WO2012086809A1 - Accurate and highly sensitive method for measuring activity of human fgf19 and agent for regulating activity of human fgf19 - Google Patents

Accurate and highly sensitive method for measuring activity of human fgf19 and agent for regulating activity of human fgf19 Download PDF

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WO2012086809A1
WO2012086809A1 PCT/JP2011/079927 JP2011079927W WO2012086809A1 WO 2012086809 A1 WO2012086809 A1 WO 2012086809A1 JP 2011079927 W JP2011079927 W JP 2011079927W WO 2012086809 A1 WO2012086809 A1 WO 2012086809A1
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human
fgf19
betaklotho
fgfr4
activity
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PCT/JP2011/079927
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French (fr)
Japanese (ja)
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鈴木 理
今村 亨
真男 中村
眞弘 浅田
明子 倉持
絵美 本田
ゆり子 上原
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独立行政法人産業技術総合研究所
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/13Amines
    • A61K31/135Amines having aromatic rings, e.g. ketamine, nortriptyline
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/13Amines
    • A61K31/135Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726Glycosaminoglycans, i.e. mucopolysaccharides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/06Gastro-intestinal diseases

Abstract

Provided is a method for accurately measuring the activity of human FGF19 through human FGFR in an in vivo human environment. Also provided are a screening method for a human FGF19-like substance or a substance which selectively enhances or suppresses only the activity of human FGF19 to activate human FGFR4 in the coexistence of human beta-Klotho, a kit therefor, and an agent for selectively enhancing or suppressing the activity of human FGF19 to activate human FGFR4 in the coexistence of human beta-Klotho.

Description

Human FGF19 accurate and sensitive measurement method as well as human FGF19 activity controlling agents active

The present invention relates to a method for measuring the accurate and sensitive bioactive exerting human FGF19, using this method, selective human FGF receptor only when there is a human betaKlotho by human FGF19 4 (FGFR4) activity mineralization to enhance or suppress substance or screening methods and kits of human FGF19-like substance, and to selective control agent of the resulting human FGF19 activity by this method.

Human FGF19 is a endocrine factors synthesized in the human body. Considered to be an important factor regulating metabolism such as regulation of synthesis and glucose of bile acids from the administration experiments in mice, utilizing the activity, enhancing the inhibition to the useful in the treatment of various diseases related to metabolism It has been considered. Therefore, use of the activity of human FGF19, enhancement, have been actively developed in drug discovery and therapeutic methods aimed at inhibiting.
At that time, as a tool of research and development of human FGF19, at the medical pharmaceutical research is most frequently used as models of human disease, mouse of extensive experimental animal research tools have been used.
In mice, it has a human FGF19 and physiologically equivalent functions, a position also on the chromosome corresponding, but ortholog genes are present, reasons research history in this gene, given the name Fgf15 are (i.e., the human does not exist FGF15, the mice no FGF19) (non-Patent Document 1). Therefore, experiments or administering human FGF19 in animals mice (Non-Patent Document 9,22,23), by or to express a gene of FGF19 (Non-Patent Document 8, 11), which analyzes the effect. Also, by inhibiting the FGF15 protein from Fgf15 genes within the body of the mouse it is physiologically produced, and by analyzing or estimating the physiological activity of FGF19 in humans (Non-Patent Document 4), even at the cellular level, mouse Ya reaction (non-patent literature when expressed a co-receptor of mouse from the reaction (non-Patent Document 22, 25) and human cells when given human FGF19 against cultured cells derived from an animal other than human such as rat We are analyzing the 21).
Normally, when to study the physiological function of the ortholog molecules between human and mouse, it was often not to the species difference is much of a problem. For this reason, even at this stage, in the many studies of the human FGF19, research was aware of the difference in species is not performed. The present inventors have also previously have been developed selective activation method and method for suppressing an FGFR4 human FGF19, as the FGFR / betaKlotho expression system using at that time, the FGF receptor from a mouse with mouse betaKlotho a expressed allowed cell lines, experiments were conducted adding human FGF19 against the cell lines (Patent Document 6). Thus, human FGF19 is, with respect to each of the various FGF receptors humans, what environment exert any organism acting under strictly had never been confirmed.
Further, in the many studies of human FGF19, the term human FGF19 activity, the concentration of the human FGF19 given to cultured cells are measured in a concentration of not less than about 10 nM (non-patent document 22), also experimental animal when administered is analyzed the effect at a concentration of about 50nM and in [9]. Measurements at concentrations greater than 0.5nM in the above patent documents present inventors is the lowest concentration, eg human FGF19 activity of the following trace 0.5nM was determined no. On the other hand, since the blood concentration of human is about 0.03 nM ~ 0.1 nM (non-patent literature 20, 24), considered abundance of FGF19 in the human body the organization is very small to the same extent, human FGF19 is a method of measuring with high sensitivity biological activity accurate to exhibit at a concentration as low as 0.03 nM ~ 0.1 nM is demanded. The control agent selected human FGF19 activity using this method has been demanded.

WO2003011213 WO200118210 US public 2002155543 US public 2007248604 JP 2006-158339 JP JP 2010-150232 JP JP 2008-99837 JP

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Tsai SP, Stephan JP, Stinson J, Stewart T, French DM A mouse model of hepatocellular carcinoma: ect-opic expression of fibroblast growth factor 19 in skeletal muscle oftransgenic mice Am J Pathol 2002 Jun; 160 (6):. 2295 -307. Desnoyers LR, Pai R, Ferrando RE, Hotzel K, Le T, Ross J, Carano R, D'Souza A, Qing J, Mohtashemi I, Ashkenazi A, French DM.Targeting FGF19 inhibits tumor growth in colon cancer xenograft . and FGF19 transgenic hepatocellular carcinoma models Oncogene 2008 Jan 3; 27 (1):... 85-97 Epub 2007 Jun 25. Pai R, Dunlap D, Qing J, Mohtashemi I, Hotzel K, FrenchDM Inhibition of fibroblas. t growth factor 19 reduces tumor growth bymodulating beta-catenin signaling Cancer Res 2008 Jul 1; 68 (13):.. 50 86-95 Jones S.Mini-review: endocrine actions of fibroblast growth factor 19. Mol Pharm. . 2008 Jan-Feb; 5 ( 1): 42-8 Epub 2008 Jan 8. McWhirter JR, Goulding M, Weiner JA, Chun J, Murre CA nov-el fibroblast growth factor gene expressed in the developing nervous system is a downstream target . of the chimeric homeodomain oncoproteinE2A-Pbx1 Development 1997 Sep; 124 (17):.. 3221-32 Nishimura T, Utsunomiya Y, Hoshikawa M, Ohuchi H, Itoh N. Structure and expression of a novel human FGF, FGF-19, . expressed in the fetal brain Biochim Biophys Acta 1999 Jan 18; 1444 (1):.. 148-51 Yu S, Zheng L, Asa SL, Ezzat S. Fibroblast growth fact-or receptor 4 (FGFR4) mediates signaling to the prolactin .... but not theFGFR4 promoter Am J Physiol Endocrinol Metab 2002 Sep; 283 (3): E490-5 Ito S, Kinoshita S, Shiraishi N, Nakagawa S, Sekine S, Fujimori T, Nabeshima YI Molecular cloning and expression analyses o. f mouse betaklotho, which encodes a novel Klotho family protein Me-ch Dev 2000 Nov; 98 (1-2):.. 115-9 Ito S, Fujimori T, Furuya A, Satoh J, Nabeshima Y, Nabeshima Y .. 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The present inventors have found that, recently, so far biological activity has been reported as a biological activity of human FGF19 in a number of studies, actually strongly suggestive result different from the activity of human FGF19 is demonstrated in the human body Obtained. This use of the activity of conventional human FGF19, enhancement, measurement of human FGF19 activity has been widely used in the development of drug discovery and therapeutic methods aimed at inhibiting strongly that there was a problem with the evaluation method It is suggested. The present invention is directed to an object to establish a method of measuring the accurate and sensitive bioactive human FGF19 exerts in the human body.
And using the measurement method to find a specific human FGF19-like substance exhibiting similar activity activating action only selectively enhance or inhibit materials, and the original human FGF19 to human FGFR human FGF19 it is intended to establish a method.

The present inventors have doubts about that have used mouse system as a research tool to analyze the phenomenon caused by human FGF19 activity in conventional in the human body. All ortholog genes involved in receiving system of the human FGF19 with human cells (human FGF receptor and the human betaKlotho coreceptors) is present also in mice. Therefore, similar to the model experiments in vivo of other human FGF, also for human FGF19 activity, analysis using a receiving system with mouse cells (murine FGF receptor and mouse betaKlotho coreceptors), or screening with respect to that of the system is to function as a fully model experiment tool, researchers in the field is conventional, including the present inventors have found is that nobody had not doubted.
The present inventors have found that the binding to human FGF19 and human FGF receptors are co-receptor of human betaKlotho mandatory, although the ortholog genes, that there are differences in amino acid sequence from the murine FGF15 and human FGF19 high probability of having a slight conformational distortion from, led thought to the possibility of deviation in coupling condition occurs between the two receiving systems for the protein to form that mouse FGF receptor via the mouse betaKlotho .
Therefore, the previously developed in Patent Document 6, a manufacturing method of the FGF receptor and expression systems betaKlotho using BaF cells, adapted to the human FGF receptor and the human betaKlotho gene, various human FGF receptor and was constructed systems transformed BaF cells expressing human betaKlotho. According to experiments using the human FGF19 receptor system, surprisingly, found significantly different from the bioactivity analysis of human FGF19 according to conventional mouse receptor system, were estimated using the conventional mouse-receiving system human physiological activity of FGF19 has demonstrated that actually differs significantly from the activity of human FGF19 exerted in vivo in humans.
This activating effect through human FGF receptor of human FGF19 has demonstrated that a function of selectively activating the human FGF receptor 4 (human FGFR4) only in the presence of human betaKlotho it is also an.
In addition, the use of the transformed BaF cell lines were successfully also to accurately measure or lower concentrations of active human FGF19 0.1 nM. The found control agent selected human FGF19 activity using this method.
By obtaining the above findings, the present invention has been completed.

Specifically, the present invention includes the following.
[1] A method for screening enhancing or inhibiting substance activating effect of selective human FGFR4 by human FGF19 human betaKlotho is exerted only when present, the following steps (1) - (8) said method comprising:
(1) FGF receptor and the step of plurality preparing a host cell that does not endogenously express betaKlotho,
(2) constructing a human FGF receptor 4 (human FGFR4) gene and other human FGF receptor gene is introduced by one in each host cell, the cell lines expressing each different type of human FGFR,
(3) Human betaKlotho gene with one of the human FGF receptor 4 (human FGFR4) gene and other human FGF receptor gene is introduced into the host cell, the human betaKlotho with each different type of human FGFR constructing a expression to cell lines,
(4) Step (2) and for all the cell lines obtained in step (3), step of reacting human FGF19 in the presence and absence of a test substance,
(5) by human FGF19 in each cell line, comparing the human FGFR activation action,
(6) a step of the test substance, to confirm that shows the activating effect of human FGF19 only in a cell line expressing human FGFR4 with human betaKlotho,
(7) when the activating effect of human FGF19 in the presence of the test substance were compared with the case of absence of the test substance, the step of selecting a test substance that enhances or inhibit the activation effect,
(8) Step a test substance selected in (7), step determines that only activating effect of the biological activity via human FGFR4 by human FGF19 is selectively enhance or inhibit material.
Comparison of the presence and absence of the test substance in [2] the step (7), in said step (4), in medium of human FGF19 concentration in multiple in the range of 0.001 ~ 0.5 nM are prepared is a system adjusted to a concentration conditions, characterized in that it is carried out by observing the to concentration-dependent variation measured activity value of human FGFR4 in each system, the [1] the screening method according to.
[3] the screening method is for searching a candidate substance bile acid diarrhea therapeutic agents having no tumorigenicity activity The screening method according to the above [1] or [2].
[4] A kit for use in the screening method according to any one of [1] to [3], along with human FGF19, characterized in that it comprises a set of cell lines of the following (1) and (2) kit to be;
(1) FGF receptor and betaKlotho to endogenously not expressing host cell surface, the set of transformed cell lines that exogenous human FGFR4 and other human FGFR is expressed by one,
(2) (1) of the response to transformed cell lines, transformed cell lines set of human betaKlotho with each different type of human FGFR is expressed.
[5] 0.001 including the concentration of human FGF19 solution of 0.5 nM, the kit according to the above [4].
[6] the an active ingredient retrieved glycosaminoglycan by the screening method of [2], selective biological activity via human FGF4 human FGF19 exerted only when the person betaKlotho exists enhancing agent.
[7] a method of screening for substances having only selectively human FGF19-like receptor activating action to activate human FGFR4 when a person betaKlotho exists, including the following steps (1) to (7) Method:
(1) FGF receptor and the step of plurality preparing a host cell that does not endogenously express betaKlotho,
(2) a step of each host cell into a human FGFR4 gene and other human FGF receptor gene, one type is introduced, each of which construct a cell line expressing a different type of human FGFR,
(3) a step of one type with introduced human betaKlotho gene, to construct a cell line expressing human betaKlotho with each different type of human FGFR out of the host cell in the human FGFR4 gene and other human FGF receptor gene ,
(4) (2) and (3) for all cell lines obtained in step exerting a test substance in the presence and absence of a test substance,
(5) comparing the human FGFR activation effects in each cell line,
(6) the test substance, the step of selecting a substance showing only human FGFR4 activation activity in a cell line expressing human FGFR4 with human betaKlotho,
(7) a test substance selected in (6), step determines that human FGF19-like receptor active substance.
In the step of adding a test substance in [8] above (4), characterized in that the addition of glycosaminoglycan in the medium, the method described in the above [7].
[9] A kit for screening according to the above [7] or [8] A kit comprising a set of cell lines of the following (1) and (2);
(1) FGF receptor and betaKlotho to endogenously not expressing host cell surface, the set of transformed cell lines that exogenous human FGFR4 and other human FGFR is expressed by one,
(2) (1) of the response to transformed cell lines, transformed cell lines set of human betaKlotho with each different type of human FGFR is expressed.
[10] Furthermore, the kit according to the above [9] comprising (3) glycosaminoglycan.
[11] A method of human FGF19 can be measured accurately with high sensitivity biological activity value for selectively activating the human FGFR4 only in the presence of human betaKlotho the body concentration of human, endogenous FGF receptors and betaKlotho constructs a cell line expressing the exogenous human FGFR4 and human betaKlotho not express, with the addition of glycosaminoglycan in the medium of the cell lines, human FGF19 concentration in the medium becomes less 0.5nM adjusting, measuring method comprises measuring a biological activity of human FGFR4 as.
[12] The activity of human FGFR4 in each case the human FGF19 concentration was adjusted to a plurality of density conditions within the range of 0.001 ~ 0.5 nM in the medium is measured, characterized by observing the concentration-dependent change in the amount of to, measuring method according to the above [11].
[13] A kit for use in measuring method according to the above [11] or [12], the kit characterized by including the following (1) to (4);
(1) Human FGF19 solution for adjusting the human FGF19 concentration into a plurality of density conditions within the range of 0.001 ~ 0.5 nM in the medium,
(2) glycosaminoglycan,
(3) do not express endogenous FGF receptors and betaKlotho, transformed cell lines that express the exogenous human FGFR4,
(4) (3) transformed cell lines further human betaKlotho in cell lines expressing the.

Human FGF Receptor 4 provided by the present invention (human FGFR4) alone or even by using cell lines that expressed human betaKlotho, human can not be obtained in the experiment using conventional frequently used has been mouse cells and mouse individuals were induced by abnormal disease physiological effects associated with FGF19, for example a therapeutic candidate for bile acid diarrhea, can be re-evaluated in the human system, resulting truly of efficacy against human high human FGF19 related It can provide a pharmaceutical composition.

Mouse betaKlotho and glycosaminoglycans various mouse FGF receptor activation by human FGF19 under glycan presence (A) mouse FGFR (mFGFR1c, R2c, R3c, R4) and BaF3 cells expressing the mouse betaKlotho (mFGFR / mbetaKlotho expressing cells) glycosaminoglycan (heparan sulfate ●, chondroitin sulfate B △, D ▲, were stimulated with various concentrations of human FGF19 in the presence (final concentration 5 [mu] g / ml) of E □), thymidine uptake It was measured. A similar experiment was performed with (B) the presence of heparin 5 [mu] g / ml in (■) (A). (For Receiver System mouse FGFR, specifically point to activate, and nonspecific human FGF19 is that the addition of heparin in the presence mice betaKlotho the mFGFR4 only when heparin was added to human FGF19 in the absence of mouse betaKlotho only common with the receiving system of human FGFR all mFGFR in that activated. However, there are significant differences in mice betaKlotho presence of at recipient system mouse FGFR, nonspecific activity upon addition of heparin the exception human FGF19 is not possible to activate the mouse FGFR4, sometimes activate mouse FGFR2 及 mouse FGFR3 rather in the case of addition of glycosaminoglycans.) Human betaKlotho and glycosaminoglycans various human FGF receptor activation by human FGF19 under glycan presence (A) human FGFR (human FGFR1c, R2c, R3c, R4) expression vectors for human betaKlotho in BaF3 cells expressing the introduced, the human FGFR / person betaKlotho expressing cells glycosaminoglycan (heparan sulfate ●, chondroitin sulfate B △, D ▲, E □) the presence of various concentrations (final concentration 5 [mu] g / ml) human FGF19 in to stimulate, to measure the uptake of thymidine. ○ Control (glycosaminoglycans additive-free). (Human FGF19, in the presence of human betaKlotho, human FGFR4 specifically activated, heparan sulfate, chondroitin sulfate B and E to enhance the activation effect of human FGF19 in that case. Human betaKlotho absence is any be added glycosaminoglycan human FGFR also can not be activated.) was subjected to the same experiment as (in B) the presence of heparin 5 [mu] g / ml (■) (B). (In the case of addition of heparin, but it would specifically activate the human FGFR4 in the presence nonhuman betaKlotho, in the presence of human betaKlotho, activate all human FGFR.) Low dose activate human FGFR4 human FGF19 is glycosaminoglycan addition amount of impact traces on human FGF19 is the presence of human betaKlotho or absence of time to specifically activate human FGFR4 of (0.5 nM or less) Effect of amount of glycosaminoglycan when to have on the activation. (Hereinafter amount of human FGF19 is 0.5 nM, particularly in the case of 1 pM ~ 0.1 nM, heparan sulfate, with chondroitin sulfate B and E, heparin enhances the human FGFR4 activation activity in the presence of human betaKlotho in a dose-dependent manner . human FGF19 is below 0.5nM the activating effect in humans FGFR4 in the presence of a non-human betaKlotho, including heparin, neither a glycosaminoglycan.) The low dose (0.5 nM or less) of human FGF19 bovine liver-derived impact human FGFR4 / person betaKlotho expressing BaF3 cells glycosaminoglycan addition on the time that specifically activate human FGFR4, liver-derived glycosaminoglycan were stimulated with human FGF19 in various concentrations in the presence (final concentration 5 [mu] g / ml) of (●), it was measured uptake of thymidine. ■ heparin 5μg / ml positive control of the addition, ○ glycosaminoglycan without the addition of a negative control. (Glycosaminoglycans from bovine liver, following the addition of human FGF19 is 0.5 nM, especially in the case of 1 pM ~ 0.1 nM, enhances the human FGFR4 activation activity in the presence of human betaKlotho to the same extent as heparin. on the other hand, no activation effect on human FGFR4 in the presence of a non-human betaKlotho.

Hereinafter, present invention will be described in more detail.
[1] for the action of the human FGF19
1. Human FGF19 and its bile acid synthesis inhibition
Human FGF19 is discovered substance as a homologous factor on the basis of the sequence of the probes designed based on the nucleotide sequence of DNA encoding mouse FGF15 (Non-Patent Document 15) (Non-Patent Document 16). Human FGF19 consists 216 amino acids including a 22 amino acid signal sequence, FGF19 human is thought to correspond to FGF15 mouse. It was initially reported expression in the brain. From the analysis, such as knockout mice knockout mice and Fgfr4 gene then Fgf15 gene, FGF15 is transported to the liver via the secreted portal from the small intestine, the synthesis of bile acids in the liver, is the rate-limiting enzyme of bile acid synthesis It is controlled cholesterol 7-alpha hydroxylase of (CYP7A1) to negative has been revealed. Treatment of cholestyramine resin blood FGF19 levels of absorbing and bile acids to reduce significantly by the administration of similar compounds also bile acids for human FGF19, the blood FGF19 levels significantly increased, further cultured human liver cells from such that expression of the stimulation dose-dependent manner CYP7A1 is inhibited by FGF19, FGF19 in humans has been shown to have physiological effects that modulate the bile acid synthesis (non-Patent Document 26). Also, Chronic idiopathic bile acid diarrhea, which is a type of diarrhea, has been reported and can be caused by lack of FGF19, and lead to the development of effective treatment QOL improved many chronic diarrhea patients expected It is (non-Patent Document 20).
The term "activating action through human FGF receptor of human FGF19" or "biological activity of human FGF19 through human FGF receptor" in the present invention, the human FGF receptor 4 only in the presence of human betaKlotho ( action to selectively activate human FGFR4), the human betaKlotho absence More specifically, as shown in FIG. 2, neither the human FGFRl ~ 4 without activating only human FGFR4 in the presence of human betaKlotho It refers to action of activating the. As such, this "in humans betaKlotho absence, without any activation of human FGFRl ~ 4, in the presence of human betaKlotho action of activating only the human FGFR4" materials having a a human live since it can be said that a substance that causes an effect similar to the "biological activity of human FGF19 through human FGF receptor" in the body, in the present invention is referred to substances with such properties as "human FGF19-like substance" there is also.
Conventionally, among the activities referred to as "biological activity of human FGF19 through human FGF receptor", preferred examples of the biological activity, activity that reduce expression of CYP7A1 in liver cells are typical, bile acid diarrhea Although the effect of improving is expected, following four. In such detail, conventional human FGFR4 tumor inducing activity via even despite human FGF19 was acting to induce in the presence nonhuman betaKlotho, were counted in one of the human FGF19 activity.
However, the present invention, it is human FGF19 activity or human FGF19-like activity via the human FGF receptor, as described above, "action to selectively activate human FGFR4 only if the person betaKlotho exists" There due to be determined, be understood as "tumor inducing activity" is not included in the term human FGF19 activity or human FGF19-like activity. Therefore, human FGF19-like active substance (hereinafter also referred to as "human FGF19-like receptor active agent".) Search things, and human FGF19 activity or to search for substances that enhance human FGF19-like activity is extremely useful it can be said that.
In the present invention, a technique for measuring a human FGF19 activity or human FGF19-like activity "action of activating only the human FGFR4 in the presence of human betaKlotho" is among the activating effect, the activating effect is desirable for the human body enhanced, inhibiting only undesirable effects selectively substance, i.e. it is applied to screening methods for obtaining enhancers or inhibitors of action of activating only human FGFR4 by human FGF19 in the presence of human betaKlotho it can.
As mentioned above, considered to blood concentration of human is about 0.03 nM ~ 0.1 nM (non-patent literature 20, 24), the abundance of FGF19 concentration in the body each tissue of humans is very small to the same extent . Accordingly, in the present invention, human FGF19 activity in an environment in the human body, human FGF19 concentration at that human FGF19 biological activity to exert in vivo, etc., 0.5 nM or less, preferably 1 pM ~ 0.3 nM, more preferably 1 pM ~ 0.1 nM, and most preferably refers to the concentration of 0.03 nM ~ 0.1 nM. Method of measuring human FGF19 activity in the present invention can be measured with high sensitivity biological activity accurate human FGF19 exerts in the case of low concentration of about 0.03 nM ~ 0.1 nM.
The human FGF19 according to the present invention, naturally derived human FGF19 (e.g., GenBank accession numbers: NM_005117 reference) is preferred, not limited thereto, may be a recombinant FGF19, further said human FGF19 activity if a, or may be a part of the amino acid sequence has been altered. To produce recombinant human FGF19 can be used an ordinary transformed host / vector system such as E. coli or mammalian cells appropriately.

2. For FGF receptor
FGF receptor (FGFR) is a protein of the membrane once transmembrane present on the cell surface. FGFR mammals are ortholog molecules with high homology, typically to the knowledge of the mouse FGFR becomes excellent human FGFR model. FGFR used in the present invention is a human FGFR, hereinafter simply the term FGFR, refers to human FGFR and mouse FGFR common findings. Currently, in humans with mouse, five FGFRl ~ 5 have been identified. Of which the FGFRl ~ 4 tyrosine kinase receptor is activated by dimerization, autophosphorylation by FGF binds. Activated FGFR interacts with signaling molecules in cells and activates multiple signaling pathways. FGFRl ~ two main isoforms by alternative splicing in 3 (FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c) is present.
Each receptor-specific functions, activities has not been completely elucidated, R1 primarily mesenchymal, neuroectodermal tissues, R2 expression in tissues from ectodermal are reported in addition ing. R3 is seen many expression in bone and cartilage, the central nervous system. R4 is the liver, muscle, lung, the expression of the pancreas, such as high. Of splicing form from R1 to R3, "c" form, with mesenchymal tissue, "b" form is specifically expressed respectively epithelial tissue.
The present inventors have found that the presence mouse FGFR1c human FGF19 mouse betaKlotho in Patent Document 6, a mouse FGFRc, have found that activating the mouse FGFR3c. Further, when the mouse FGF15 suppresses the synthesis of bile acids act in the liver, to act through the presence FGFR4 the betaKlotho has been revealed. Also it has been shown that the expression of CYP7A1 by FGF19 from the analysis of mice expressing human FGFR4 is required human FGFR4. Thus, as the current specific embodiment, when observing the selectivity of the human FGF receptor activating action by human FGF19, human FGFR1c, human FGFR2c, and human FGFR3c, important physiological actions as human FGF19 above using human FGFR4 that are believed to strongly related to tumor-inducing activity of human FGF19. At that time, while having a respective extracellular portion which is involved in ligand binding of each human FGF receptors, and in order to facilitate comparison of about activation of DNA synthesis by intracellular kinase moiety, intracellular portions human FGFR1 4 types in terms of the exchanged and integrated with the intracellular portion of the used as human FGF receptor. Incidentally, human FGFR1c, human FGFR2c, human FGFR3c, gene sequence of the human FGFR4 is GenBank Accession No. NM_015850.3 (human FGFR1c), NM_000141.3 (human FGFR2c), NM_000142.2 (human FGFR3c), NM_002011.3 (human available at FGFR4).

3. For co-receptor betaKlotho
For human FGF19 exerts effect is insufficient only FGF receptor, one membrane is a transmembrane protein betaKlotho has been reported to be required to function as a coreceptor (Non-patent Document 6). There is also a report that can activate FGFR4 in betaKlotho independent (non-patent document 17).
betaKlotho is cloned material as homologues of Klotho (Non-Patent Document 18), mouse-derived betaKlotho gene sequences, for example GenBank accession numbers: in NM_031180, corresponding amino acid sequences, for example GenBank accession numbers: a NP_112457 shown, the base sequence of human betaKlotho for example GenBank accession numbers: in NM_175737, corresponding amino acid sequences, for example GenBank accession numbers: shown in NP_783864. It is known to be expressed in such adipose tissue in mice developing fetus, the gene acquisition method from other species, various variants, manufacturing methods such mutants have also been described (non-patent literature 18, 19, Patent Document 5).
In an embodiment of the present invention, although using the full length of the cDNA was cloned betaKlotho gene derived from human, as long as they do not impair the human betaKlotho activity, the first portion may be deleted or modified.

3. Measurement of human FGF19 activity via the human FGF receptor
In general, each FGF binds to FGF receptors on the cell surface, activate the FGF receptor, by activity of various signal transduction mechanisms in cells, would cause some effects and functions to the cell Therefore, the measuring operation and features that are the observed as FGF activity.
The general measurement, Ornitz, DM, Xu, J., Colvin, JS, McEwen, DG, MacArthur, CA, Coulier, F., Gao, G. and Goldfarb, M. (1996) Receptor specificity of the fibroblast growth factor family. J.Biol.Chem., 271,15292-15297. It includes cell growth stimulating activity assay as described in. The outline is as follows. The FGF to be measured in the culture solution in the culture cells having FGF receptors in addition to the surface, performing the further predetermined time culture additive in a culture solution of the title thymidine after a certain incubation time. In this case, for easy comparison of the degree of activation of various FGF receptors to each other, FGF receptors other than FGFR1c also to the cells as a hybrid molecules intracellular kinase moiety has been replaced and integrated into intracellular kinases FGFR1 It has been forced expression. Then, by measuring the incorporation into the polymer DNA of labeled thymidine occurring during the culture, assessing the degree activation of DNA synthesis by FGF. In the present invention, employing this technique in measuring the activity of human FGF19 through human FGF receptor. Human FGF19 in an environment in the human body is at a concentration at which exert bioactivity, 0.5 nM or less, for example, 1 pM ~ 0.3 nM, human preferably 1 pM ~ 0.1 nM, more preferably 0.03 nM ~ 0.1 nM FGF19 activity value can also be measured in a similar manner.
That is, the specific measurement method for human FGF19 measures accurate and sensitive biological activity value for selectively activating the human FGFR4 only in the presence of human betaKlotho the body concentration of human are as follows.
Do not express endogenous FGF receptors and betaKlotho build a cell line expressing the exogenous human FGFR4 and human betaKlotho, with the addition of glycosaminoglycan in the medium of the cell lines, human in the medium FGF19 concentration 0.5nM or less (e.g., 1 pM ~ 0.3 nM, preferably from 1 pM ~ 0.1 nM, more preferably 0.03 nM ~ 0.1 nM) was adjusted to be, the measurement method comprises measuring a biological activity of human FGFR4 , and the example, to measure the activity value of human FGFR4 in each case the human FGF19 concentration was adjusted to a plurality of density conditions within the range of 0.001 ~ 0.5 nM in the culture medium, to observe the concentration-dependent variation.
The kit therefor, set comprising in combination the following (1) to (4) are preferred.
(1) Human FGF19 solution for adjusting the human FGF19 concentration in the medium into a plurality of density conditions within the range of 0.001 ~ 0.5 nM.
(2) glycosaminoglycan.
(3) do not express endogenous FGF receptors and betaKlotho, transformed cell lines that express the exogenous human FGFR4.
(4) (3) transformed cell lines further human betaKlotho in cell lines expressing the.

4. For tumor-inducing effect of human FGF19
In the above-mentioned human FGF19-expressing transgenic mice it has also been reported that hepatocellular carcinoma is formed in about 10 months (Non-Patent Document 14). Also in mice administered with recombinant-human FGF19 protein it has been observed proliferation of hepatocytes (Non-Patent Document 9). Human FGFR4 High expression is observed colon cancer, liver cancer, breast cancer, in various types of cancers such as pancreatic cancer in humans. There is also that human FGF19 and human FGFR4 expression both is observed (Non-Patent Document 12). Further, RNA interference method the expression of the human FGFR4, when inhibited with antibodies, and the like that the cancer growth is inhibited, by human FGF19 activates human FGFR4, believed likely tumor is induced They are (non-patent literature 12, 13, 14). Recently mouse betaKlotho has been found that the cancer growth is inhibited by expressing with mouse FGFR4 (Non Patent Document 21).
Conventionally, the tumor-inducing activity through such human FGFR4 were also counted in one of the human FGF19 activity, the present invention, human FGF19 activity or human FGF19-like activity via the human FGF receptor described above included as due to "human betaKlotho only human FGFR4 selective action of activating when present" be determined to be in, when that person FGF19 activity or human FGF19-like activity, rather "tumor inducing activity" It is understood to be not. In particular to eliminate the tumorigenicity through human FGFR4, for use by using only bile acid synthesis inhibitory effect in the prevention or treatment of such bile acid diarrhea, the human FGFR4 only when "human betaKlotho exists controlling substance enhances receptor characteristic of human FGF19 of action "of selectively activated or selectively induce effects on human FGFR4 dependent on human betaKlotho, and to human FGFR4 that is not dependent on human betaKlotho action is required to find a control substance of human FGF19-like does not induce. And, as such a control substance, in particular, human FGF19 in an environment in the human body is at a concentration range that exhibits biological activity, 0.5 nM or less, for example, 1 pM ~ 0.3 nM, preferably from 1 pM ~ 0.1 nM, still more preferably a substance having a selective action, such as described above with 0.03 nM ~ 0.1 nM.
In the present invention, it was examined for various glycosaminoglycans, in the following human FGF19 concentration conditions 0.5 nM, activating effect in humans FGFR4 heparin at any of glycosaminoglycans human betaKlotho absence included from that none it can be said that the use of the glycosaminoglycans tumorigenicity activity by human FGF19 can be eliminated.

Previous application by the present inventors in (Patent Document 6), where in a system using a mouse FGF receptor and mouse betaKlotho reacted combining various glycosaminoglycans non-human FGF19 and heparin, chondroitin sulfate (particularly B and when the E) was allowed to act, human FGF19 mouse FGFR4 of coexistence mouse FGF receptor mice betaKlotho is not activated, was found to selectively activate only mouse FGFR2c. Then, when the human FGF19 to act in conjunction with heparan sulfate (derived from bovine kidney) in the presence of mouse betaKlotho the mouse FGF receptor R1c, R2c, although R3c is activated, the mouse FGF receptor R4 is quite It had found that does not activate.
In contrast, in the system using the human FGF receptor and the human betaKlotho In the present invention, first, was observed with the addition of further heparin above system, the human is a non-presence of human betaKlotho like the mouse system FGF19 activates only human FGF receptor R4, any human FGF receptor in the coexistence of human betaKlotho also activated. However, human FGF19, was reacted by combining various glycosaminoglycan other than heparin, any glycosaminoglycan human FGF19 even when combining glycans which human FGF receptors activity in non-coexistence of human betaKlotho Although not of, in the presence of human betaKlotho Surprisingly, quite unlike the mouse system, chondroitin sulfate B, and the case of E and heparan sulfate, human FGF receptor R1c, R2c, R3c is not activate not been found to enhance the activity of the human FGF receptor R4.
On the other hand, match the physiological environment in the human body, low dose (0.5 nM or less, in particular 1 pM ~ 0.1 nM) in the case of adding human FGF19 result of investigation of the, be combined either glycosaminoglycan including heparin in a non-presence of human betaKlotho not activate the human FGFR4, in the presence of human betaKlotho, with heparin, among the glycosaminoglycans, chondroitin sulfate B, and when combined with any of the E and heparan sulfate, It found that a dose-dependent manner activate human FGFR4.

This means that in the original human FGF19 concentration range under human in vivo environment, chondroitin sulfate B, not only the E and heparan sulfate, the activity of heparin against human FGF19 to human FGFR4 under coexistence human betaKlotho it can be said that a substance that selectively enhance only of. In particular, chondroitin sulfate B, E and heparan sulfate, excellent human FGF19 activity controlling agent be a human FGF19 concentration is high it holds the selective activation effect on human FGFR4 under coexistence human betaKlotho it is shown that there is.
Then, as described above, the human betaKlotho gene is essential for cell lines that expressed a cell line or a human betaKlotho gene and human FGFR4, expressing only human FGFR4 without introducing, more preferably expression system in other human FGFR used as a set that combines by performing together experiments at low doses (e.g., about 0.05 nM), it can be screened enhance or substance that inhibits specific human FGF19 activity to the original human FGFR4. For example, the above-mentioned chondroitin sulfate B, if it is possible to E and materials behave similarly and heparan sulfate searches, the substance excellent human FGF19 activity control agent for selectively activating the human FGFR4 in the coexistence human betaKlotho , and the said that can be expected to have only a bile acid synthesis inhibiting action no tumorigenicity, it is a candidate for prophylactic or therapeutic agent such as a bile acid diarrhea.

[2] for the glycosaminoglycans
Among sugar chains, it had the most structural diversity, one of the substances a significant impact on the various cell activities is a glycosaminoglycan. As typical of this molecule, with heparin, heparan sulfate, glycosaminoglycans sulfated such chondroitin sulfate and the like.
Heparin and heparan sulfate contained uronic acid residue and a glucosamine residue, and can by repeating structure of the disaccharide unit. The uronic acid configuration of C-5, α-D- glucuronic acid (GlcUA) and beta-L-iduronic acid and (IdoA) are distinguished, some of IdoA is 2-O-sulfate group (IdoA (2S )) with a. Glucosamine residue has from N- acetyl group (GlcNAc) or N- sulfate group (GlcNS). Glucosamine residues, some of which have a sulfate group to the C-6. Compared to these, content low but 2-O-sulfated alpha-D-glucuronic acid (GlcUA (2S)) and 3-O-sulfated GlcNS (GlcNS (3S)) or 3-O, 6-O - di-sulfated GlcNS (GlcNS (3,6S)) is present. The repeating structure of disaccharide exceeding several tens of hundreds consisting of these residues, heparin and heparan sulfate has. Therefore, the combination of different disaccharide units mentioned above, structural diversity of heparin and heparan sulfate are formed.
Meanwhile chondroitin sulfate containing uronic acid and galactosamine residues are made by repeating structure of the disaccharide unit. There are the following different types depending on the position to be sulfated, both are already commercially available.
A Type: 4-position of galactosamine are sulfated.
Type B: 4-position of galactosamine are sulfated, glucuronic acid (also referred to dermatan sulfate) has become iduronic acid epimerized.
C Type: 6-position of galactosamine are sulfated.
D-type: 6-position and 2-position of glucuronic acid galactosamine are sulfated.
E Type: 4-position and 6-position of galactosamine are sulfated.
Having a repeating structure of disaccharide exceeding several hundreds to several tens consisting of these residues. (Note that uniformly all disaccharide units that may not be repeated in the same structure.)
Heparin in the present invention, glycosaminoglycan (heparan sulfate, chondroitin sulfate) generally as, including those with the structure of the hundreds of polymer from having a low molecular repeating disaccharide is one, this study glycosaminoglycan used in the examples of a mixture of those having a structure in which disaccharide units are repeated about 20 to 100.
Glycosaminoglycans are used in the form of a pharmaceutically acceptable salt thereof. For example, alkali metal salts salts (sodium salt, lithium salt, potassium salt, etc.), alkaline earth metal salts, salts with inorganic bases such as ammonium salt or diethanolamine salt, cyclohexylamine salt, and organic bases such as amino acid salt of, it can be used pharmaceutically acceptable salts. It is preferred among them is sodium salt.
In embodiments of the present invention, in the original human FGF19 concentration range, glycosaminoglycans only activated selectively enhanced to human FGFR4 under coexistence human betaKlotho is chondroitin sulfate B, E and heparan sulfate, and It was heparin. In particular, chondroitin sulfate B, E and heparan sulfate, was demonstrated to be human FGF19 activity control agent having excellent selectivity even at a concentration range such as man FGF19 throat.
Other glycosaminoglycans, or even in such sulfated glycosaminoglycan, materials exhibiting similar behavior and chondroitin sulfate B, E and heparan sulfate are considered many present, screening of the present invention by applying the law, it can be easily retrieved. Thus, a typical material of the target substance in the screening methods of the present invention are various glycosaminoglycans.

[3] The pharmaceutical composition according to bile acid diarrhea treatment of the present invention:
Agents of the present invention, chondroitin sulfate B, glycosaminoglycan is selected from the E and heparan sulfate or by the screening method of the present invention, in the original human FGF19 concentration range, human under human betaKlotho coexist with human FGF19 FGFR4 material only activated retrieved as selectively enhancing substance into, or human betaKlotho enable searching substance only activation of the human FGFR4 under coexistence selectively as human FGF19-like substance activating and components, including a pharmaceutically acceptable carrier. Specifically, the therapeutic composition of bile acid diarrhea no tumorigenicity activity and the like. Pharmaceutically acceptable carriers, e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, excipients such as calcium carbonate, cellulose, methyl cellulose, hydroxypropyl cellulose, polypropyl pyrrolidone , gelatin, gum arabic, polyethylene glycol, sucrose, binders such as starch, starch, carboxymethyl cellulose, hydroxypropyl starch, sodium - glycol - starch, sodium bicarbonate, calcium phosphate, disintegrating agents such as calcium citrate, magnesium stearate , Aerosil, talc, lubricants such as sodium lauryl sulfate, citric acid, menthol, Gurishirurishin ammonium salt, glycine, fragrances orange powder, etc., benzoic Sanna Potassium, sodium bisulfite, methylparaben, preservatives such as propylparaben, citric acid, sodium citrate, stabilizers such as acetic acid, methyl cellulose, polyvinylpyrrolidone, suspending agents aluminum stearate, dispersing agent such as a surfactant, water, saline, diluent such as orange juice, cocoa butter, polyethylene glycol, etc. based waxes such as kerosene and the like, but is not limited to them.

Formulations suitable for oral administration include water, capsules containing liquids obtained by dissolving an effective amount of an agent in a diluent, such as saline, an effective amount of an agent as a solid or granular, sachets or tablets, suitable suspensions prepared by suspending an effective amount of a substance in Do dispersion medium, a emulsion or the like is charged and emulsified in an effective amount of a substance a solution of a suitable dispersant.
Parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, etc.) Suitable formulations, there are injection solutions of aqueous and non-aqueous isotonic sterile, including antioxidants , buffers, bacteriostatic, may contain an isotonizing agent and the like. Suspensions can also be mentioned aqueous and non-aqueous sterile suspensions thereto, solubilizers, thickening agents, stabilizers, may be contained preservatives, and the like. The preparation can be enclosed in a container by a unit dose or multiple doses as ampules and vials. The effective ingredients and a pharmaceutically acceptable carrier and freeze-dried, may be stored in or state be dissolved or suspended in an appropriate sterile vehicle just before use.
The dosage of the agent of the present invention, the activity and the type of the active ingredient, severity of the disease, animal species to be the subject of administration, drug acceptability of the administration subject, the body weight, but not be generalized age and the like, usually, amount of active ingredient per day for an adult is from about 0.001 to about 500 mg / kg.

[4] the screening method and kit of the present invention
Screening methods and kits of the present invention, by observing the activating effect for a plurality of human FGFR including humans FGFR4 in the presence of non or presence of human betaKlotho, if there is a similar "human betaKlotho human FGF19 only it is for the search of a substance having an action "to selectively activate human FGFR4, also selectively active human FGFR4 only if there is a" human betaKlotho human FGF19 has inherent also intended to search for enhancing or inhibiting substance effect "only to reduction.
Then, following the method screening, and will be described in detail as a kit, the methods and kits associated with substances to enhance or inhibit human FGF19 activity via conventional specific FGFR, and / or human FGF19 active pharmaceutical the substance was identified as an active ingredient of the composition, it is understood that it is also possible to use a method or a kit for reevaluation.

In the screening method of the present invention, it is necessary to use a system of cells not expressed endogenously at all FGF receptors not human origin but also betaKlotho.
The cells FGF receptors betaKlotho is also not endogenously expressed, typically because BaF3 cells are used a murine leukemia ProB cells will be described with reference to BaF3 cells below. However, any of these genes in other cells are also not expressed on the cell surface, and as long as the cell can be expressed on the cell surface when transformed with these genes, it is possible to use as well , the present invention is not limited to BaF3 cells.
Traits with respect to the BaF3 cells, (. By techniques of Patent Document 6) introducing the expression vector containing human FGFR1c, R2c, a gene encoding R3c and R4 respectively, each expressing human FGFR1c, R2c, and R3c and R4 it is possible to obtain the conversion BaF3 cell line. Then, by further introducing human betaKlotho gene, respectively, transformed BaF3 cell line human betaKlotho also expressed with the human FGF receptor can be produced. At that time, the human FGFR1c against BaF3 cells transfected with human betaKlotho gene, R2c, be introduced a gene encoding a R3c and R4, or both genes may be introduced at the same time.
Thus with the system of BaF3 cells expressing respectively each human FGF receptor obtained, with each human FGF receptor human betaKlotho also be provided as a set system of BaF3 cells expressing, original it can be used to screen for enhanced or substance that suppresses the effect of selectively activating the human FGFR4 only if the person betaKlotho by human FGF19 in the state in vivo environment is present. Also By using this system, it is possible to screen a substance having a selective action of human FGF19-like activating the human FGFR4 only in the presence of human betaKlotho.

In screening of a substance having a potentiation or human FGF19-like receptor activity of human FGF19 activity for only selectively human FGFR4 in the presence of human betaKlotho, each expressing a plurality of different types of human FGFR a containing human FGFR4 further also using a set of cell lines expressing human betaKlotho, evaluated by comparing each human FGFR activation activity of human FGF19 in the presence of the test substance corresponds to the cell line and the cell line is .
Analyte screening methods of the present invention, although the highest reliability for retrieval from various glycosaminoglycans, not limited thereto, proteins, lipids, and any materials such as sugars the target. When added to the medium of human FGF19 simultaneously transformed cell lines, preferred water-soluble substances. Further, the test substance is as long as the form of gene, prior to the action of human FGF19, by introducing the gene into the transformed cell lines, it is necessary to stabilize expression.
Specifically, for example, firstly human FGF receptor expressing human betaKlotho on the cell surface BaF3 cells of the system together with the bodies, then 0.5nM below together with the test substance with respect to systems expressing only human FGF receptor , preferably 1 pM ~ 0.3 nM, more preferably 1 pM ~ 0.1 nM, and most preferably it is possible to use a method of reacting the human FGF19 of 0.03 nM ~ 0.1 nM. At that time, the order of addition of the test substance with human FGF19 can be simultaneous, either may be the first. In addition, it is preferable to take data in the case of adding human FGF19 high concentrations above 1 nM.

Specific procedures of the screening methods of enhancing or inhibiting substance activating effect of selective human FGFR4 by human FGF19 exerted only when the person betaKlotho exists is as follows.
By the screening method, it is possible to find a candidate substance bile acid diarrhea therapeutic agents having no tumorigenic activity.
(1) FGF receptor and betaKlotho endogenously expressed non step of the host cell plurality prepared. As the typical host cell used in the process, there is a human BaF3 cells.
(2) constructing a human FGF receptor 4 (human FGFR4) gene and other human FGF receptor gene is introduced by one in each host cell, the cell lines expressing each different type of human FGFR. As the human FGF receptor gene non-human FGFR4 gene used in the process, it is preferable to use human FGFR1c gene, human FGFR2c gene, and a human FGFR3c gene, the human FGFR genes used in step (2), these it is most preferred to use all of the genes of four.
(3) Human betaKlotho gene with one of the human FGF receptor 4 (human FGFR4) gene and other human FGF receptor gene is introduced into the host cell, the human betaKlotho with each different type of human FGFR constructing a expression to cell line.
(4) Step (2) and for all the cell lines obtained in step (3), step of reacting human FGF19 in the presence and absence of the test substance. Incidentally, in this step, the concentration 0.001 to in medium of human FGF19 0.5 nM, preferably it is preferred to provide a system which is adjusted to a plurality of density conditions within the range of 0.001 to 0.1 nM.
(5) by human FGF19 in each cell line, comparing the human FGFR activation action,
(6) a step of the test substance, to confirm that shows the activating effect of human FGF19 only in a cell line expressing human FGFR4 with human betaKlotho,
(7) when the activating effect of human FGF19 in the presence of the test substance were compared with the case of absence of the test substance, the step of selecting a test substance that enhances or inhibit the activation effect. Since the above-mentioned step (4) the system was adjusted to a plurality of density conditions is prepared in the case where the activation effect of human FGF19 have been identified in the plurality of systems, the activity value of human FGFR4 in each system by observing the concentration-dependent change amount is measured, compared in the presence and absence of the test substance is preferably performed.
(8) Step a test substance selected in (7), step determines that only activating effect of the biological activity via human FGFR4 by human FGF19 is selectively enhance or inhibit material.
As the kit above screening, specifically, with human FGF19, may be prepared a set of cell lines of the following (1) and (2). Incidentally, human FGF19, for example 1 pM ~ 0.5 nM, preferably 1 pM ~ 0.1 nM, more preferably combined as the human FGF19 aqueous solution at a concentration of 0.03 nM ~ 0.1 nM.
(1) on the host cell surface that is not endogenously express the FGF receptor and betaKlotho, the set of transformed cell lines that exogenous human FGFR4 and other one or more human FGFR is expressed respectively. As the typical host cells used here, there is a human BaF3 cells.
(2) (1) of the response to transformed cell lines, transformed cell lines set of human betaKlotho with each different type of human FGFR is expressed.

Also, above, in each step of the selectively only screening methods enhancing or inhibiting substance activating effect of human FGFR4 by human FGF19 exerted when the person betaKlotho exists, in step (4), human FGF19 without adding to the culture medium and the cells by comparing the human FGFR activation action in system only selectively human FGF19-like receptor activation to activate human FGFR4 when a person betaKlotho exists it is possible to screening a substance having an action. Specific methods of screening a substance having only selectively human FGF19-like receptor activating action to activate human FGFR4 when a person betaKlotho exists, performs the following procedures (1) to (7).
(1) to a plurality prepared Host cells not endogenously express FGF receptors and betaKlotho.
(2) the human FGFR4 gene and other human FGF receptor gene is introduced by one in each host cell, each of which construct a cell line expressing a different type of human FGFR.
(3) Human betaKlotho gene with one of the human FGFR4 gene and other human FGF receptor gene is introduced into the host cell to construct a cell line expressing human betaKlotho with each different type of human FGFR .
(4) (2) and for all the cell lines obtained in (3), the action of the test substance in the presence and absence of the test substance. Incidentally, in the step, it is effective to add a previously glycosaminoglycans such in the medium, since the measurement sensitivity of the FGF19-like activation action of the test substance increases preferable.
(5) comparing the human FGFR activation effects in each cell line.
(6) a test substance, selecting a material which only human FGFR4 activation activity in a cell line expressing human FGFR4 with human betaKlotho.
(7) a test substance selected in (6), determines that the human FGF19-like receptor active substance.

As the kit therefor, except that no combination of human FGF19 can be the same cell line sets the screening kit of enhancing or inhibiting substance activating effect of selective human FGFR4 by the human FGF19 . Specifically, it may be prepared a set of cell lines of the following (1) and (2). Furthermore, preferably added to the set (3) glycosaminoglycan.
(1) FGF receptor and betaKlotho to endogenously not expressing host cell surface, transformed cell lines set of exogenous human FGFR4 and other human FGFR is expressed by one. As the typical host cells used here, it is the same in that there is a human BaF3 cells.
(2) (1) of the response to transformed cell lines, transformed cell lines set of human betaKlotho with each different type of human FGFR is expressed.

Note that when the actual screening, the proliferative capacity of BaF3 cells, can be assessed relative to control systems, such as systems without addition of the test substance. BaF3 proliferation ability of cells is measured by a conventional method such as the measurement of the uptake of labeled thymidine. The same applies to the other screening methods.
Further, in actual screening can be the activation of proliferation signaling BaF3 cells is assessed relative to control, such as system without addition of the test substance. BaF3 activation of cell growth signaling, measures tyrosine phosphorylation of FGF receptor, tyrosine phosphorylation of FGF receptor substrate 2 (FRS2), by a conventional method such as phosphorylation of the map kinase (ERK1 / 2). Were the same in other screening methods.
Each human FGF receptor and the human betaKlotho of BaF3 cells expressing the cell surface system, the system expressing only the human FGF receptor combined with human FGF19, Koshin according to regulatory effect of human FGF19 activity it can be a kit for the screening of substances or inhibitors. Also it is possible to only human FGFR4 selectively kit for screening substances that exert the action of human FGF19 when a person betaKlotho exists by this system.
The resulting material by these screening, all of which can be used as a pharmaceutical composition according to regulators or bile acid diarrhea therapy, human FGF19 activity exerted by human FGFR4 in the presence of human betaKlotho.

Hereinafter, the present invention will be described in detail with reference to examples, but the technical scope of the present invention is not limited to the following examples.
Incidentally, the techniques used to implement the present invention is particularly except the techniques explicitly their origin, are easily and reliably be carried out by persons skilled in the art based on known literature such as, for example, a gene engineering techniques J.Sambrook, EFFritsch & T.Maniatis, "Molecular Cloning: A Laboratory Manual (2 nd edition)", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); DMGlover et al.ed. , "DNA Cloning", 2 nd ed., Vol.1 to 4, (the Practical Approach Series), IRL Press, Oxford University Press (1995) method, or their substantially the same manner as and modifications method described in such as it can be carried out by. Also, the description of the prior art or patent application cited in the present invention shall be incorporated as described herein with reference.

[Reference Example] analysis using "mouse FGFR / mouse betaKlotho system" selective activation effect due to the presence of glycosaminoglycans to FGF receptors of human FGF19 (additional test of Patent Document 6)
Patent four mouse FGF receptor prepared in Example 1 of document 6 using 4 kinds of BaF3 cells respectively forcibly expressed with mouse betaKlotho or alone (mouse FGFR1c, FGFR2c, FGFR3c or FGFR4), these reactivity of human FGF19 against cells were analyzed how the change in the presence of various glycosaminoglycans.
Four mice FGF receptor (mouse FGFR1c, R2c, R3c or R4) concrete construction method for a total of eight BaF3 cells forced to express with mouse betaKlotho or alone each is as follows.
The BaF3 cells are cells without FGF receptor original, first each mouse FGFR (mFGFR1c, R2c, R3c, R4) cDNA animal cell expression vector (Ornitz, DM, Xu, J., Colvin, JS, McEwen, DG, MacArthur, CA, Coulier, F., Gao, G.and Goldfarb, M. (1996) Receptor specificity of the fibroblast growth factor family.J.Biol.Chem., are described in 271,15292-15297. built in and is), which was introduced into mouse BaF3 cells by electroporation. It performed about 2 weeks in culture in the presence G418, after obtaining a drug-resistant clones were obtained as a clone which can be grown by human FGF1 stimulation. BaF3 cells expressing both the murine FGFR and mouse betaKlotho, after a full-length cDNA of mouse betaKlotho cloned into an animal cell expression vector was introduced into a respective mouse FGFR-expressing BaF3 cells, approximately in Puromycin and presence G418 2 performs a weekly culture, was acquired as resistant clones. These genes introduced BaF3 cells 10% FBS and interleukin 3 (IL3, supplied by adding WEHI-3 10% of the conditioned medium of cells), were maintained in RPMI1640 medium containing Puromycin and G418. When growth stimulation experiment in screening were seeded in culture plates were suspended in RPMI1640 medium containing 10% FBS cells were washed with RPMI1640 medium.
Then, each cell of various glycosaminoglycans (heparin, Sigma product number H3393; heparan sulfate, Seikagaku product number 400700; chondroitin sulfate B, Seikagaku product number 400660; chondroitin sulfate D, Seikagaku Inc. product number 400676; chondroitin sulfate E, in Seikagaku product number 400678) presence, stimulated by changes as shown in FIG amount of human FGF19, DNA synthesis was measured. The analysis results are shown in Figure 1. For cells expressing the murine FGFR alone, as shown in FIG, human FGF19 in the presence of either glycosaminoglycan did not activate mouse FGFR1c, R2c, and R3c, heparin to mice FGFR4 It was activated only in the presence of. Whereas for the mouse FGFR and mouse KLB co-expressing cells show activity against cells Human FGF19 is expressing either murine FGFR in the presence of heparin, in the presence of heparan sulfate and mouse FGFR4 only mice betaKlotho (mR4 / mbetaKlotho) expressing cells revealed that not activated.
In the presence of chondroitin sulfate B or chondroitin sulfate E, only mR2c / mbetaKlotho expressing cells revealed to be activated.
Compared to the case of Example 2 below of human FGFR receptor system, for Receiver System mouse FGFR, in mice betaKlotho absence point to specifically activate mouse FGFR4 only if human FGF19 was added heparin, and human FGF19 the addition of heparin in the presence of mouse betaKlotho is in terms of activating all mice FGFR nonspecifically common with the receiving system of the human FGFR. However, there are significant differences in the presence of mouse betaKlotho, human FGF19 except nonspecific activation upon addition of heparin can not activate the mouse FGFR4, in the case of adding the glycosaminoglycan In some cases, activating mouse FGFR2 and mouse FGFR3 rather.

Construction of Screening System Using Example 1 BaF3 cells (human FGFR / person betaKlotho system)
Out created a cells forced to express human FGF receptor and the human betaKlotho in BaF3 cells using the same method as in Reference Example 1, was constructed a system for detecting the reactivity and enhancing or suppressing substance that of a human FGF19 .
Each FGFR and betaKlotho expressing BaF3 cells were prepared as follows. First the human FGFR (human FGFR1c, hR2c, hR3c, hR4) built into the same animal cell expression vector as used in cDNA Reference example, was introduced into mouse BaF3 cells by electroporation. It performed about 2 weeks in culture in the presence G418, after obtaining a drug-resistant clones were obtained as a clone which can be grown by human FGF1 stimulation. BaF3 cells expressing both the human FGFR and human betaKlotho, after a full-length cDNA of human betaKlotho cloned into an animal cell expression vector was introduced into a respective human FGFR-expressing BaF3 cells, approximately in Puromycin and presence G418 2 performs a weekly culture, was acquired as resistant clones. These genes introduced BaF3 cells 10% FBS and interleukin 3 (supplied by adding human IL3, WEHI-3 10% of the conditioned medium of cells), were maintained in RPMI1640 medium containing Puromycin and G418. When growth stimulation experiment in screening was used seeded in culture plates were suspended in RPMI1640 medium containing 10% FBS cells were washed with RPMI1640 medium.

Example 2 Analysis of selective activation effect due to the presence of glycosaminoglycans to human FGF receptor of human FGF19 (human FGFR / person betaKlotho system)
Example 1 4 kinds of human FGF receptor created by using four types of BaF3 cells respectively forcibly expressed with human betaKlotho or alone (human FGFR1c, human FGFR2c, human FGFR3c or human FGFR4), these reactivity of human FGF19 in respect cells were analyzed how the change in the presence of various glycosaminoglycans. First the cells were seeded in suspended culture plates RPMI1640 medium containing 10% washed FBS in RPMI1640 medium, in the presence various glycosaminoglycans, stimulated with varying amounts of human FGF19 as in FIG. 2, DNA the synthesis was measured. The analysis results are shown in Figure 2. As the cells expressing the human FGFR alone shown, human FGF19 in the presence of either glycosaminoglycan human FGFR1c, R2c, but did not activate R3c, heparin for human FGFR4 It was activated only in the presence of. These results were similar to the results of a system of the mice shown in Reference Example. Meanwhile, as for the human FGFR human betaKlotho co-expressing cells shown in (reference example shown activity against cells Human FGF19 is expressing either human FGFR in the presence of heparin, which is also the mouse systems as for generally similar) that, in the presence glycosaminoglycan other than heparin, non-human FGFR4 / person betaKlotho expressing cells was found to be not activated.
In other words, human FGF19, in the presence of human betaKlotho, human FGFR4 specifically activated, heparan sulfate, chondroitin sulfate B and E to enhance the activation effect of human FGF19 in that case. Human betaKlotho absence can not be activated nor any human FGFR added glycosaminoglycans. On the other hand, the addition of heparin, but would specifically activate the human FGFR4 in the presence nonhuman betaKlotho, in the presence of human betaKlotho, activate all human FGFR.

EXAMPLE 3 Low-dose human FGF19 human FGF receptor 4 by the effect of human betaKlotho and a glycosaminoglycan for activating effect (human FGFR4)
The human FGFR4 created in Example 1 with human betaKlotho, or using a BaF3 cells expressing human FGFR4 alone, in the same manner as Example 2, various glycosaminoglycans human under glycans presence FGF19 amounts were stimulated by changing at low doses (physiologically) area as shown in the illustration, DNA synthesis was measured. The analysis results are shown in Figure 3. Incidentally, chondroitin sulfate A is Seikagaku Corporation (product number 400658), chondroitin sulfate C was used Seikagaku Corporation (product number 400670). Other glycosaminoglycans, using the same as those used in Reference Examples and Examples 2.
As shown in FIG. 3, 0.5 nM or less, especially at low doses of 1pM to about 0.1 nM, human FGF19 even in the presence of any glycosaminoglycan, but does not activate the cells expressing the human FGFR4 alone for human FGFR4 / person betaKlotho expressing cells, chondroitin sulfate E, chondroitin sulfate B, heparan sulfate, in the presence of heparin, was found to activate the cell. Similar results in the presence of a glycosaminoglycan derived from the liver were obtained. Enhancing effect of the action of glycosaminoglycans low dose human FGF19 glycans, chondroitin sulfate E, chondroitin sulfate B, to heparan sulfate, heparin was more potent.
As described above, a method of measuring the accurate and sensitive bioactive exerting human FGF19 was obtained. In particular the presence action of human FGF19 is human betaKlotho in the liver, since it is action to suppress bile acid synthesis from cholesterol by activating human FGFR4, screening systems enhancer of action of this method human FGF19 by applying as has been shown to be applicable to the development of pharmaceuticals to improve the condition of the bile acid diarrhea.

Example 4 Low-dose human FGF19 effect of glycosaminoglycan prepared from liver to biologically active (human FGFR4 / person betaKlotho system)
Glycosaminoglycan than beef liver was prepared as follows. Liver tissue minced after boiling was ground liver cells were homogenized. Thereafter, it crushed liver acetone, chloroform - degreasing by methanol treatment. After removal of the solvent under reduced pressure, 2 mM CaCl 2 and 50mM Tris-HCl (pH8.0) 50 ℃ in Digestion was performed processing of the protein by 48 hours actinase. Then, heat-inactivated the actinase, were collected by centrifugation and the supernatant. The supernatant was dialyzed against deionized water and applied to a DEAE Sepharose Fast Flow column to separate the fraction containing the glycosaminoglycan by stepwise salt gradient of sodium chloride. The obtained fraction was treated with sodium borohydride solution, and the glycosaminoglycan chains were released from the beta elimination by protein. Glycosaminoglycan chains obtained, was neutralized with hydrochloric acid and precipitating and removing protein mixed by adding perchloric acid. Thereafter, the dialyzed against pure water, was subjected to sterile filtration by 0.22μm filter. The concentration of the prepared glycosaminoglycan was determined by the carbazole-sulfuric acid method.
The screening system created in Example 1 - Example 3, was applied to a liver-derived glycosaminoglycan prepared as described above was evaluated enhancing effect on the biological activity of human FGF19.
As shown in FIG. 4, the liver-derived glycosaminoglycan concentration of 5 [mu] g / ml, to the human FGFR4 / person betaKlotho expressing cells, human FGF19 0.5 nM or less, low dose especially 1pM of about 0.1nM whereas enhancing significantly activated heparin comparable even when added, for the human FGFR4 alone expressing cells, activation potentiation in the case of addition of the same human FGF19 in the same low doses it has been shown not.
As a result, the present screening system (human FGFR4 / person betaKlotho expression system), to be effective in screening for activators that enhance the biological activity via selective human FGFR4 human FGF19 was demonstrated.

Claims (13)

  1. A selective screening methods enhancing or inhibiting substance activating effect of human FGFR4 by human FGF19 exerted only when the person betaKlotho exists, the method including the following steps (1) to (8):
    (1) FGF receptor and the step of plurality preparing a host cell that does not endogenously express betaKlotho,
    (2) constructing a human FGF receptor 4 (human FGFR4) gene and other human FGF receptor gene is introduced by one in each host cell, the cell lines expressing each different type of human FGFR,
    (3) Human betaKlotho gene with one of the human FGF receptor 4 (human FGFR4) gene and other human FGF receptor gene is introduced into the host cell, the human betaKlotho with each different type of human FGFR constructing a expression to cell lines,
    (4) Step (2) and for all the cell lines obtained in step (3), step of reacting human FGF19 in the presence and absence of a test substance,
    (5) comparing the human FGFR activation activity by human FGF19 in each cell line,
    (6) a step of the test substance, to confirm that shows the activating effect of human FGF19 only in a cell line expressing human FGFR4 with human betaKlotho,
    (7) when the activating effect of human FGF19 in the presence of the test substance were compared with the case of absence of the test substance, the step of selecting a test substance that enhances or inhibit the activation effect,
    (8) Step a test substance selected in (7), step determines that only activating effect of the biological activity via human FGFR4 by human FGF19 is selectively enhance or inhibit material.
  2. Comparison of the presence and absence of the test substance in the step (7), in said step (4), the concentration in the medium of human FGF19 into a plurality of density conditions within the range of 0.001 ~ 0.5 nM adjusted systems are prepared, characterized in that it is carried out by observing the concentration-dependent change in the amount by measuring the activity of human FGFR4 in that each system, screening according to claim 1 Method.
  3. The screening method is for searching a candidate substance bile acid diarrhea therapeutic agents having no tumorigenicity activity The screening method according to claim 1 or 2.
  4. A kit for use in the screening method according to any one of claims 1 to 3, with human FGF19, the kit characterized in that it comprises a set of cell lines of the following (1) and (2);
    (1) FGF receptor and betaKlotho to endogenously not expressing host cell surface, transformed cell lines set of exogenous human FGFR4 and other human FGFR is expressed by one.
    (2) (1) of the response to transformed cell lines, transformed cell lines set of human betaKlotho with each different type of human FGFR is expressed.
  5. Containing 0.001 to concentration of human FGF19 solution of 0.5 nM, the kit of claim 4.
  6. As an active ingredient glycosaminoglycan retrieved by the screening method according to claim 2, selective enhancer of biological activity via human FGF4 human FGF19 exerted only when the person betaKlotho was present.
  7. A screening method for materials having only selectively human FGF19-like receptor activating action to activate human FGFR4 when a person betaKlotho exists, the method including the following steps (1) to (7):
    (1) FGF receptor and the step of plurality preparing a host cell that does not endogenously express betaKlotho,
    (2) constructing a cell line of human FGFR4 gene and other human FGF receptor gene is introduced by one in each host cell, expressing the respective different types of human FGFR,
    (3) Human betaKlotho gene with one of the human FGFR4 gene and other human FGF receptor gene is introduced into the host cell to construct a cell line expressing human betaKlotho with each different type of human FGFR process,
    (4) (2) and (3) for all cell lines obtained in step exerting a test substance in the presence and absence of a test substance,
    (5) comparing the human FGFR activation effects in each cell line,
    (6) the test substance, the step of selecting a substance showing only human FGFR4 activation activity in a cell line expressing human FGFR4 with human betaKlotho,
    (7) a test substance selected in (6), step determines that human FGF19-like receptor active substance.
  8. In the step of adding a test substance in the (4), characterized in that the addition of glycosaminoglycan in the medium A method according to claim 7.
  9. A kit for screening according to claim 7 or 8, a kit comprising a set of cell lines of the following (1) and (2);
    (1) FGF receptor and betaKlotho to endogenously not expressing host cell surface, the set of transformed cell lines that exogenous human FGFR4 and other human FGFR is expressed by one,
    (2) (1) of the response to transformed cell lines, transformed cell lines set of human betaKlotho with each different type of human FGFR is expressed.
  10. Further, the kit of claim 9 comprising (3) glycosaminoglycan.
  11. A method of human FGF19 can be measured accurately with high sensitivity biological activity value for selectively activating the human FGFR4 only in the presence of human betaKlotho the body concentration of human, not express endogenous FGF receptors and betaKlotho without constructing a cell line expressing the exogenous human FGFR4 and human betaKlotho, with the addition of glycosaminoglycan in the medium of the cell line, adjusted to human FGF19 concentration in the medium is equal to or less than 0.5nM and, measuring method comprises measuring a biological activity of human FGFR4.
  12. Measures the activity of human FGFR4 in each case the human FGF19 concentration was adjusted to a plurality of density conditions within the range of 0.001 ~ 0.5 nM in the medium, characterized by observing the concentration-dependent variation, the measuring method according to claim 11.
  13. A kit for use in measuring method according to claim 11 or 12, a kit characterized in that it comprises the following (1) to (4);
    (1) Human FGF19 solution for adjusting the human FGF19 concentration into a plurality of density conditions within the range of 0.001 ~ 0.5 nM in the medium,
    (2) glycosaminoglycan,
    (3) do not express endogenous FGF receptors and betaKlotho, transformed cell lines that express the exogenous human FGFR4,
    (4) (3) transformed cell lines further human betaKlotho in cell lines expressing the.
PCT/JP2011/079927 2010-12-24 2011-12-22 Accurate and highly sensitive method for measuring activity of human fgf19 and agent for regulating activity of human fgf19 WO2012086809A1 (en)

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US9089525B1 (en) 2011-07-01 2015-07-28 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for reducing glucose levels in a subject
US9580483B2 (en) 2011-07-01 2017-02-28 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for treatment of diabetes
US9670260B2 (en) 2011-07-01 2017-06-06 Ngm Biopharmaceuticals, Inc. Compositions comprising fusion variants of FGF19 polypeptides
US9751924B2 (en) 2011-07-01 2017-09-05 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising fusion variants of FGF19 polypeptides for reducing glucose levels in a subject
US9963494B2 (en) 2012-11-28 2018-05-08 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for reducing glucose levels in a subject
US9290557B2 (en) 2012-11-28 2016-03-22 Ngm Biopharmaceuticals, Inc. Compositions comprising variants and fusions of FGF19 polypeptides
US9878009B2 (en) 2012-12-27 2018-01-30 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for modulating bile acid homeostasis in a subject having error of bile acid synthesis
US9878008B2 (en) 2012-12-27 2018-01-30 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for modulating bile acid homeostasis in a subject having bile acid diarrhea or bile acid malabsorption
US9889178B2 (en) 2012-12-27 2018-02-13 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for modulating bile acid homeostasis in a subject having nonalcoholic steatohepatitis
US9889177B2 (en) 2012-12-27 2018-02-13 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for modulating bile acid homeostasis in a subject having primary sclerosing cholangitis
US9895416B2 (en) 2012-12-27 2018-02-20 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for modulating bile acid homeostasis in a subject having cholestasis
US9925242B2 (en) 2012-12-27 2018-03-27 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for treatment of nonalcoholic steatohepatitis
US9273107B2 (en) 2012-12-27 2016-03-01 Ngm Biopharmaceuticals, Inc. Uses and methods for modulating bile acid homeostasis and treatment of bile acid disorders and diseases
US9974833B2 (en) 2012-12-27 2018-05-22 Ngm Biopharmaceuticals, Inc. Methods of using compositions comprising variants and fusions of FGF19 polypeptides for modulating bile acid homeostasis in a subject having pregnancy intrahepatic cholestasis
US10093735B2 (en) 2014-01-24 2018-10-09 Ngm Biopharmaceuticals, Inc. Beta-klotho binding proteins

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