyperplasia, inflammation, and fibrosis with moderate matrix deposition in the fibrotic synovium. . Articular cartilage had no or minimal proteoglycan loss or fibrillation. The central area of the joint where the cross was attached and the intraarticular injection was performed had minimal to high fibrosis and cartilage / bone damage. All knees had mild to moderate marginal zone cartilage formation. One animal had cartilage formation in the area associated with articular cartilage.

Conclusion These results demonstrate that repeated intra-articular injection of CG53135-05 induces synovial fiber proliferation. Vehicle injection resulted in mild inflammation and fibrosis, thus suggesting that this vehicle has some stimulating potential. In animals that received protein injection, an increased concentration responsiveness of the synovial proliferative response and marginal zone cartilage components developed. The area of the cruciform attachment where the injection was performed had areas of bone resorption and fibroproliferation that increased in severity with increasing protein concentration as well as synovial inflammation. The observed adverse effects of synovial fiber growth and bone resorption are due to either FGF-20 activity or endotoxin levels within the nonclinical grade hyaluronic acid used to formulate the protein. It is thought that it was what. Furthermore, intra-articular inflammation can induce bone resorption and marginal zone cartilage formation, so these results need to be interpreted in light of the possibility that the inflammatory response to protein injection contributed to the proliferative response. is there. The morphological appearance of proliferative changes and cartilage formation clearly indicates that the biological activity of this protein (CG53135-05) is important in generating a response.

  The results of the experiments reported in this document indicate that repeated intra-articular injection of CG53135-05 induces synovial fiber proliferation and cartilage formation.

5.7. Example 7: Intra-articular injection of CG53135-05 in a meniscal tear model of rat osteoarthritis: Prophylactic and therapeutic dosing Example 6 identifies normal rats in order to identify effects on relevant cell populations by histomorphometric analysis Administration of CG53135-05 into the joint. At a dose of 100 μg per joint, CG53135-05 induces significant marginal zone cartilage formation similar to that seen with other growth factors such as TGF-beta, and pluripotency within the marginal zone This suggested an effect on sex stem cells. There was no apparent effect on mature chondrocytes as evidenced by the lack of response within the mature cartilage area of the joint. Observed potentially adverse effects of synovial fiber growth and bone resorption are attributable to either FGF-20 activity or endotoxin levels within the nonclinical grade hyaluronic acid used to formulate the protein. It is thought that it was caused.

  Further studies performed on animals with osteoarthritis related to: 1) Synergy with anti-inflammatory drugs (standard approach for osteoarthritis patients) 2) Whether CG53135-05 (FGF-20) can induce functional repair or protection of the articular cartilage layer and 3) lubrication Was membrane fiber growth and bone resorption induced by FGF-20 or due to contaminating endotoxin within the formulation?

  Thus, one aspect of this study was to evaluate the protective and therapeutic effects of intra-articular injection of CG53135-05 against joint damage in osteoarthritis within a meniscal tear model of rat osteoarthritis. This relatively new model of OA has been shown to have morphological alterations in cartilage degeneration and osteophyte formation that mimic changes that occur in sudden and surgically induced diseases in other species ( Bendele AM, Animal Models of Osteoarthritis, J. Musculoskel. Neuron Interact. 2001; 1: 363-376, Bendele A.M. and Hulman ) JF, Spontaneous cartilage degeneration in guinea pigs, Arthritis and Rheumatism, 19 8; 31: 561-565). The model can be used to evaluate the potentially beneficial effects of anti-degeneration as well as regenerative therapy.

Animals housed at a ratio of 2 per experimental design (10 per group) were anesthetized with isoflurane and the right knee area was pretreated for surgery. A skin incision was made over the entire medial side of the knee, the medial collateral ligament was exposed by blunt dissection and then severed. The inner half-moon was then moved symmetrically with thin scissors and cut across the entire thickness to simulate a complete laceration. The skin was closed with sutures.

  Prophylactic dosing: Intra-articular administration of the right knee joint (CG53135-05) was started on the day of surgery, with the rats under isoflurane anesthesia for 2 weeks after surgery on Thursday, Saturday and Monday (0, 2 This was continued by intra-articular injection on days 4, 7, 9 and 11). Indomethacin, a non-steroidal anti-inflammatory drug, was dosed daily (1 mg / kg / day) by the oral route starting from the day of surgery to reduce any potential inflammation resulting from the injection. Body weights were recorded on days 0, 7 and 14. After disposal of the animals on day 14 after surgery, both knees were collected for histopathologic evaluation. The study design is shown in Table 10.

^ａ 毎週３回で２週間の投与（１００μｇ／関節、関節内）
^ｂ 毎日で、２週間の投与（０．５ｍｇ／ｋｇ、ＰＯ）

^a 3 times weekly for 2 weeks (100 μg / joint, intra-articular)
^b Daily, 2 weeks of administration (0.5 mg / kg, PO)

  Therapeutic dosing: Intra-articular dosing of the right knee joint (CG53135-05) started on day 21 after surgery and the rats are under isoflurane anesthesia for 2 weeks on Friday, Sunday and Tuesday (22, 25, This is continued by intra-articular injection on days 27, 29, 32 and 34). Indomethacin is dosed daily by oral route starting from the day of surgery. Body weight is recorded on days 0, 7, 14, 21, 28, and 35. On day 35, both knees are collected for histopathologic evaluation. The study design is shown in Table 11.

^ａ 毎週３回で２週間の投与（１００μｇ／関節、関節内）
^ｂ 毎日で、２週間の投与（０．５ｍｇ／ｋｇ、ＰＯ）

^a 3 times weekly for 2 weeks (100 μg / joint, intra-articular)
^b Daily, 2 weeks of administration (0.5 mg / kg, PO)

  Results of the prophylactic dosing study: The observations made included standards followed for this model. Numerous sections (3 levels) of the right knee were analyzed with a microscope and evaluated according to the following method. In assessing the three sections, the worst case scenario for the two halves on each of the three slides representing the three levels was determined for cartilage degeneration and osteophyte formation. This value of each parameter for each slide was then averaged to determine an overall subjective cartilage degeneration score for the tibia and femur and osteophyte score for the tibia.

Cartilage degeneration was rated with a rating from none to severe (numerical value 0-5) for depth and area (surface area divided by 3) using the following criteria.
0 = No degeneration 1 = Minimal degeneration with or without fibrillation involving surface zones, chondrocyte and proteoglycan loss 2 = Mild degeneration with or without fibrillation involving the top third , Chondrocyte and proteoglycan loss 3 = moderate degeneration, chondrocyte and proteoglycan loss, where fibrillation has spread well into the central zone and generally affects half of the total cartilage thickness.
4 = high degeneration, chondrocyte and proteoglycan loss, with no complete matrix loss (up to the highest point), but with fibrillation fully extended into the deep zone,
5 = severe denaturation, matrix loss to the highest point.

  In this rating method, strict attention was paid to the zones (outer, middle, inner third). The total score reflects a comprehensive sum of the severity of tibial degeneration.

  In addition to this overall subjective analysis of cartilage degeneration, additional subjective using similar criteria to assess degenerative severity, paying attention to specific regional differences across the tibial plateau Evaluation was performed. In this OA model, in general, the outer third of the tibia suffers from the most severe meniscal tear injury, with the lesion often extending to the highest point by 3 weeks after surgery. The middle 1/3 is usually a transition zone where the change in severity or elevation is moderate or mild and the interior 1/3 has a change that is mild or greater than the minimum. Each of these areas was assessed separately in an attempt to determine potential treatment differences for the outer 1/3 severe lesions versus the intermediate 1/3 and milder lesions of the inner 1/3. The sum of the area values was calculated and expressed as the sum of the three zones.

  In addition to the subjective assessment described above, any osteophyte with adjacent cartilage degeneration (outside 1/3) to the point where the tangential layer and underlying cartilage appear histologically normal across the surface If not present, micrometric measurements (total tibial cartilage degeneration width μm) of the full range of tibial plateaus suffering from any severity of degeneration extending from the origin of the osteophyte or marginal zone.

  Additional measurements (significant cartilage degeneration width μm) reflected areas of tibial cartilage degeneration where chondrocyte and matrix loss had spread over more than 50% of cartilage thickness.

  Finally, the micrometer depth of any type of lesion expressed as a ratio of the depth of the altered area to the depth to the highest point (cell / proteoglycan loss, metachromia changes, but excellent retention of the collagen matrix. And may have no fibrillation) and was taken over four equally spaced points on the tibial surface. These measurements are (No. 1) matrix adjacent to the osteophytes, (No. 2) 1/4 of the distance across the tibial plateau, (No. 3) 1/2 of the distance across the tibial plateau, (No 4) Performed at 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 the highest point (denominator) also provides an indication of the cartilage thickness across the tibial plateau, so groups can be selected when trying to determine whether hypertrophy or hyperplasia has occurred. Allows cross-sectional comparisons.

  A single growth tibial plate measurement was performed for each section within the area that is believed to best represent the overall thickness in the non-tangential plane of the section.

Using an eyepiece micrometer, osteophyte evaluation and small, medium and large classification were performed.
None = 0, no measurable proliferative response in the marginal zone.
Small bone spine = 1 (up to 299μm)
Medial spine = 2 (300-399 μm)
Large spine = 3 (> 400μm)

  The score (0-3) was included in the overall joint score. In addition, the mean ± SE for actual osteophyte measurements (on average of three sections) was determined as well.

  In general, in performing surgery, attempts have been made to dissect the collateral ligament at a location that results in the meniscus moving symmetrically proximally toward the femur. The cut was then made by inserting the tip of the scissors toward the femur instead of the tibia. Although some mechanical damage can be detected in the condylar cartilage of the femur at this time, it is rarely seen on the tibia, thus making the tibia the most appropriate site for assessment of cartilage protection. ing.

  Although a proteoglycan likely to be the result of physical trauma to the femoral cartilage and a small focal area of cell loss has been described, it is a larger and broader area that undergoes a subjective rating according to the method described for the tibia It was not included in the rating about. These larger areas were more consistent with atraumatic degeneration. Due to the potential for iatrogenic lesions on the femur, the overall joint score was expressed with and without the femoral cartilage degeneration score.

The following criteria were used to assess damage to the calcified cartilage layer and subchondral bone.
0 = no change 1 = increased basophilia at the highest point: no fragmentation or bone marrow change at the highest point.
2 = Increased basophilia at the highest point: minimal or mild fragmentation of calcified cartilage at the highest point, and 1/4 of the total area involved in mesenchymal changes in the bone marrow However, it is generally limited to the subchondral region under the lesion.
3 = Increased basophilia at the highest point: mild to high fragmentation of calcified cartilage, mesenchymal changes in the bone marrow are up to 3/4 of the total area, and the area of bone marrow cartilage formation is obvious There may be no collapse of the articular cartilage into the epiphyseal bone.
4 = Increased basophilia at the highest point: high to severe fragmentation of calcified cartilage, up to 3/4 area is involved in bone marrow mesenchymal change, articular cartilage is in the epiphysis It collapsed from the highest point to a depth of 250 μm or less.
5 = Increased basophilia at the highest point: high to severe fragmentation of calcified cartilage, up to 3/4 area is involved in bone marrow mesenchymal change, Collapsed from the highest point to a depth exceeding 250 μm.

  Descriptive comments were made on the extent of fibrotic overgrowth with or without synovitis, synovial fibrosis, marginal zone cartilage formation, bone resorption, chondrogenesis / incorporation into existing cartilage.

  Statistical analysis: Statistical analysis of histopathological parameters was performed by comparing group means using Student's two-tailed t-test with significance set at P ≦ 0.05. Due to the nature of the data, the non-parametric ANOVA (Kruskal Wallis test was used to analyze the assessed parameters, and parametric ANOVA was used to analyze the measurements. The post-test was a Dunnett's multiple comparison test for parametric data and Dunn's test was used for non-parameter data, and significance was set at P ≦ 0.05 for all parameters.

  Results: 100 μg CG53135-05 internode injection with or without simultaneous indomethacin administration resulted in significant inhibition (39%) of tibial cartilage degeneration over the middle 1/3 (40-43% for zone 1), And resulted in an overall insignificant inhibition of 3 zones totaling 41% (FIG. 12). Total cartilage degeneration width was significantly reduced by 35-37% (FIG. 13), significant degeneration was reduced by 70-89%, and this inhibition was only significant within the protein and indomethacin treated groups (FIG. 14).

  Results of prophylactic dosing studies: The data described are the result of both intrachondral administration of 100 μg CG53135-05 into the knee joint of a rat with a medial meniscal tear resulting in both inhibition of cartilage degeneration and stimulation of cartilage repair. It is shown that the cartilage protection effect is brought about. Some joints had a layering of new cartilage that grew over existing normal or damaged cartilage. This observation is particularly interesting because it demonstrates the potential for surface reconstruction.

  These beneficial effects have always been associated with increased synovial fiber proliferation, bone resorption and increased synovitis. Concomitant indomethacin treatment (1 mg / kg / day) does not affect any of the disease process in the knee that received the injection of Synvisc alone or the response to the protein and the disease process in the knee that received the injection of protein containing Synbisc. Even if it was effective, it was minimal. A single exception to this statement is reflected in the data for osteophyte measurements where all groups had similar measurements except the group treated with protein and vehicle po. The group had larger measurements, thus suggesting a larger marginal zone stimulation that is a common occurrence in inflamed joints.

  The morphological changes induced by 100 μg injection of this protein demonstrate the potential for CG53135-05 to be effective in the cartilage repair process. It has the ability to induce the growth of fibrous tissue with cartilage differentiation and, importantly, the incorporation of the newly grown tissue. The proliferative process is somewhat uncoordinated and counterproductive within areas such as the marginal zone and subchondral bone. However, rodents certainly have a much greater tendency to show marginal zone, periosteum and bone marrow proliferation from a variety of stimuli, including inflammatory mediators, and thus the excessive and counterproductive seen in rats Some responses may not occur in dogs or primates. Similarly, there has been some induction of antibody responses, thus leading to enhanced knee inflammation that would not occur in humans or other animals that did not have an antibody response.

  Additional studies useful in depicting the potential efficacy of CG53135-05 in osteoarthritis include the following:

  1. Assessment in a dog model of OA-this would allow assessment in larger joints with cartilage and bone structures that are more similar to humans, which are hyperproliferative such as those occurring in rodents A species with a relatively low tendency to show sexual response.

  2. It would be advantageous to evaluate a 3-4 week intra-articular injection with a recovery period to see if more aggressive anti-inflammatory systemic therapy and subsequent new tissue remodeling if possible Seem. Remodeling the joint in the absence of any further proliferative stimuli may result in a more favorable morphological endpoint. A treatment cycle with a remodeling period may be the way to achieve the most satisfactory repair. Studies like these will also answer the question of whether the repair tissue will last for a long time. In general, fibrocartilage is less prone to do this.

  Results: Intra-articular injection of 100 μg CG53135-05 with or without simultaneous oral indomethacin administration did not result in significant inhibition of the tibial cartilage degeneration score (FIG. 15). The total or significant cartilage degeneration width did not decrease (FIGS. 16 and 17).

  Results of therapeutic dosing study: The data described demonstrated the potential chondroproliferative activity of CG53135-05 administered intra-articularly. However, joints injected with protein had significantly increased inflammation, fibrosis, and connective tissue resorption processes.

  The most important difference between prophylactic and therapeutic dosing was the nature of the OA lesion at the start of dosing. Rats in therapeutic dosing studies had areas of severe matrix loss in the outer to middle 1/3 of cartilage, thus exposing calcified cartilage / subchondral bone to protein. Thus, effective repair required filling this defect with newly grown tissue derived from the marginal zone or exposed bone marrow pluripotent cells. In prophylactic dosing studies, beneficial effects required inhibition of matrix degradation and stimulation of repair on degenerative scaffolds with repair tissue derived only from the marginal zone. Since filling a defect appears to be much more difficult than repairing a damaged scaffold, longer treatment times may be required in a therapeutic model to obtain a beneficial effect.

  Indomethacin treatment was not effective in reducing inflammatory changes and had no beneficial effect in inhibiting the resorption process that is occurring in bone. In order to achieve effective growth and differentiation into cartilage in the absence of inflammation and tissue destruction, the following modifications to the therapeutic dosing study can be attempted. That is, increasing the dosing interval once or twice a week and / or extending the duration of the study to allow time for the proliferative tissue to fill a large cartilage defect induced by this disease process. . Investigating the effects of CG53135-05 in larger species such as dogs, because dogs are less prone to connective tissue proliferation and bone resorption in response to various stimuli than rodents One possibility.

  The results detailed in this document (both prophylactic and therapeutic dosing studies) show that CG53135-05 is particularly useful in joints suffering from severe osteoarthritis for joint replacement surgery Yes. These types of agents will be injected into joints requiring little or no normal cartilage and requiring surface reconstruction. Under this circumstance, the repair may be sourced from pluripotent cells in the marginal zone or bone marrow. Repairs starting from these locations are more likely to result in the production of fibrocartilage rather than hyaline cartilage. However, some cartilage may be preferred over no cartilage and is injectable to maintain the cartilage surface even though there is a high probability of having to repeat treatment over time to continue repair The method may be acceptable. Treatment with an injectable anabolic agent is likely to require some sort of periodic process in conjunction with continuous passive exercise rather than sustained dynamic load bearing exercise.

6). Equivalents and cited references All references cited herein are specifically and individually directed to each individual publication or patent or patent application to be incorporated by reference in its entirety for all purposes. To the same extent, are incorporated herein by reference in their entirety for all purposes.

  Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are provided for purposes of illustration only. Thus, we are not wishing to be limited to the precise terms set forth herein, but have made changes and modifications that can be made to adapt the invention to various applications and conditions. Someone who wants to be available. Such modifications and changes include different compositions for administration of a polypeptide according to the invention to a subject; different polypeptide amounts; different administration times and means of administration; eg different peptide combinations or different biologically active It includes, but is not limited to, different materials that are included within a dosage including compound and peptide combinations. Such changes and modifications likewise do not alter the functionality of the polypeptide, such that the solubility of the peptide in the composition to be administered to the subject, the absorption of the peptide by the body, the life of the peptide during or in the body Of a particular polypeptide described herein that alters the sequence in such a way as to alter the protection of the polypeptide and the alteration of such similar modifications to the point where the biological activity can produce the desired effect. It is intended to include modifications in the amino acid sequence. Accordingly, such changes and modifications are suitably intended to fall within the full scope of equivalents of the present invention.

2 shows liquid chromatography and mass spectrometry of CG53135-05. CG53135-05 was injected onto a phenyl-hexyl column in an aqueous mobile phase containing 95% water, 5% acetonitrile and 0.1% trifluoroacetic 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% trifluoroacetic acid. Each of the four peaks was characterized using LC / ESI / MS, MALDI-TOF MS and N-terminal amino acid sequencing. 1 represents a peptide map of CG53135-05. The top trace in each plate represents the peptide map of CG53135-05, and the bottom trace in each plate represents the same sample treated in the same manner but without CG53135-05. Detection at 214 nm to monitor CG53135 peptide. 1 represents a peptide map of CG53135-05. The top trace in each plate represents the peptide map of CG53135-05, and the bottom trace in each plate represents the same sample treated in the same manner but without CG53135-05. Detection at 295 nm to monitor tryptophan-containing peptides. The receptor binding specificity of CG53135 is shown. NIH3T3 cells are serum-starved and incubated with the indicated factors (green square = platelet-derived growth factor; blue triangle = FGF-1; red circle = CG53135) alone or with the indicated soluble FGFR, and CG53135 Responsive DNA synthesis was measured in a BrdU incorporation assay. Data scores represent the average obtained from triplicate wells and are expressed as percent BrdU incorporation for cells receiving the factor alone. The result of a forelimb repositioning test is shown. The mean and standard error of the scores are expressed over time for the group receiving vehicle (diamond), 1.0 μg CG53135-05 (square) and 2.5 μg CG53135-05 (triangle) per injection. The asterisk indicates a significant difference from the vehicle control as assessed by one-way ANOVA. The result of a rear limb repositioning test is shown. The mean and standard error of the scores are expressed over time for the group receiving vehicle (diamond), 1.0 μg CG53135-05 (square) and 2.5 μg CG53135-05 (triangle) per injection. The asterisk indicates a significant difference from the vehicle control as assessed by one-way ANOVA. The result of a body swing test is shown. The mean and standard error of the scores are expressed over time for the group receiving vehicle (diamond), 1.0 μg CG53135-05 (square) and 2.5 μg CG53135-05 (triangle) per injection. A scoring range of about 50% rocking to the right represents no dysfunction, while 0% rocking to the right represents maximum dysfunction. The asterisk indicates a significant difference from the vehicle control as assessed by one-way ANOVA. The result of a cylinder test is shown. The mean and standard error of the scores are expressed over time for the group receiving vehicle (diamond), 1.0 μg CG53135-05 (square) and 2.5 μg CG53135-05 (triangle) per injection. The result of a weight test is shown. Mean and standard errors of body weight are expressed over time for the group receiving vehicle (diamond), 1.0 μg CG53135-05 (square) and 2.5 μg CG53135-05 (triangle) per injection. FIG. 5 shows the effect of CG53135-05 on Pro-MMP production in SW1353 cells in the presence of IL-1beta. Figure 3 shows the effect of CG53135-05 on Pro-MMP production in SW1353 cells in the presence of TNF-alpha. Figure 3 shows the effect of CG53135-05 on TIMP production in SW1353 cells. FIG. 5 shows the relationship of intra-articular injection effects of CG53135-05 (prophylactic dosing) versus medial tibial cartilage degeneration in a meniscal tear model of rat osteoarthritis. Figure 3 shows the results of intra-articular injection of CG53135-05 in a meniscal tear model of rat osteoarthritis: (prophylactic dosing) vs. total cartilage degeneration width. FIG. 6 shows the results of intra-articular injection of CG53135-05 in a meniscal tear model of rat osteoarthritis: (prophylactic dosing) versus significant tibial cartilage degeneration width. FIG. 5 shows the relationship between the results of intra-articular injection of CG53135-05 (therapeutic medication) versus medial tibial degeneration in a meniscal tear model of rat osteoarthritis. FIG. 5 shows the relationship between the results of intra-articular injection of CG53135-05 (therapeutic dosing) versus the total cartilage degeneration width in a meniscal tear model of rat osteoarthritis. FIG. 9 shows the relationship between the results of intra-articular injection of CG53135-05 (therapeutic medication) in a meniscal tear model of rat osteoarthritis versus a significant tibial cartilage degeneration width.

Claims (9)

  A method of treating an osteoarthritis-related disorder in a subject comprising administering to the subject a composition comprising a CG53135-05 polypeptide.   The method of claim 1, wherein the subject is a human.   2. The method of claim 1, wherein a prophylactically effective dose of the polypeptide is administered to the subject.   2. The method of claim 1, wherein a therapeutically effective dose of the polypeptide is administered to the subject.   The method of claim 1, wherein the polypeptide is administered by a parenteral route.   6. The method of claim 5, wherein the route of administration comprises intra-articular, intravenous, intramuscular, subcutaneous, or intradermal administration.   2. The method of claim 1, wherein a therapeutically effective amount of the polypeptide is formulated in the form of a pharmaceutical composition.   A method of reducing cartilage degeneration in a subject, comprising administering to the subject a composition comprising a CG53135-05 polypeptide.   A method of stimulating cartilage repair in a body of a subject comprising administering to the subject a composition comprising a CG53135-05 polypeptide.

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