WO2011158655A1 - Utilisation d'un mécansime de formation de complexes de klotho, fgf23- et fgfr dans des tissus calcifiés - Google Patents

Utilisation d'un mécansime de formation de complexes de klotho, fgf23- et fgfr dans des tissus calcifiés Download PDF

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WO2011158655A1
WO2011158655A1 PCT/JP2011/062639 JP2011062639W WO2011158655A1 WO 2011158655 A1 WO2011158655 A1 WO 2011158655A1 JP 2011062639 W JP2011062639 W JP 2011062639W WO 2011158655 A1 WO2011158655 A1 WO 2011158655A1
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calcification
fgf23
klotho
hard tissue
solubilized
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Japanese (ja)
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裕二 吉子
朋子 南崎
広陽 吉岡
憲彦 前田
克之 香西
和晃 渡邉
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国立大学法人広島大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

  • the present invention relates to a screening method for a calcification regulating agent, a calcification regulating agent, and bone mineralization, vascular calcification, and pathological progression associated therewith, associated with signal transduction common to calcification of hard tissue and / or blood vessels. And a test kit for the same.
  • bone diseases such as osteoporosis correlate with circulatory diseases such as vascular calcification (bone vascular correlation). Furthermore, it has been reported that bone mass loss is associated with a high risk of death.
  • the preventive / therapeutic agents for diseases involving bone or blood vessel calcification include calcium, vitamin D preparation, estrogen, ipriflavone, calcitonin, bisphosphonate, vitamin K2 preparation, phosphate adsorbent or statin. Including. However, more preferably, establishment of a novel mechanism-oriented therapeutic method, therapeutic agent, and prophylactic method that directly affects bone or blood vessel calcification is desired.
  • Fibroblast growth factor 23 is a protein identified as a causative factor of hereditary and neoplastic rickets / osteomalacia (see Non-Patent Document 1 to Non-Patent Document 3). Furthermore, in recent years it has been recognized as a physiological hormone that regulates phosphorus and vitamin D metabolism. Biological responses to the FGF family, including FGF23, take place through the binding of specific cell surface receptors to tyrosine kinase type receptors (FGFR, in particular FGFR1). However, the affinity of FGF23 for FGFR is low.
  • FGF23 is secreted mainly by osteoblasts and bone cells. After secretion, expression of type II sodium-phosphorous cotransporter (NaPiII) and expression of 25 hydroxyvitamin D 3 -1 ⁇ hydroxylase are suppressed targeting renal tubular epithelium. Therefore, the production of excess FGF23 suppresses reabsorption of phosphorus, and as the blood 1 ⁇ , 25 dihydroxyvitamin D 3 (1,25D) concentration decreases, absorption of phosphate from the intestinal tract is suppressed. Medium phosphorus concentration decreases. Furthermore, it has been clarified that FGF23 plays a central role in phosphorus homeostasis since Fgf23-deficient mice exhibit high phosphate and hypervitamin Demia.
  • FGF23 regulates the physiological mineralization of hard tissues including bone and the mineralization associated with pathological conditions such as blood vessels. ing. Specifically, FGF23 has been shown to suppress physiological calcification of bones and blood vessels (see Patent Document 1, Non-Patent Documents 1 and 2).
  • Klotho which encodes a type I membrane protein with a molecular weight of about 130,000, has been identified as a responsible gene for mice that cause various human aging phenomena by mutation of a single gene. Klotho is expressed mainly in the main cells of renal tubules, brain choroid and parathyroid glands.
  • Klotho is involved in phosphorus reabsorption and vitamin D metabolism in the kidney and parathyroid hormone production in the parathyroid gland. Moreover, coupled with Na + / K + -ATPase in a cell, in response to a decrease in calcium concentration outside the cell recruit Na + / K + -ATPase to the cell membrane.
  • the extracellular domain of membrane-type Klotho is cleaved and solubilized Klotho (hereinafter sometimes abbreviated as KLe) released from the extracellular domain in the blood circulates in the blood and is a secretor involved in development. It has been reported that it binds to the protein Wnt and inhibits the Wnt signal, or regulates the sugar chain modification of TRPV5 to regulate the function as a calcium channel.
  • Patent Document 3 describes an invention related to a fusion protein (a fusion polypeptide) comprising a polypeptide containing such KLe and FGF (specifically, FGF23) (active fragment). Including a polypeptide consisting of: In detail, it describes about the invention of the method of treating or preventing the various disease state and metabolic disease state accompanying an aging phenomenon by using the said fusion protein.
  • membrane type Klotho is expressed only slightly in the proximal tubule, and it is involved in intracellular transport and degradation of NaPiIIa independently of FGF23 by its own ⁇ -glucuronidase activity, and reabsorbs phosphorus.
  • Klotho a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.
  • Hu MC Shi M, Zhang J, Pastor J, NakataniazzT, Lanske Bazz Baum MG, Kuro-O M, Moe OW. FASEB J. 2010 May 21. http://www.fasebj.org/cgi/rapidpdf/fj.10-154765v2.pdf [Epub ahead of print]).
  • FGF23 is not necessarily involved in the action of Klotho in the kidney.
  • Patent Document 2 and Patent Document 3 there is a description that the two of FGF23 and KLe form a complex, but there is evidence that the two form a complex in a living body and other than muscle cells. Absent. Further, for example, when considering the case where such a complex is used in a living body, it binds nonspecifically to FGFR widely distributed in the living body, and the characteristics of FGF23 described above cannot be explained.
  • the present invention has been made in view of the above circumstances, and a screening method for a calcification regulator, a calcification regulator, and bone calcification, which are associated with signals common to hard tissue and / or blood vessel calcification.
  • An object of the present invention is to provide a method and a test kit for testing the degree, vascular calcification, and the progression of pathological conditions associated therewith.
  • KLe forms a functional complex with FGF23 and FGFR in Obc cells (see Examples), and is used for specific signal transduction in Obc cells (including suppression of bone mineralization by FGF23 associated with signal transduction). It is essential.
  • the action of (1) is specific to bone and not seen in the kidney.
  • the effect of (1) is also seen in calcification of vascular smooth muscle.
  • the screening method for a calcification regulating agent for hard tissue and / or blood vessels comprises at least solubilized Klotho, FGFR, and FGF23 in a specimen in the absence and presence of a test compound.
  • a step of contacting Selecting a compound that changes FGF23-specific signaling in the sample in the presence of the test compound as compared to FGF23-specific signaling in the sample in the absence of the test compound; It is characterized by including.
  • the change in specific signaling is characterized by FGFR phosphorylation, ERK phosphorylation, or Egr-1 expression level as an index.
  • the screening for the calcification regulating agent is performed in vivo.
  • the hard tissue and / or blood vessel calcification regulating agent according to the second aspect of the present invention is characterized by containing a compound that suppresses or promotes complex formation of solubilized Klotho, FGFR, and FGF23.
  • the compound is a solubilized Klotho.
  • the compound is a ligand having binding affinity for solubilized Klotho, FGFR, or a complex of the solubilized Klotho, FGFR, and FGF23.
  • the ligand is an antibody.
  • the method for testing calcification of hard tissue and / or blood vessels includes a concentration of solubilized Klotho, formation of a complex of solubilized Klotho, FGFR, and FGF23 in the specimen, or formation of the complex. And a step of measuring FGF-specific signal transduction associated with.
  • the specimen is animal blood, plasma, serum, bone marrow, bone tissue or cell, or blood vessel tissue or cell.
  • the animal is a human.
  • the calcification test kit for hard tissue and / or blood vessels contains a ligand having binding affinity for solubilized Klotho or a complex of solubilized Klotho, FGFR and FGF23. It is characterized by.
  • a new calcification regulating agent that promotes or suppresses calcification in hard tissues and / or blood vessels can be screened.
  • calcification in hard tissue and / or blood vessels can be suppressed or promoted.
  • calcification inspection method and the calcification inspection kit according to the third and fourth aspects of the present invention calcification in a hard tissue and / or blood vessel can be inspected.
  • FIG. 6 shows data of real-time RT-PCR results at the Egr-1 mRNA level according to Example 2. It is a figure which shows the mode of the result of the Western blotting of ERK1 / 2 which concerns on Example 2, and phosphorylated ERK1 / 2. It is a figure which shows the data of the result of the mineralization parameter
  • FIG. 6 shows data of real-time RT-PCR results at the Egr-1 mRNA level according to Example 2. It is a figure which shows the mode of the result of the Western blotting of ERK1 / 2 which concerns on Example 2, and phosphorylated ERK1 / 2. It is a figure which shows the data of the result of the mineralization parameter
  • FIG. 6 shows data of real-time RT-PCR results at the Egr-1 mRNA level according to Example 4. It is a figure which shows the mode of the result of the Western blotting of ERK1 / 2 which concerns on Example 4, and phosphorylated ERK1 / 2. It is a figure which shows the mode in the fluorescence microscope of the plastic
  • FIG. 6 shows data of real-time RT-PCR results at the Egr-1 mRNA level according to Example 4. It is a figure which shows the mode of the result of the Western blotting of ERK1 / 2 which concerns on Example 4, and phosphorylated ERK1 / 2. It is a figure which shows the mode in the fluorescence microscope of the plastic
  • FIG. 10 is a view showing a state of a plastic section of a skull stained with toluidine blue / von Kossa according to Example 5 under a microscope.
  • FIG. It is a figure which shows the data of the thickness of the osteoid in the mode in the microscope of FIG. It is a figure which shows the result of the electronic probe microanalyzer which concerns on Example 5.
  • FIG. It is a figure which shows the data of the result of the calcium content measurement which concerns on Example 6.
  • FIG. It is a figure which shows the mode in the stereomicroscope of the aorta of the kl / kl mouse which concerns on Example 7.
  • FIG. It is a figure which shows the mode of the result of the Western blotting of ERK1 / 2 which concerns on Example 8, and phosphorylated ERK1 / 2.
  • FIG. 9 It is a figure which shows the mode of the alizarin red / toluidine blue dyeing
  • FIG. It is a figure which shows the data of the alizarin red dyeing
  • FGF23 “FGFR1”, and “Klotho” of humans (Homoapisapiens) are registered under the accession numbers AAG09997, AAH15035, and BAA23382 in GenBank, respectively.
  • Embodiment 1 of the present invention relates to a screening method for a calcification regulating agent for hard tissue and / or blood vessels.
  • the step of contacting solubilized Klotho (KLe) with FGF23 and FGFR in a sample in the absence and presence of the test compound is compared with specific signal transduction in the sample in the absence of the test compound.
  • a method comprising a step of selecting a compound that changes the specific signal transduction in the specimen in the presence of the test compound.
  • the change in specific signaling can be indicated by FGFR phosphorylation, ERK phosphorylation, or Egr-1 expression level (see Examples).
  • the sample in the presence of the test compound is compared with the step of measuring the concentration of KLe in the sample in the absence and presence of the test compound and the concentration of KLe in the sample in the absence of the test compound. And a step of selecting a compound that increases or decreases the concentration of KLe.
  • the formation of a complex of KLe, FGF23, and FGFR (hereinafter referred to as “complex containing KLe” or “complex”) may be measured and compared in a specimen. In this case, it is possible to compare the degree, presence, concentration, etc. of the formation of a complex containing KLe directly. These screenings are more preferably performed in vivo.
  • “solubilized Klotho (KLe)” means Klotho blood in mammals such as humans, mice, rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats or dogs. Refers to the portion of the extracellular domain that circulates in Of these, KLe of human and non-human mammals (eg, rat, mouse, monkey or rabbit) having high homology with the amino acid sequences of human and human KLe are preferable. “High homology” means having a homology of 60% or more, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more. Most preferably, it refers to human KLe.
  • “solubilized Klotho (KLe)” is used as a meaning containing all of them.
  • “regulation” may include “promotion” or “activation” for positively controlling calcification, and “suppression” or “inhibition” for negative control.
  • “positive” control it is possible to show the inhibition of calcification due to the formation of KLe or a complex containing KLe and the activation of specific signal transduction associated therewith.
  • controlling to “negative” it is possible to show the formation of KLe or a complex containing KLe and the promotion of calcification by suppressing the specific signal transmission associated therewith.
  • the increase or decrease in the formation of a complex containing KLe can include, for example, promotion or inhibition of binding of KLe to FGFR in hard tissue and / or blood vessels.
  • specimen means all target samples that can be used by those skilled in the art to measure KLe or a complex containing KLe and the accompanying FGF-specific signal transduction.
  • a biological sample in vivo or in vitro refers to a non-human animal that is a target in vivo. Examples thereof include mice, rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats and dogs.
  • hard tissue means living hard tissue, for example, bone tissue forming the skeleton of the skull and trunk, alveolar bone and cementum constituting periodontal tissue, or dentin (which comprises these) Including cells etc.). Most preferred is mammalian bone tissue. More preferably, it is a bone tissue of a non-human mammal (for example, rat, mouse, monkey or rabbit) having high gene homology with human.
  • a non-human mammal for example, rat, mouse, monkey or rabbit
  • test compound may be any substance capable of evaluating the formation of a complex containing KLe or KLe and the accompanying increase or decrease in specific signal transduction.
  • a low molecular compound, a nucleic acid, or a polypeptide can be used.
  • the low molecular weight compound, nucleic acid, polypeptide or the like may be extracted and purified from a natural product, or may be artificially synthesized.
  • purified For example, even an unpurified cell extract etc. can be used as a test compound.
  • an existing therapeutic or preventive drug or derivative thereof can be evaluated by the screening method of the first embodiment.
  • FGF23 fibroblast growth factor 23
  • FGF23 fibroblast growth factor 23
  • FGFR also includes FGFR of a non-human mammal having high homology with the amino acid sequence of human FGFR.
  • FGFR1, 3 and 4 that have been confirmed to bind to FGF23 (Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o M. J Biol Chem. 2006 Mar 10; 281 (10): 6120-6123.), Most preferably FGFR1 (Non-patent Document 3 (Urakawa I., et al., Nature 444, 770-). 774, 2006)).
  • any method for measuring and comparing KLe or KLe-containing complex formation in the specimen and the accompanying FGF23-specific signal transduction any method for measuring and comparing them known to those skilled in the art can be used. can do.
  • a test compound is administered or injected into a non-human mammal as described above.
  • the formation of KLe or a complex containing KLe in blood or the like of the mammal is measured.
  • FGF23 and KLe are directly brought into contact with a hard tissue or a part of hard tissue expressing FGFR, and complex formation in the hard tissue and the accompanying phosphorylation of FGFR, phosphorylation of ERK, or Egr-1 Expression levels can also be measured.
  • an experimental system in which KLe, FGF23, or FGFR is forcibly expressed in an osteoblast, bone cell, or vascular smooth muscle cell as a specimen may be constructed so that measurement and screening can be performed more easily.
  • Such cell configuration and DNA introduction are easy for those skilled in the art, and examples thereof include methods using DNA introduction plasmids or viral vectors, electroporation, lipofection, or microinjection. .
  • the screening method according to the first embodiment can be performed in vivo or in vitro.
  • test compound is likely to be a calcification regulator that suppresses or promotes calcification of the hard tissue and / or blood vessels, respectively.
  • the thus screened hard tissue and / or vascular calcification regulating agent that suppresses calcification is osteoarthritis, ectopic calcification, arteriosclerosis or as a disease / condition in which calcification is promoted. It can be used as a means for preventing or treating vascular calcification in renal failure.
  • a calcification regulating agent that promotes calcification it can be used as a prophylactic / therapeutic agent for osteoporosis, rickets or osteomalacia as a disease / condition in which calcification is inhibited or suppressed.
  • use in fields such as other hard tissue diseases or regenerative medicine is also conceivable.
  • Embodiment 2 describes a hard tissue and / or blood vessel calcification regulator.
  • the regulator that suppresses calcification of hard tissue and / or blood vessels contains, as an active ingredient, a compound that promotes complex formation containing KLe, such as KLe.
  • the regulator that promotes calcification of hard tissue contains KLe, a complex containing KLe, or a ligand having binding affinity for FGFR.
  • the ligand is an antibody.
  • a more effective calcification regulator can be obtained by simultaneously containing other inhibitors / promoters of specific signal transduction associated with the formation of a complex containing KLe as active ingredients.
  • inhibitors of ERK signals such as U0126 are readily available and may contain other modulators of specific signaling associated with complex formation involving such KLe.
  • KLe can be easily obtained, manufactured or purified. As shown in the examples, KLe can be easily purchased and obtained at, for example, R & D Systems.
  • a protein / peptide having the amino acid sequence shown in SEQ ID NO: 1 can be produced using a known protein / peptide synthesis method (eg, including a solid phase synthesis method, a liquid phase synthesis method, etc.).
  • proteins and peptides can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.
  • the “antibody” may be a monoclonal antibody or a polyclonal antibody, and is not particularly limited as long as it specifically binds to a complex containing KLe, FGFR or KLe. This also includes an antibody that promotes the formation of a complex of KLe, or an antibody that promotes the formation of a complex containing KLe. That is, any antibody may be used, either an agonist antibody or an antagonist antibody.
  • the “neutralizing antibody” of KLe means an antibody that inhibits the function of specific signal transduction by KLe, such as an antibody that inhibits complex formation with FGFR and FGF23.
  • an anti- Klotho monoclonal antibody is mentioned, for example.
  • the “neutralizing antibody” of FGFR means an antibody that inhibits the function of specific signal transduction by FGFR.
  • a “neutralizing antibody” of a complex containing KLe means an antibody or the like that inhibits the function of specific signal transduction caused by the complex containing KLe.
  • the regulator for suppressing calcification of hard tissue and / or blood vessels in osteoarthritis, ectopic calcification, or arteriosclerosis, which are diseases / conditions in which calcification is promoted It can be used as a means for preventing the progression of vascular calcification.
  • the modulator may be administered directly to the patient, or it may constitute a compound that also contains these other components and be administered to the patient.
  • known methods such as oral, intravenous, intraarterial or subcutaneous injection can be used.
  • the regulator that promotes calcification of hard tissue it can be used as a means for prevention / treatment of osteoporosis, rickets or osteomalacia.
  • the administration method and the like are the same as those of the regulator for suppressing calcification described above, but it is also conceivable to administer directly to a specific site (for example, a site where bone is decreased or excessively formed) by injection or the like.
  • a method for examining calcification of hard tissue and / or blood vessels which includes a step of measuring the concentration of KLe in a specimen can be mentioned.
  • the degree of calcification can be estimated by measuring not only the concentration of KLe but also the formation of a complex containing KLe or the accompanying FGF23-specific signal transduction.
  • the measurement step is blood, plasma, serum, bone marrow, bone tissue or cell, or Kle concentration in blood vessel tissue or cell, formation of a complex containing KLe
  • the concentration of KLe or the formation of a complex containing KLe may be measured.
  • methods such as immunoprecipitation, Western blotting, ELISA, RIA, immunohistochemical staining, immunofluorescence, or flow cytometry can be used.
  • Measurement of FGF23-specific signal transduction associated with complex formation can be performed by measuring FGFR phosphorylation, ERK phosphorylation, or Egr-1 expression level, as described above.
  • the measured concentration of KLe in blood or the like can be evaluated by, for example, comparing the degree of calcification and the progression of the pathological condition accompanying it, and screening for active substances, by comparing with a normal value that has been previously measured and averaged. It is considered possible.
  • vascular calcification in osteoarthritis, ectopic calcification, or arteriosclerosis is achieved. There is a high possibility that the disease state is grasped and monitored, or the disease state is grasped and monitored for bone mineralization failure in osteoporosis, rickets or osteomalacia.
  • Hard tissue and / or blood vessel calcification test kit As described in Embodiment 3, it is considered that the measurement of the concentration of KLe in blood and the formation of a complex containing KLe can serve as a measure of the degree of calcification of hard tissues and / or blood vessels. Therefore, as a fourth embodiment, hard tissue and / or blood vessel containing a ligand having binding affinity for KLe or a complex containing KLe for use in the calcification testing method described in the third embodiment. Mention may be made of a calcification test kit.
  • the ligand means a substance that specifically binds to a measurement target (KLe or a complex containing KLe).
  • KLe or a complex containing KLe a measurement target
  • an antibody of KLe or a complex as described above part of which can be a Klotho antibody
  • the antibody Refers to the secondary antibody against.
  • various reagents, enzymes, buffers, reactor materials, and / or instructions for comparing and evaluating the degree of calcification failure may be included. The method of using these is the same as the method described in the third embodiment.
  • Example 1 In Example 1, an example relating to formation of a complex of KLe with FGF23 and FGFR1 in osteoblasts / bone cells will be described in detail.
  • Osteoblast / bone cell the mature osteoblast / bone cell (Obc cell) and the method for growing and culturing the Obc cell used in Examples 1 to 3 will be described.
  • Cells were isolated from calvaria derived from embryonic day 21 fetal rats (see Yoshiko Y, et al., Endocrinology 144, 4134-4143, 2003), and the calvarial cells were separated from 10% FBS (fetal bovine serum, Fetal Bovine Serum ) (HyClone) and ⁇ -MEM (Modified Essential Medium) medium containing 50 ⁇ g / ml ascorbic acid.
  • FBS fetal bovine serum, Fetal Bovine Serum
  • ⁇ -MEM Modified Essential Medium
  • osteoid-like nodules cells were selectively removed from the nodules by collagenase (type I) treatment. Thereafter, the removed cells were cultured under the above-described conditions and used as Obc cells.
  • Ocn osteocalcin
  • Dmp-1 distal endogen activator-1
  • Sost Sost was determined using real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction). Measured by the method (not shown).
  • Obc cell cultures cultured under conditions of serum deficiency of 0.1% FBS were used. Furthermore, the cells contain both recombinant KLe (rKLe) ( ⁇ 500 ng / ml (R & D & Systems)) (described in SEQ ID NO: 2) and recombinant FGF23 (rFGF23) ( ⁇ 500 ng / ml (R & D Systems)). The cells were cultured for 15 minutes under any of the following conditions: conditions containing only rKLe, conditions containing only rFGF23, or conditions containing none.
  • KLe recombinant KLe
  • rFGF23 recombinant FGF23
  • IP Immunoprecipitation
  • ⁇ Klotho Mab anti-Klotho monoclonal antibody
  • ⁇ FGFR1 Mab anti-FGFR1 monoclonal antibody
  • IB immunoblotting with ⁇ FGFR1 Mab or anti-FGF23 polyclonal antibody ( ⁇ FGF23) (R & D Systems) ( IB) was performed.
  • solubilized cell extract was incubated with protein G coupled to magnetic microbeads ( ⁇ MACS protein G microbeads, Miltenyi Biotec).
  • ⁇ Klotho Mab or ⁇ FGFR1 Mab was added to obtain an immune complex (MACS (registered trademark) Separator User Manual, Miltenyi Biotec). Thereafter, this sample was subjected to SDS-PAGE (Poly-Acrylamide Gel Electrophoresis), immunoblotting (IB) with ⁇ FGFR1 Mab or ⁇ FGF23 (Santa Cruz) was performed, and a signal was detected by chemiluminescence.
  • MCS registered trademark
  • SDS-PAGE Poly-Acrylamide Gel Electrophoresis
  • IB immunoblotting
  • FIG. 1 is a view showing the results of immunoprecipitation and immunoblotting according to Example 1.
  • FGFR1 and FGF23 formed a complex with Klotho and FGFR1 only in the presence of both rKLe and rFGF23, respectively. From these results, it was revealed that KLe and FGF23 and FGFR1 form a complex in osteoblasts / bone cells.
  • Example 2 In Example 2, an example relating to the correlation between FGF23-specific signal transduction and KLe in Obc cells will be described in detail.
  • Egr-1 is a gene whose expression is promoted by FGF23-specific signal transduction (see Non-Patent Document 3). Therefore, the present inventors measured the mRNA level of Egr-1 in Obc cells by real-time RT-PCR.
  • RNA of cells was prepared using TRIzol reagent (Invitrogen). Real-time RT-PCR was performed using the prepared RNA (Y. Yoshiko, GA Candeliere, N. Maeda, JE Aubin, Mol Cell Biol 274, 465-4474, 2007, and H. Wang, Y. Yoshiko, R Yamamoto, T.Minamizaki, K.Kozai, K.Tanne, JE Aubin, N.Maeda, J Bone Miner Res 23, 939-948, 2008). Ribosomal protein L32 was used as an internal standard.
  • the Egr-1 forward primer is the one described in SEQ ID NO: 3
  • the reverse primer is the one described in SEQ ID NO: 4
  • the L32 forward primer is the one described in SEQ ID NO: 5
  • the reverse primer is the sequence SEQ ID NO: 4. The thing of No. 6 was used.
  • phosphorylation of ERK1 / 2 is also an index of FGF23-specific signal transduction (see Non-Patent Document 3). Therefore, the present inventors performed Western blotting in order to examine ERK1 / 2 activity.
  • FIG. 3 shows the results of Western blotting of ERK1 / 2 and phosphorylated ERK1 / 2 according to Example 2.
  • phosphorylation of ERK1 / 2 was detected for those treated for 10 minutes under conditions including both rFGF23 and rKLe.
  • increase of mRNA level of Egr-1 and activation of ERK1 / 2 were confirmed only in the presence of rFGF23 and rKLe.
  • Example 3 In Example 3, an example relating to the correlation between calcification failure due to FGF23-specific signaling in Obc cells and KLe will be described in detail.
  • the present inventors detect the activity of alkaline phosphatase (ALP), which is an osteoblast marker enzyme, and calcification. Kossa staining was performed. In addition, since the present inventors have confirmed that 1 ⁇ , 25 dihydroxyvitamin D 3 (1,25D) strongly promotes the expression of FGF23, not only rFGF23 but also 10 nM 1,25D was added. The cultured ones were also examined. FBS was confirmed to contain KLe (not shown, confirmed by Western blotting), so that 5% of FBS was removed from FBS by ⁇ MACS protein G microbeads and ⁇ Klotho Mab. To the culture medium.
  • ALP alkaline phosphatase
  • rKLe was used for recovery experiments.
  • the culture was terminated 36 hours after the addition of the reagent, washed with PBS, fixed with 10% neutral buffered formalin, and then stained.
  • the culture supernatant (conditioned media) was collected, and the level of the added rKLe was confirmed by Western blotting.
  • 3 mM ⁇ -glycerophosphoric acid was added. Thereafter, the number of ALP-positive calcified nodules was counted under a stereomicroscope. The numerical value was expressed with the negative control (solvent only) as 100.
  • FIG. 4 is a diagram showing data of the result of the calcification index by addition of rKLe in ALP / von Kossa staining according to Example 3. In addition, there is a significant difference compared to the solvent alone ( ⁇ ).
  • * (significance level): p ⁇ 0.05, ** (significance level): p ⁇ 0.01, and n 4.
  • calcification index (Mineralized foci) in FIG. 4 in both 1,25D and rFGF23, calcification is not suppressed, but rather promoted, as compared with the control when rKLe is not added. However, it has returned to the state of calcification suppression again by adding rKLe.
  • FIG. 4 is a diagram showing data of the result of the calcification index by addition of rKLe in ALP / von Kossa staining according to Example 3. In addition, there is a significant difference compared to the solvent alone ( ⁇ ).
  • FIG. 5 is a diagram showing the result of Western blotting of rKLe according to Example 3. As shown in FIG. 5, the rKLe concentration of the culture supernatant was consistent with the concentration dependency of the calcification inhibiting action shown in FIG. These results in FIGS. 4 and 5 suggest that the degree of calcification depends on the rKLe concentration.
  • Obc cells obtained by culturing under the condition of serum deficiency of 0.1% FBS in the same manner as described in Example 1 were used for 2 hours with 10 ⁇ M of ME01 / 2 inhibitor U0126 or solvent alone for 2 hours. Pretreatment was performed. After washing, only rKLe and rFGF23 or a solvent were added, and 36 hours later, ALP / von Kossa staining was performed as described above.
  • FGFR neutralizing antibody ( ⁇ FGFR) 2 ⁇ g / ml (CHEMICON) or its solvent alone (negative control) was added when rKLe and rFGF23 were added. Further, as described above, ⁇ -glycerophosphate was added simultaneously with rKLe and rFGF23.
  • FIG. 6 is a diagram showing data on the result of calcification index by adding rKLe and rFGF23 in ALP / von Kossa staining according to Example 3.
  • ⁇ the solvent alone
  • ** (significance level): p ⁇ 0.01 and n 4.
  • the suppression of calcification by rKLe and rFGF23 is largely recovered by the FGFR neutralizing antibody or U0126, so that signal transduction based on the formation of a complex containing KLe is strongly involved in the suppression of calcification. It became clear to do. Therefore, they are effective as therapeutic agents for suppressing calcification based on the formation of a complex containing KLe.
  • KLe is effective as a regulator that suppresses calcification
  • a neutralizing antibody of a complex containing KLe and KLe is effective as a regulator that promotes calcification.
  • KLe forms a functional complex with FGF23 and FGFR in Obc cells, and FGF23-specific signaling in Obc cells (of bone by FGF23) (Including calcification failure).
  • Wnt and TRPV5 that react with KLe may be affected, but Wnt was similarly stained with ALP / von Kossa and confirmed that it was not affected (not shown). TRPV5 is not present in the bone and therefore has no effect.
  • Example 4 In Example 4, an example relating to the correlation between FGF23-specific signal transduction and calcification failure and KLe in bone and kidney will be described in detail.
  • RKLe was administered to Klotho mutant mice lacking Klotho (kl / kl mice) in order to confirm whether the action of KLe shown in the above-mentioned Examples was also observed in the living body.
  • the present inventors have confirmed that the concentrations of phosphate, calcium, 1,25D and FGF23 are significantly higher in kl / kl mouse serum than in WT mouse serum. (Not shown, 1 and 25D were measured by RIA kit (TFB), and the concentration of FGF23 was measured by FGF23 ELISA kit (Kainos).
  • Phosphoric acid and calcium were Phospha C-Test Wako and Calcium C-Test Wako, respectively.
  • real-time RT-PCR primers and other detailed methods are the same as in Example 2 described above.
  • FIG. 7 is a diagram showing data on the results of real-time RT-PCR at the Egr-1 mRNA level according to Example 4.
  • wild type mice WT
  • WT wild type mice
  • n 9
  • administration of rKLe increases Egr-1 mRNA levels.
  • the kidney (Kidney) of the same mouse the mRNA level of Egr-1 is hardly changed by administration of rKLe.
  • Example 2 Western blotting was also performed.
  • the mice used for the samples were kl / kl mice administered in the same manner as described above, and samples of parietal bone and kidney cells of the mice were used.
  • the Western blotting method and other conditions are the same as in Example 2 described above.
  • FIG. 8 shows the results of Western blotting of ERK1 / 2 and phosphorylated ERK1 / 2 according to Example 4.
  • a figure shows the representative example of the individual of 9 examples implemented for each group.
  • promotion of ERK1 / 2 phosphorylation was detected in the bones of mice administered with rKLe.
  • activation of ERK1 / 2 was not detected in the group administered with rKLe. That is, the results shown in FIGS. 7 and 8 suggest that FGF-specific signal transduction by administration of rKLe to kl / kl mice is specific to bone and not found in the kidney. That is, it can be said that KLe does not show a cooperative action with FGF23 in the kidney.
  • Example 5 In this Example 5, a specific example in vivo relating to the correlation between calcification inhibition by FGF23-specific signal transduction in Kbc cells and KLe will be described in detail.
  • FIG. 9 is a diagram showing a state of a plastic section of the parietal bone double-labeled with calcein according to Example 5 under a fluorescence microscope. The figure shows a representative example of the 9 individuals in each group implemented.
  • FIG. 10 is a diagram showing data of the result of the label interval in the state of the fluorescence microscope of FIG.
  • FIG.10, FIG.12, FIG.13, FIG.14 and FIG. 16 which show the data in this Example 5 have a significant difference compared with the WT mouse
  • FIG. 11 is a view showing a state of a paraffin section of the skull subjected to H & E staining according to Example 5 under a microscope.
  • a figure shows the representative example of the individual of 9 examples implemented for each group.
  • the portion “]” indicates an osteoid.
  • FIG. 12 is a diagram showing data on the number of osteoblasts in the state of the microscope of FIG.
  • FIG. 13 is a diagram showing data of the number of bone cavities in the state of the microscope of FIG.
  • FIG. 14 is a diagram showing bone thickness data in the state of the microscope of FIG.
  • the kl / kl mice did not show any significant changes regardless of whether or not rKLe was administered.
  • FIG. 15 is a view showing a state of a plastic section of a skull stained with toluidine blue / von Kossa according to Example 5 under a microscope.
  • FIG. 16 is a diagram showing data on the thickness of the osteoid in the state of the microscope of FIG.
  • the parietal bone of kl / kl mice administered rKLe is markedly more osteoid (noncalcified bone) than that of kl / kl mice not administered rKLe. Had increased.
  • the figure shows a representative example among 9 individuals in each group.
  • the electronic probe microanalyzer used JXA-8200 (JEOL) according to the analyzer's instruction manual (irradiation current value 20 nA, acceleration voltage 15 kV, measurement time 0.05 sec / 1 pixel).
  • FIG. 17 shows the results of the electronic probe microanalyzer according to Example 5.
  • the figure shows a representative example of the 9 individuals in each group implemented.
  • element mapping of magnesium (Mg), calcium (Ca), and phosphorus (P) is shown from the left.
  • Mg, Ca, and P are decreased compared to the case of kl / kl mice not administered with rKLe. That is, the results of the electron probe microanalyzer also showed that administration of rKLe decreased bone mineralization.
  • FGF23 in Obc cells acts cooperatively with rKLe in vivo.
  • Example 6 The present inventors considered that the function of KLe as described above is also involved in the calcification of blood vessels from the bone-vessel correlation. Therefore, in this sixth embodiment, an embodiment relating to the measurement of calcium content in aortic smooth muscle cells will be described in detail.
  • Rat aortic smooth muscle cells were cultured in 15% FBS DMEM (Dulbecco's Moodified Eagle's Medium) in the incubator in the same manner as described in Example 1. After the cells became confluent, the culture was continued under the condition of 0.1% FBS serum deficiency and loaded with 2 mM sodium hydrogen phosphate. Two days after the start of culture under the condition of serum deficiency, rKLe and rFGF23 were added individually or simultaneously, and cultured for another week. The medium was replaced with fresh medium every 2-3 days. After completion of the culture, the cells were washed with PBS and treated with 0.6N hydrochloric acid overnight. The amount of calcium in the supernatant was measured by the aforementioned calcium C-test Wako.
  • Example 7 In this Example 7, an example relating to calcification of the aorta in vivo in order to confirm whether or not the involvement of the complex for inhibiting vascular calcification shown in Example 6 is also observed in the living body is detailed. Explained.
  • aorta was collected from kl / kl mice administered with rKLe and kl / kl mice administered with solvent (PBS-Atelocollagen) in the same manner. did.
  • the collected aorta was fixed with 4% paraformaldehyde, washed with water, and permeated with 0.5% potassium hydroxide. Thereafter, the solution was stained in a solution in which 20 ⁇ g / ml alizarin red was dissolved.
  • FIG. 19 is a diagram showing a state of an aorta of a kl / kl mouse according to Example 7 with a stereomicroscope.
  • the figure shows a representative example of the 9 individuals in each group implemented.
  • the left is a diagram of the aorta of a kl / kl mouse administered only with a solvent
  • the right is a diagram of the aorta of a kl / kl mouse administered with rKLe.
  • Each figure shows a representative example of nine examples.
  • the calcified nest stained with alizarin red is clearly reduced in the mice administered with rKLe compared to the mice administered with solvent alone. That is, it is considered that a complex containing KLe suppresses vascular calcification even in in vivo.
  • FIG. 20 shows the results of Western blotting of ERK1 / 2 and phosphorylated ERK1 / 2 according to Example 8.
  • the figure shows a representative example of 5 individuals in each group implemented.
  • enhanced phosphorylation of ERK1 / 2 in the parietal bone was observed depending on the dose of rKLe. That is, in consideration of the result of the dependence between the rKLe concentration and the degree of calcification briefly shown in Example 3, the rKLe concentration and lime in cells, tissues, etc. of mouse blood, plasma, serum, bone marrow, bone and blood vessels This means that the dependence on the degree of conversion has been strongly suggested.
  • Example 9 In this Example 9, an example relating to alizarin red / toluidine blue staining of calcification of the aortic blood vessel wall in vivo will be described in detail.
  • rKLe was administered directly into the male kl / kl mouse at the age of 4 weeks (day 28) using an Alzette (registered trademark) osmotic pump (flow rate of 150 ng / h).
  • Alzette registered trademark
  • osmotic pump flow rate of 150 ng / h.
  • sampling of the thoracic aorta of kl / kl mice was performed at 6 weeks of age (day 42).
  • the mouse thoracic aorta was placed in 4% paraformaldehyde / PBS for 16 hours at 4 ° C. and then lavaged.
  • FIG. 21 is a view showing a state of alizarin red / toluidine blue staining of a paraffin section of a kl / kl mouse thoracic aorta according to Example 9.
  • the figure shows a representative example of the kl / kl mouse thoracic aorta performed.
  • -RKLe is a paraffin section of the thoracic aorta of a kl / kl mouse administered with solvent
  • + rKLe is a paraffin section of the thoracic aorta of a kl / kl mouse administered with rKLe.
  • FIG. 21 is a view showing a state of alizarin red / toluidine blue staining of a paraffin section of a kl / kl mouse thoracic aorta according to Example 9.
  • the figure shows a representative example of the kl / kl mouse thoracic
  • FIG. 22 is a diagram showing data on the results of alizarin red staining range of kl / kl mice and wild-type mice according to Example 9. That is, it is a figure which shows the calcification range (Calcification area).
  • the value of the kl / kl mouse (kl / kl (+)) administered with rKLe when the kl / kl mouse (kl / kl ( ⁇ )) administered with the solvent is taken as 100, and the same
  • the figure shows the value of a wild-type mouse (WT ( ⁇ )) administered with a solvent prepared and stained for paraffin sections.
  • the kl / kl mouse (+) is significantly different from the kl / kl mouse ( ⁇ ), and in FIG. 22, * (significance level): p ⁇ 0.05.
  • * signalificance level
  • Example 10 In Example 10, an example relating to immunostaining of calcification of the aortic blood vessel wall in vivo will be described in detail.
  • This Example 10 is performed on kl / kl mice administered with a solvent and kl / kl mice administered with rKLe, and is the same as Example 9 described above until the preparation of paraffin sections in the experimental method. Thereafter, blocking was performed using Protein® Block (DAKO) for 1 hour at room temperature, followed by washing. Anti-Runx2, Anti-phosphorylated phosphoERK, Anti-FGF23 and Anti-Klotho (both are SantauzCruz, dilution factor ⁇ 50) were used as primary antibodies and placed at 4 ° C. for 16 hours. Next, a Cy2 / 3-labeled secondary antibody (Jackson Immunoresearch Lab., Dilution factor ⁇ 400) was used for 1 hour at room temperature. Then, each was observed using the fluorescence microscope.
  • DAKO Protein® Block
  • FIG. 23 is a view showing a state of immunostaining of a paraffin section of a kl / kl mouse thoracic aorta according to Example 10.
  • the figure shows a representative example of the kl / kl mouse thoracic aorta performed.
  • -RKLe is a paraffin section of the thoracic aorta of a kl / kl mouse administered with solvent
  • + rKLe is a paraffin section of the thoracic aorta of a kl / kl mouse administered with rKLe.
  • FIG. 23 is a view showing a state of immunostaining of a paraffin section of a kl / kl mouse thoracic aorta according to Example 10.
  • the figure shows a representative example of the kl / kl mouse thoracic aorta performed.
  • -RKLe is a paraffin section of
  • the portion L delimited by a line indicates the lumen of the blood vessel
  • the arrow M indicates the portion of the media of the blood vessel
  • the arrow A indicates the portion of the outer membrane of the blood vessel.
  • the primary antibody used is shown in the upper right of each figure.
  • the rKLe used in each example is also verified for boiling and deactivated, and it is added that the result was the same as when only the solvent was used in each example (not shown).
  • a calcification regulating (suppression or promotion) agent capable of suppressing or promoting hard tissue (for example, bone) and / or vascular calcification by FGF23, a screening method thereof, and hard tissue by FGF23
  • a calcification inspection method and a calcification inspection kit capable of inspecting calcification in blood vessels and / or blood vessels are provided.
  • the mineralization regulator is an important means for preventing and treating osteoporosis, rickets, osteomalacia, osteoarthritis, ectopic calcification, or arteriosclerosis.
  • a screening method for a calcification regulating agent is an important means for developing new preventive / therapeutic agents for these medical conditions. Providing a calcification test method / test kit is likely to prevent these symptoms associated with calcification inhibition.

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Abstract

L'invention concerne un procédé de criblage d'un agent permettant de contrôler la calcification, à laquelle est liée la transduction de signal, qui est commune dans les tissus durs et/ou les vaisseaux sanguins. L'invention concerne également un agent de contrôle de calcification, un procédé d'examen de la calcification d'un os, de la calcification d'un vaisseau sanguin et du développement pathologique d'une maladie l'accompagnant, ainsi qu'un kit d'examen à cette fin. Le procédé de criblage d'un agent permettant de contrôler la calcification de tissus durs et/ou d'un vaisseau sanguin selon un premier aspect, comprend une étape consistant, dans un spécimen en l'absence et en présence d'un composé de test, à mettre en contact un Klotho soluble avec du FGFR et du FGF23, et une étape consistant à choisir un composé entraînant un changement de la transduction de signal spécifique. L'agent de contrôle de calcification selon un second aspect comprend un composé capable d'inhiber ou de promouvoir la formation d'un complexe contenant du Klotho soluble. Le procédé d'examen de calcification selon un troisième aspect de l'invention comprend une étape consistant à mesurer la concentration de Klotho soluble, etc. Selon un quatrième aspect, le kit d'examen de calcification comprend un ligand ayant une affinité de liaison envers le Klotho soluble, etc.
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