JP2000217595A - Screening of cd14 agonist or antagonist - Google Patents

Abstract

PROBLEM TO BE SOLVED: To screen a compound capable of changing the affinity of a lipopolysaccharide and CD14 and effective for treating diseases caused by lipopolysaccharide of bacteria by using a labeled lipopolysaccharide and a cell containing expressed membrane-bonded CD14. SOLUTION: A compound capable of changing the affinity of a lipopolysaccharide and CD14 is screened by using a lipopolysaccharide labeled with fluorescein isothiacyanate, biotin, enzyme, radioactive isotope, etc., and a cell containing about >=100,000 molecules of expressed membrane-bonded CD14 based on one cell and measuring, by flow cytometry, the mean fluorescence intensity(MFI) generated by contacting the labeled lipopolysaccharide with the cell containing expressed membrane-bonded CD14, the MFI generated by contacting the labeled lipopolysaccharide with the test compound and the cell containing expressed membrane-bonded CD14 and the MFI generated by contacting the labeled lipopolysaccharide with an anti-CD14 antibody and the cell containing expressed CD14.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to lipopolysaccharide and CD1
And a high-sensitivity screening method for efficiently finding a compound that changes the binding property of No. 4.

[0002]

2. Description of the Related Art A systemic inflammatory response syndrome (SIR) caused by an inappropriate or excessive biological response to a severe invasion.
Lipopolysaccharide (LPS), an endotoxin released from lysed cells when an antibacterial agent is administered to septic shock, the most prominent condition among S)), especially severe infections caused by gram-negative bacteria The septic shock caused by is a major problem in antimicrobial treatment. Septic shock is caused mainly by the activation of monocyte cells by bacterial components released during bacterial infection and the overproduction of various mediators from these cells. In order for bacterial components released during bacterial infection to activate target cells such as macrophages, the receptor on the target cell side needs to recognize the bacterial components. CD which is differentiation antigen of macrophages and monocytes
14 was found to play an important role (Science, 1990, 249: 1431). CD14 is membrane-bound
(mCD14) and solubilized form (sCD14). mCD14 has no transmembrane domain and is an anchor protein anchored on the cell surface by glycosylphosphatidylinositol. On the other hand, sCD14 is present in serum and is synthesized in the liver, or is excised from the surface of macrophages or monocytes by the action of phosphatidylinositol-specific phospholipase (Mo).
l. Immunol., 1989 26: 657). In the binding of bacterial components to mCD14, in the case of Gram-negative bacteria, LPS, one of the bacterial components, forms a complex with LPS-binding protein (LBP), which is an acute phase protein in serum. LBP has a catalytic action and promotes the binding of LPS to mCD14 (Annu. Rev. Immunol., 1995, 13: 437). From cells activated by the bacterial cell components, TNF-α, IL-1,
Cytokines such as IL-6 and IL-8, nitric oxide (N
O), superoxide, platelet activating factor (PA
F) and various mediators such as arachidonic acid cascade products are produced (Clin. Infect. Dis., 1994)
18 (Suppl. 2) S205). Each of these mediators expresses a plurality of activities, and since these mediators are related to each other, the expression of the activities is diversified, and when the activities are overexpressed, they exert a detrimental effect on the living body. As a result, hypotension, generalized intravascular coagulation (DI)
C), adult respiratory distress syndrome (ARDS), respiratory failure,
Serious and diverse clinical symptoms such as multiple organ failure (MODS) occur (Clin. Infect. Dis., 1994, 18 (Suppl. 2) S2
05). As described above, septic shock is caused mainly by the activation of monocyte cells by bacterial components released during bacterial infection and the overproduction of various mediators from these cells (Clin. Infect. Dis.,
1994, 18 (Suppl. 2, p. S205), a method of blocking bacterial factors and a method of blocking host factors have also been studied. As a treatment method by blocking bacterial side factors derived from Gram-negative bacteria, (1) a method using an anti-endotoxin monoclonal antibody (Infect.
Immun., 1993, 613863, JAMA, 1991, 266 1097
Page), (2) BPI (Bactericidal Permeability-Incre)
asing Protein) (Antimicrob. Agents Che
mother., 1996, 40, 65), (3) Method using an endotoxin antagonist (Science, 1995, 268, 80, W
O9639411), (4) Method using polymyxin B (No. 36)
Times ICAAC), (5) HDL (High-density Lipoprotein)
(J. Exp. Med., 1996, 184, 1601),
(6) Method by inhibition of LPS biosynthesis (Science, 1996, 2
74, p. 980). In addition, host-side factors (TNF overproduced by macrophage activation)
The treatment method by blocking various mediators such as -α, IL-1, NO, PAF, etc. includes (1)
a) Method using anti-TNF-α antibody (Nature, 1987, 330)
Volume 662, Lancet, 1998, Volume 351 929), (1b) Method using soluble TNF-α receptor (36th ICAAC), (1)
c) Method by suppressing TNF-α production (Nature, 370, 1994)
558, Nature, 1994, 370, 218), (2) IL-
1 receptor antagonist (J. Exp. Med.,
1991, 173, 1029, JAMA, 271, 1836, 1994) or a method by inhibiting IL-1β converting enzyme (J. Antibiot.,
1996 49: 333, (3) Method using anti-E-selectin antibody (Inflamm. Res., 1996 45: 448, Crit. Care.
Med., 1996, 24: 229). Septic shock drugs that control host factors can treat both Gram-positive and negative bacterium septic shock because of their mechanism of action, but septic shock involves multiple mediators However, since they exhibit a wide variety of activities, none of them has been effective in clinical trials by a method that suppresses a single mediator as described above. In addition, most of septic shock drugs that control bacterial side factors are targeted by LPS of Gram-negative bacteria as described above, and host cell activating factors differ between Gram-positive bacteria and negative bacteria. However, it is not possible to treat both gram-positive and negative bacterium septic shock by controlling the bacterial factor itself. However, it was revealed that both bacterial components released by infection with Gram-positive bacteria and negative bacteria both bind to mCD14 present on the surface of the target cell of the host and activate the cells (Im
munity, 1994, vol. 1, p. 509). Therefore, an agent that inhibits the binding of bacterial side factors to mCD14 and suppresses cell activation may be able to treat septic shock of both Gram-negative and positive bacteria. Infection and
Immunity, June 1997, pages 2272-2277, describes a binding test between FITC-labeled LPS and mononuclear cells in which the binding ratio of fluorescein isothiocyanate (FITC) to LPS (FITC / LPS) is 1: 0.9. Disclosed and Infect
ion and Immunity, Feb. 1998, pages 486-491
A binding test of THP-1 cells in which mCD14 is highly expressed by C-labeled LPS and 1,25-dihydroxyvitamin D ₃ is disclosed.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides (1) a method for screening a compound that changes the binding property between lipopolysaccharide and CD14, (2) a kit for the screening,
(3) Compounds that alter the binding between lipopolysaccharide and CD14 that can be obtained using the screening method or the screening kit (for example, inhibit the binding between bacterial components released by bacterial infection and membrane-bound CD14) Thus, a compound that suppresses cell activation, that is, C
D4 antagonist etc.) and (4) Lipopolysaccharide and CD
It is intended to provide a drug or the like containing a drug (for example, a therapeutic agent for septic shock or the like containing the antagonist) containing a compound that alters the binding to 14 or the like.

[0004]

Means for Solving the Problems In view of the above problems, the present inventors have conducted intensive studies and as a result, have found that human monocyte cells THP
-1 was cultured in the presence of 1,25-dihydroxyvitamin D ₃ to induce mCD14 expression, LPS labeled with FITC was added to the mCD14 expression-inducing cells together with the test compound, and after the reaction, the cells were washed. LP attached to cells
By using a novel screening method of detecting S by fluorescence intensity, it has been found that a compound that changes the binding property between LPS and CD14, that is, a compound with changed fluorescence intensity, can be efficiently screened. Reached.

That is, the present invention provides (1) labeled lipopolysaccharide (hereinafter abbreviated as LPS) and membrane-bound CD14
(Hereinafter, abbreviated as mCD14), a method for screening a compound that changes the binding property between LPS and CD14, and (2) LPS is fluorescein isothiocyanate, biotin, an enzyme or a radioisotope. The screening method according to (1), wherein the LPS is an element-labeled LPS, (3) the screening method according to (1), wherein the LPS is a rough LPS, (4) LP.
L in which S is labeled with fluorescein isothiocyanate
The screening method according to (1), which is PS.
(5) Bonding ratio (fluorescein isothiocyanate /
LPS labeled with fluorescein isothiocyanate having an LPS of about 0.03 to about 1.0, and m.
(1) The screening method according to the above (1), which uses cells in which CD14 is highly expressed;
The screening method according to the above (1) or (5), wherein a cell expressing not less than 000 molecules is used,
(7) The screening method according to (1), wherein the cell is a cell in which mCD14 expression is induced by 1,25-dihydroxyvitamin D ₃ , (8) a method wherein the cell is a mononuclear cell or a polymorphonuclear cell. (1) the screening method according to the above,
(9) the screening method according to (1), wherein the cells are monocytes, macrophages or neutrophils; (10) the screening method according to (1), wherein the cells are human cells;
(11) The screening method according to (1), wherein the cells are THP-1, (12) about 1 ng per about 10 ⁶ cells.
The screening method according to (1), wherein from about 30 ng of labeled LPS is used, (13) (i) labeled L
The case where PS and mCD14-expressing cells were brought into contact with each other, and (ii) labeled LPS and a test compound and mCD14.
14. The screening method according to item (1), wherein the binding between the labeled LPS and mCD14 is measured when the cells are brought into contact with cells that have expressed 14;
4) The mean fluorescence intensity (Mean fluorescence intensity (M) when the labeled LPS and the cells expressing mCD14 were brought into contact with each other using the flow cytometry method.
FI)) value, (ii) MF upon contact of labeled LPS and test compound with cells expressing mCD14
I value, and (iii) labeled LPS and anti-CD1
The MFI value when the antibody 4 and mCD14-expressing cells were contacted was measured, and the following formula (I) inhibition rate (%) = (1−MFI ¹ / MFI ² ) × 100 (I) (MFI ¹ = MFI when test compound was added-MFI when anti-CD14 antibody was added MFI ² = MFI when test compound was not added-anti-CD14
(15) a method for screening a compound that alters the binding between LPS and CD14, wherein the inhibition rate is determined by MFI when an antibody is added, (15) LPS and CD14
(1) The screening method according to (1), wherein the compound that alters the binding to CD1 is a CD14 antagonist.
6) The screening method according to (1), (17) or (14), wherein the compound that alters the binding between LPS and CD14 is a CD14 agonist. The compound does not bind to a CD14 agonist, a CD14 antagonist, or mCD14, but does not bind to LPS, CD14, or LPS-binding protein (hereinafter, abbreviated as LBP).
A method for screening a compound that inhibits the binding to CD14 or a compound that promotes the binding between LPS and CD14, (1
8) LPS-like action is TNF-α, IL-1, IL-6,
(17) The screening method according to (17), which is a production of IL-8, NO, superoxide, platelet activating factor or arachidonic acid cascade product, (19) LPS containing labeled LPS and cells expressing mCD14 Kit for screening for a compound that alters the binding between CDS and CD14, (20) a compound or a derivative thereof that alters the binding between LPS and CD14 obtainable by the screening method described in (1), (21) C obtained by the screening method described in (17).
D14 agonist, CD14 antagonist, mC
Does not bind to D14, but does not bind to LPS, CD14 or LBP
, A compound that inhibits the binding between LPS and CD14, a compound that promotes the binding between LPS and CD14, or a derivative thereof; (22) LPS and CD14 that can be obtained by the screening method described in (1). (23) a compound for preventing or treating septic shock, which comprises a compound that changes the binding property of CD1 or a derivative thereof;
(24) The preventive or therapeutic agent for septic shock according to (22), wherein the compound is mCD1
(2) a compound which does not bind to 4, but acts on LPS, CD14 or LBP to inhibit the binding between LPS and CD14.
A therapeutic agent, (25) an immunostimulant comprising a compound capable of changing the binding property between LPS and CD14 or a derivative thereof obtainable by the screening method according to (1),
(26) The compound (25) wherein the compound is a CD14 agonist
Item 27, wherein the compound is LPS and CD1
(28) (A) (i) a labeled LPS which is a compound that promotes binding to mCD14 and a cell that expresses mCD14,
i) Labeled L in the case of contacting cells expressing mCD14 with labeled LPS and a test compound.
The binding between PS and mCD14 is measured, or (B) the average fluorescence intensity (Mean fluorescence) when contacting (i) a cell expressing mCD14 with labeled LPS using flow cytometry. intensity
(MFI)) value, (ii) the MFI value when contacting cells expressing mCD14 with labeled LPS and test compound, and (iii) labeled LPS and anti-C
The MFI value when the D14 antibody was contacted with the cells expressing mCD14 was measured, and the following equation: Inhibition rate (%) = (1−MFI ¹ / MFI ² ) × 100 (I) (MFI ¹ = MFI when added-MFI when anti-CD14 antibody was added MFI ² = MFI when test compound was not added-anti-CD14
The compound that alters the binding between LPS and CD14 is selected by determining the inhibition ratio by MFI when an antibody is added, and then TNF-α, IL
-1, IL-6, IL-8, NO, superoxide, platelet activating factor or arachidonic acid cascade product production-inducing activity was measured. (A) A compound having the production-inducing activity was identified as a CD14 agonist or LPS and C
Selected as a compound that promotes the binding to D14, (b)
The compound having no production-inducing activity does not bind to CD14 antagonist or mCD14,
A CD14 agonist, a compound that promotes the binding of LPS to CD14, a CD14 antagonist or a compound that does not bind to mCD14, wherein the compound acts on D14 or LBP to inhibit the binding of LPS to CD14. The screening method according to (28), out of a method for screening a compound that acts on LPS, CD14 or LBP to inhibit the binding between LPS and CD14, and (29) a compound library that can be produced by combinatorial chemistry technology. A CD14 agonist selected using the method,
Compound that promotes binding between LPS and CD14, CD1
4 antagonist, does not bind to mCD14, but LP
Acting on S, CD14 or LBP, LPS and CD1
A compound that inhibits binding to No. 4 or a derivative thereof.

Further, the present invention relates to (30) a CD14 agonist, LPS selected from the compound library which can be produced by the combinatorial chemistry technique using the screening method described in (28).
(31) an immunostimulant containing a compound or a derivative thereof that promotes the binding between CD14 and CD14, and (31) a compound library that can be produced by combinatorial chemistry technology, using the screening method described in (28). A CD14 antagonist, selected from
a prophylactic / therapeutic agent for septic shock, comprising a compound or a derivative thereof that does not bind to mCD14 but acts on LPS, CD14 or LBP to inhibit the binding of LPS to CD14; (33) a method for preventing or treating septic shock, which comprises administering an effective amount of a compound or a derivative thereof that alters the binding between LPS and CD14, which can be obtained by the screening method described in (1). (34) an immunostimulatory method, which comprises administering to a mammal an effective amount of a compound or a derivative thereof that alters the binding between LPS and CD14, which can be obtained by the screening method described in (1), or (34) ) LPS and C obtainable by the screening method according to (1) for producing a prophylactic / therapeutic agent for septic shock Use of a compound or a derivative thereof that alters the binding to CD14, and (35) alters the binding between LPS and CD14 obtainable by the screening method described in (1) for producing an immunostimulating agent. It relates to the use of the compounds or derivatives thereof.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Preparation of labeled LPS In the present invention, any LPS derived from Gram-negative bacteria such as Salmonella and Escherichia coli can be used. Are preferred. The rough type has no O-glycan, and has a characteristic that the molecular weight is smaller than that of the smooth type. Specifically, Salmonella bacteria, especially those derived from Salmonella minnesota, are preferred, and Re595 (Sigma) is particularly preferred. In addition, not only LPS itself, but also various derivatives such as Lipid A (Lipid A), isomers and the like can be used. In the present invention, LPS needs to be labeled with a labeled compound that can be detected by a chemical or biochemical method, a radioisotope, or the like. As the labeling compound, for example, FITC, biotin, an enzyme and the like are used. As FITC, not only FITC itself but also various derivatives (for example, fluorescein-5-isothiocyanate (FIT
Cisomer I), fluorescein-6-isothiocyanate (FITC isomer II) and the like can also be used, but are simply abbreviated as FITC below. In the present invention, FITC Isomer I (Dojinsha) is preferably used. As biotin, for example, biotin-LC-hydrazide
(biotinamido hexanoyl hydorazide), biotin-LC-ASA
(1- (4-azidosalicylamide) -6- (biotinamido) -hexene)
Are used. As the enzyme, for example, alkaline phosphatase, horseradish peroxidase, and the like are used. As the radioisotope, for example, [ ³ H], [
⁵¹ Cr], [ ¹²⁵ I] and the like.

[0008] The labeling of LPS can be performed by a method known per se. For example, when labeling LPS with FITC, Infection and Immunity, June 1997, 227
The method can be performed by the method described on page 2-2277 or the like or a method analogous thereto, and specifically, the method can be performed by the following method or a method analogous thereto. First, L
About 10μl of base such as triethylamine per 1mg of PS
To about 2,000 μl, preferably about 100 μl to about 1,000 μl, more preferably about 500 μl, and after sonication as desired, a chelating agent such as ethylenediaminetetraacetic acid (EDTA) (preferably 100 mM EDTA) from about 1 μl to about 200 μl
l, preferably about 10 μl to about 100 μl, more preferably about 50 μl, and if necessary, further sonication, to dissociate the aggregated LPS (preferably to a monomer). The sonication can be carried out generally at about 0 ° C. to room temperature, preferably at about 4 ° C. to about 20 ° C., particularly preferably at about 4 ° C. to 15 ° C. The sonication time is preferably about 30 seconds to about 60 minutes, more preferably about 1 minute to about 30 minutes, and particularly preferably about 5 minutes to about 20 minutes. Also,
The sonication may be performed only once after the addition of the base or after the addition of the chelating agent, or may be performed separately after each addition. In particular, it is preferable to perform the treatment for about 15 minutes after adding the base and for about 5 minutes after adding the chelating agent.

Then, the pH is adjusted to about 3 to about 7, preferably about 4 to about 6, more preferably about 5 using 1N hydrochloric acid, and then about 4 ° C. to about 40 ° C., preferably about 4 ° C. to about 40 ° C. About 30 ° C., more preferably about room temperature, about 3
Leave for 0 seconds to about 15 minutes, preferably about 1 minute to about 10 minutes, more preferably about 1 minute to about 5 minutes, particularly preferably about 2 minutes. After neutralizing the reaction solution with 1N sodium hydroxide or the like, the pH is about 8 to about 1
1, preferably about 9 to about 10, more preferably about 9.5 of a suitable buffer (eg, borate buffer or carbonate / bicarbonate buffer, preferably carbonate / bicarbonate buffer, more preferably FITC dissolved in about 0.5 M carbonate / bicarbonate buffer (FITC concentration is about 1 mg / ml to about 20
0 mg / ml, preferably about 5 mg / ml to about 100 mg / ml,
More preferably, about 25 mg / ml) is added, and sonication is performed if desired. The sonication is preferably performed at the same temperature as the sonication, and the treatment time is about 15 seconds to about 15 minutes, preferably about 30 seconds to about 5 minutes, more preferably about 1 minute to about 3 minutes. In particular, it is preferably performed for about 1 minute. After sonication, about 0.1%
To about 10%, preferably about 1.6% (w / v) of a surfactant (preferably a deoxycholate (preferably a sodium salt)) for about 1 hour to about 48 hours,
Preferably, for about 6 hours to about 24 hours, more preferably for about 18 hours, about 4 ° C. to about 40 ° C., preferably
Incubate at room temperature to about 40 ° C., more preferably about 37 ° C., preferably under shading conditions, with stirring. After incubation, optionally, distilled water or LP with a pH of about 3 to 11, preferably about 6 to about 10,
The volume of the reaction solution was adjusted by adding an appropriate buffer or the like that did not affect the binding between S and FITC, and the volume was adjusted by dialysis using a dialysis membrane or Sephadex G-25M (Pharmacia).
The so-called free FITC which is not bound to LPS is removed by gel filtration using Biotech) or the like. For example, when a PD-10 column (Pharmacia Biotech) packed with Sephadex G-25M is used, the volume of the reaction solution is adjusted to 2.5 ml with distilled water, applied to the PD-10 column, and used for elution. The eluted fraction obtained by applying 3 ml of the buffer can be used as a fraction containing FITC-labeled LPS. As the buffer for equilibration and elution of the resin for gel filtration, any buffer having a pH of about 6 to about 8 can be used, but preferably PBS, more preferably Dulbecco's PBS (Dainippon) Pharmaceutical company) is used. It is preferable to add 0.01% to 1%, preferably 0.16% (all in w / v) of a surfactant (preferably deoxycholate (preferably sodium salt)) to the buffer. The amount of FITC in the obtained FITC-labeled LPS can be determined, for example, by using a fluorometer such as Fluoroskan II (Labsystems) at an excitation wavelength of 485 nm and a measurement wavelength of 538 nm.

In the FITC-labeled LPS described above, L
The binding ratio of FITC to PS (FITC / LPS) is preferably set, for example, as follows. About 0.03 to about 1.0 about 0.1 to about 1.0 about 0.3 to about 1.0 about 0.3 to about 0.8 about 0.3 to about 0.5 about 0.3 As described above, by setting the binding ratio of FITC to LPS to a specific ratio, the measurement sensitivity is increased, and a binding experiment can be performed with a lower concentration of LPS, that is, under conditions close to the LPS concentration in vivo. . Therefore, the target compound can be efficiently screened by the screening method of the present invention described below using such FITC-labeled LPS.

(2) Preparation of cells expressing mCD14 Cells used in the present invention may be any cells as long as they express mCD14 on the cell surface.
Those that highly express D14 are preferred, and specifically,
Cells expressing mCD14 per cell of about 100,000 or more can be used.
0,000 to about 2,000,000 molecules, preferably about 100,000 to about 1,500,000 molecules.
0 molecules, more preferably from about 500,000 to about 1,500,000 molecules, particularly preferably about 1,0
Those expressing 000 molecules are preferably used. In the present invention, even those expressed more can be used without any problem. In addition, not only cells whose expression level is inherently high, but also cells in which mCD14 expression is induced can be preferably used. induction of the expression of mCD14, for example, can be performed by 1,25-dihydroxyvitamin D ₃ and the like. The inducer is usually
About 1 ng to about 500 ng, preferably about 10 ng to about 100 ng, more preferably about 30 ng to about 40 ng per 3 × 10 ⁵ cells. As the cell type, for example, mononuclear cells, polymorphonuclear cells, etc. are used, and preferably, monocytes, macrophages, neutrophils, etc. are used. Among them, monocytes are preferably used. The cell is a mammal (eg,
Guinea pigs, mice, cats, dogs, pigs, sheep, cows, monkeys, humans, etc.) can be used as long as they are derived from THP-1, HL, etc.
Human cells such as -60, U937 and MonoMac-6 are preferably used, and THP-1 is particularly preferably used.

The cells most preferably used in the present invention are, specifically, 1,25-cells obtained in Reference Example 2 described later.
This is THP-1 in which the expression of mCD14 is induced by dihydroxyvitamin D ₃ . These cells express approximately 1,000,000 molecules of mCD14 per cell (Journal of Leukocyte Biology 61 June 1997 721
-728 pages). As described above, in the present invention, cells in which CD14 expression has been induced can be used, and the induction is carried out in the Journal of Leukocyte Biology 61 June 1997, July 1997.
It can be carried out by the method described on page 21-728 or a method analogous thereto. For example, cells such as THP-1 (purchased from Dainippon Pharmaceutical Co., Ltd.) can be prepared in a suitable medium (eg, RPMI medium 1640 with L-gluta) at about 3 × 10 ⁵ cells / ml.
mine (GIBCO) with 1 inactivated fetal bovine serum (GIBCO)
At a concentration of 0% (v / v), penicillin-streptomycin (GIBCO) was suspended in sodium penicillin G and streptomycin sulfate at concentrations of 100 U / ml and 100 μg / ml, respectively, and CD14. An appropriate amount of a substance that induces the expression of is added. Specifically, for example,
1,25-dihydroxyvitamin D ₃ (VD ₃ ; Roussel Uc
laf) to a final concentration of about 10 nM to about 1000 nM, preferably about 50 nM to about 500 nM, more preferably about 50 nM to about 200 nM, particularly preferably a final concentration of about 100 nM. Add Next, for example, mCD14 expression can be induced by culturing in a CO ₂ incubator in the presence of about 5% CO ₂ at about 37 ° C. for usually about 1 to 7 days, preferably about 3 days. . mCD
The expression level of 14 can be measured by using an antibody against CD14. As the antibody against CD14, a commercially available antibody can be used, or the antibody can be produced using CD14 as an immunogen according to a method known per se. Specifically, the measurement of the expression level of mCD14 is carried out by first suspending CD14-induced cells at about 10 ⁶ cells / ml in PBS.
Usually, FITC-labeled anti-human CD14 monoclonal antibody clone.UCHM1 (Ancell) is added at a concentration of about 0.1 μg / ml to about 10 μg / ml, preferably about 1.6 μg / ml, usually at about 4 ° C. to about 25 ° C. Preferably about 4 ° C to 15 ° C,
More preferably at about 4 ° C., usually for about 15 minutes to about 2 minutes.
Incubate for 4 hours, preferably about 15 minutes to about 12 hours, more preferably about 30 minutes to about 3 hours, particularly preferably about 1 hour. Cells are usually centrifuged once to five times, preferably three times with PBS (1,000 times).
After washing at rpm, 3 minutes, 20 ° C.), the cells were suspended in an appropriate amount (for example, 1,000 μl) of PBS, and the tube with a filter (FALCON2235 [Be
cton Dickinson]). For the cell suspension obtained, the amount of FITC-labeled anti-CD14 antibody bound to 10,000 cells is usually measured using a flow cytometer Cyto ACE-300.
(JASCO Corporation). FACS for elution buffer
Flow (Becton Dickinson) is used.

(3) Screening method The screening method of the present invention comprises a method (A) labeled L
A method for screening a compound that alters the binding between LPS and CD14, characterized by using cells expressing PS and mCD14, and is preferably a method (Method B)
Labeled LPS and mCD14 in the case where (i) the cell that expressed mCD14 was contacted with the labeled LPS and (ii) the case where the cell that expressed mCD14 was contacted with the labeled LPS and the test compound. Using a method for screening a compound that alters the binding between LPS and CD14, which is characterized by measuring the binding to LPS, and more preferably using (Method C) flow cytometry.
(I) Mean fluorescence intensity (Mean fluorescence) when the labeled LPS is brought into contact with cells expressing mCD14
intensity (MFI)) value, (ii) labeled LPS and MFI value when a test compound is brought into contact with cells expressing mCD14, and (iii) labeled LPS
The MFI value was measured when the anti-CD14 antibody and the cells expressing mCD14 were brought into contact with each other, and the following formula (I) inhibition rate (%) = (1−MFI ¹ / MFI ² ) × 100 (I) (MFI ¹ = MFI with test compound added-MFI with anti-CD14 antibody added MFI ² = MFI without test compound added-anti-CD14
This is a method for screening for a compound that alters the binding between LPS and CD14, wherein the inhibition rate is determined by MFI when an antibody is added. In the method C, the obtained MFI value is defined as the amount of FITC-labeled LPS bound to the cells, and the mCD14 of each test compound is determined based on the measured value.
The activity of altering the specific binding properties of and FITC-labeled LPS is determined by the above formula (I).

Usually, a test compound is added and labeled LP
By performing a binding test between S and mCD14-expressed cells, a compound that changes the binding property between LPS and CD14 can be screened. Specifically, the method described below or a method analogous thereto can be used. You can do it. First, a test compound is added to cells expressing mCD14. The test compound is desirably dissolved in a buffer suitable for screening. As such a buffer, a buffer that does not affect the binding between LPS and CD14 is used, preferably a buffer having a pH of about 6 to about 10, more preferably PBS or the like. (Hereinafter referred to as a buffer such as PBS). The test compound solution is used in an amount of about 1 μl to about 1,000 μl, preferably about 5 μl to about 200 μl per about 10 ⁶ cells.
l, particularly preferably about 5 μl to about 100 μl, particularly preferably about 20 μl. The concentration of the test compound in the test compound solution can be appropriately selected. Then, if desired, for example, in the presence of about 5% CO ₂ ,
At about 37 ° C. for about 1 minute to about 60 minutes, preferably about 5 minutes.
The preincubation is performed for about 30 minutes to about 30 minutes, more preferably about 15 minutes. About 1n per about 10 ⁶ cells
g to about 1000 ng, preferably about 1 ng to about 10 ng.
0 ng, more preferably about 1 ng to about 30 ng, particularly preferably about 5 ng of FITC-labeled LPS are added, and (I) P
Human serum diluted about 1-fold to about 1000-fold, preferably about 2-fold to about 100-fold, more preferably about 5-fold to about 50-fold, particularly preferably about 10-fold with a buffer such as BS, or (ii) ) About 1 ng to about 1000 ng, preferably about 1 ng to about 100 ng, particularly preferably about 20 ng of LBP, for example in the presence of about 5% CO ₂ at about 37 ° C. for about 15 minutes to about 12 hours, preferably about 1 hour.
Incubate for about 5 minutes to about 2 hours, more preferably about 1 hour. The cells are washed several times using a device that can be used for cell washing such as a multi-screen filtration system vacuum manifold (Millipore) with a buffer such as PBS, preferably three times, and then washed with 300 μl of PBS. Suspend and filter. The amount of FITC-labeled LPS bound to 1,000 cells of the obtained cell suspension is measured using a flow cytometer such as Cyto ACE-300. Usually, FACS Flow can be used as the elution buffer. When the test compound is not added, that is, the maximum binding amount (negative control) can be measured by adding a buffer such as PBS instead of the test compound in the same amount as the test compound.

[0015] The amount of specific binding to mCD14 (positive control) was measured by using anti-CD1 instead of the test compound.
4 antibodies (for example, about 1 to 10 times with a buffer such as PBS)
An anti-human CD14 monoclonal antibody diluted 0-fold, preferably about 10-fold, specifically clone.MEM18 (SANBIO
Can be carried out by adding an equivalent amount of the compound (1) to the test compound. FITC obtained by binding the obtained MFI value to cells
Based on the amount of labeled LPS, based on the measured value, the activity of each test compound for changing the specific binding between mCD14 and FITC-labeled LPS can be determined by the above formula (I). A test compound having an inhibition of about 20% or more, preferably about 50% or more can be selected as a compound that inhibits the binding of LPS to CD14. On the other hand, the inhibition rate is-
Test compounds that are about 20% or more, preferably -about 50% or more, can be selected as compounds that promote binding of LPS to CD14.

As described above, the screening method of the present invention is useful as a reagent for screening a compound that inhibits or promotes the binding between CD14 and LPS.
Compounds that inhibit the binding of CD14 to LPS include the following 3
Class of compounds. A compound that binds to mCD14 and inhibits the binding of CD14 to LPS and has an LPS-like action itself (a so-called CD14 agonist). A compound that binds to mCD14 and inhibits the binding of CD14 to LPS, but does not itself have an LPS-like effect (a so-called CD14 antagonist). A compound that does not bind to mCD14 but acts on CD14, LPS, LBP, or the like to inhibit the binding between CD14 and LPS (hereinafter, abbreviated as CD14 inhibitor compound). The presence or absence of the binding of the compound to mCD14 can be determined by a general method for measuring the binding activity between a ligand and a receptor, for example, European J
The method can be performed by the method described in ournal of Pharmacology, 1991, 208, pp. 199-205, or a method analogous thereto.
The presence or absence of an LPS-like effect can be determined by a method known per se, for example, Anti
It can be measured by the method described in Microb. Agents Chemother., 1998, Vol. 42, pp. 1015-1021 or a method analogous thereto. LPS-like actions include, for example, TNF-α,
Cytokines such as IL-1, IL-6, and IL-8;
Production inducing activity of nitric oxide (NO), superoxide, platelet activating factor, arachidonic acid cascade product and the like can be mentioned.

Therefore, the screening method of the present invention is more preferably applied to the compound selected by the above (Method A) to (Method C), furthermore, the presence or absence of the binding of the compound to mCD14 and the LPS-like action of the compound. By measuring the presence or absence of the compound, a CD14 agonist, a CD14 antagonist, a CD14 inhibitory compound or a compound that promotes the binding between CD14 and LPS (hereinafter, referred to as a compound)
CD14 promoting compound). More specifically, the present invention relates to (A) (i) labeled LPS and m
Contacting cells expressing CD14, and (ii)
Labeled LPS when contacting labeled LPS and test compound with cells expressing mCD14
The binding between mCD14 and (B) using flow cytometry (i) labeled LP
M when contacting S with cells expressing mCD14
FI value, (ii) labeled LPS and test compound and m
MFI when contacted with cells expressing CD14
Values, and (iii) labeled LPS and anti-CD14
The MFI value when the antibody was contacted with cells expressing mCD14 was measured, and the following equation: Inhibition rate (%) = (1−MFI ¹ / MFI ² ) × 100 (I) (MFI ¹ = addition of test compound MFI when added-MFI when anti-CD14 antibody was added MFI ² = MFI when test compound was not added-anti-CD14
The compound that alters the binding between LPS and CD14 is selected by determining the inhibition ratio by MFI when an antibody is added, and then TNF-α, IL
-1, IL-6, IL-8, NO, superoxide, platelet activating factor or arachidonic acid cascade product production-inducing activity is measured, and (a) a compound having the production-inducing activity is converted to a CD14 agonist or a CD14 promoting compound (B) a compound having no production-inducing activity is a CD14 antagonist or CD14
CD1 characterized by being selected as an inhibitory compound
4 is a method for screening a CD14 promoting compound, a CD14 antagonist or a CD14 inhibitory compound. In particular, a CD14 agonist, a CD14 promoting compound, a CD14 antagonist or a CD14 inhibitory compound can be efficiently screened by performing the above screening method using a compound library that can be produced by combinatorial chemistry technology as a test compound. . As the combinatorial chemistry technique, a technique known per se or a technique to be developed in the future may be used.

(4) Screening Kit The screening kit of the present invention contains the above-mentioned labeled LPS, cells expressing mCD14, and the like. Examples of the screening kit of the present invention include the following. [Reagent for screening] Buffer, etc. PBS, human serum LPS FITC-labeled LP having binding ratio (FITC / LPS) of 0.3
S-cell 1,25-dihydroxyvitamin D ₃ treated T expressing approximately 1,000,000 molecules of mCD14 per cell
HP-1 Others Anti-human CD14 monoclonal antibody (MEM18) measurement The amount of FITC-labeled LPS bound to 1,000 cells was determined by Cyto
Measure using a flow cytometer such as ACE-300.

(5) Compounds that alter the binding between LPS and CD14 Compounds that can be obtained using the screening method or screening kit of the present invention include the test compounds described above, for example, peptides, proteins, non-peptide compounds, It is a compound selected from a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract, plasma, and the like, and is a compound that changes the binding between CD14 and LPS. Examples of the derivative of the compound that can be obtained by using the screening method or the screening kit of the present invention include a compound in which any substituent in the chemical structure of the compound is deleted, and an optional substituent in the chemical structure of the compound. Addition compounds, compounds in which an arbitrary substituent in the chemical structure of the compound is converted into another substituent, prodrugs of the compound, and the like are used. A prodrug of a compound (hereinafter, sometimes abbreviated as the compound of the present invention) obtainable by using the screening method or the screening kit of the present invention can be obtained by a reaction with an enzyme, gastric acid, or the like under physiological conditions in a living body. A compound that converts to the compound of the present invention, that is, a compound that changes to the compound of the present invention by enzymatic oxidation, reduction, hydrolysis, or the like, or a compound that changes to the compound of the present invention by hydrolysis or the like due to stomach acid or the like. May be.

The prodrug of the compound of the present invention includes
Compounds in which the amino group of the compound of the present invention is acylated, alkylated or phosphorylated (eg, the amino group of the compound of the present invention is eicosanoylated, alanylated, pentylaminocarbonylated, (5-methyl-2-oxo- 1,3-dioxolen-4-yl) methoxycarbonylation, tetrahydrofuranylation, pyrrolidylmethylation, pivaloyloxymethylation, tert-butylated compound, etc.); the hydroxyl group of the compound of the present invention is acylated, Alkylated, phosphorylated, and borated compounds (eg, the compound of the present invention has a hydroxyl group acetylated, palmitoylated, propanoylated, pivaloyylated, succinylated, fumarylated, alanylated,
Dimethylaminomethylcarbonylated compound, etc.); the carboxyl group of the compound of the present invention is esterified,
Amidated compounds (e.g., the carboxyl group of the compound of the present invention is ethylesterified, phenylesterified, carboxymethylesterified, dimethylaminomethylesterified, pivaloyloxymethylesterified, ethoxycarbonyloxyethylesterified, Phthalidyl esterification, (5-methyl-2-oxo-1,3-dioxolen-4-yl) methyl esterification, cyclohexyloxycarbonylethyl esterification, methylamidated compound, and the like. These compounds can be produced from the compound of the present invention by a method known per se. The prodrug of the compound of the present invention can be prepared under physiological conditions as described in Hirokawa Shoten, 1990, “Development of Pharmaceuticals,” Vol. 7, Molecular Design, pp. 163 to 198.
It may be changed to the compound of the present invention.

The compound or derivative thereof obtained by using the screening method or the screening kit of the present invention may form a salt. As such salts, pharmaceutically and physiologically acceptable salts are used, for example, salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, basic or Salts with acidic amino acids and the like are used. Preferred examples of the salt with an inorganic base include, for example, alkali metal salts such as sodium salt and potassium salt;
Alkaline earth metal salts such as calcium salts and magnesium salts; and aluminum salts and ammonium salts are used. Preferred examples of the salt with an organic base include, for example,
Trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N'-
A salt with dibenzylethylenediamine or the like is used. Preferred examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Suitable examples of salts with organic acids include, for example, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p A salt with toluenesulfonic acid or the like is used. Preferred examples of the salt with a basic amino acid include, for example, salts with arginine, lysine, ornithine, and preferred examples of the salt with an acidic amino acid include, for example, salts with aspartic acid, glutamic acid, and the like. Can be

Among the compounds or derivatives thereof that can be obtained using the screening method or screening kit of the present invention, CD14 antagonists or CD14
The inhibitory compound is useful as an agent for preventing or treating a disease caused by the binding between LPS and mCD14. Such diseases include, for example, heart disease, autoimmune disease, inflammatory disease, central nervous system disease, infectious disease, sepsis, septic shock, etc., and more specifically, for example, sepsis , Endotoxin shock, exotoxic shock, heart failure, shock, hypotension, rheumatoid arthritis, osteoarthritis, gastritis, ulcerative colitis, peptic ulcer, stress gastric ulcer, Crohn's disease, autoimmune disease, tissue damage after organ transplantation and Rejection, ischemia / reperfusion injury, acute coronary microvascular embolism, shock vascular embolism (general intravascular blood coagulation syndrome (DIC), etc.), ischemic encephalopathy, arteriosclerosis, pernicious anemia, Fanconi anemia, Sickle cell anemia, pancreatitis, nephrotic syndrome, nephritis, renal failure, insulin-dependent diabetes, non-insulin-dependent diabetes, hepatic porphyrin , Alcoholism, Parkinson's disease, chronic leukemia, acute leukemia, tumor, myeloma, anticancer reducing the side effect, infant and adult respiratory distress syndrome, emphysema, dementia,
Alzheimer's disease, multiple sclerosis, vitamin E deficiency,
Aging, sunburn, muscular dystrophy, myocarditis, cardiomyopathy, myocardial infarction, sequelae of myocardial infarction, osteoporosis, pneumonia, hepatitis, psoriasis, pain, cataract, influenza infection, malaria, human immunodeficiency virus (HIV) infection, radiation injury, Burn, in vitro fertilization efficiency, hypercalcemia, ankylosing spondylitis, osteopenia, bone Pechet disease, osteomalacia, fracture, acute bacterial meningitis, Helicobacter pylori infection, invasive staphylococcal infection , Tuberculosis, systemic fungal infection, herpes simplex virus infection, varicella-zoster virus infection, human papillomavirus infection, acute viral encephalitis, encephalitis, asthma, atopic dermatitis, allergic rhinitis, reflux esophagitis, Fever, hypercholesterolemia, hyperglyceridemia, hyperlipidemia, diabetic complications, diabetic nephropathy, diabetic god Disorders, diabetic retinopathy, gout, gastric atony, hemorrhoids, systemic lupus erythematosus, spinal cord injury, insomnia, schizophrenia, epilepsy, cirrhosis, liver failure, unstable angina, valvular heart disease, thrombocytopenia due to dialysis , Acute ischemic stroke, acute cerebral thrombosis, cancer metastasis, bladder cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, ovarian cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, Hodgkin's disease , Non-Hodgkin's lymphoma and the like. In particular, it is useful as a prophylactic / therapeutic agent for septic shock. on the other hand,
Among the compounds or derivatives thereof that can be obtained using the screening method or the screening kit of the present invention, a CD14 promoting compound or a CD14 agonist is
For example, it is useful as a medicament such as an immunodeficiency, prevention of infectious diseases upon administration of an immunosuppressant, a therapeutic agent, and an immunostimulant.

When a compound, derivative or salt thereof obtainable by using the screening method or screening kit of the present invention is used as the above-mentioned therapeutic or prophylactic agent,
It can be carried out according to conventional means. The compounds, derivatives or salts thereof can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the compound or a derivative thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration. That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se,
It contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, carriers for tablets,
As an excipient, lactose, starch, sucrose, magnesium stearate and the like are used. As compositions for parenteral administration, for example, injections, suppositories and the like are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections and the like. Is included. Such injections are
The compound is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the compound, a derivative or salt thereof in a sterile aqueous or oily liquid commonly used for injections. Examples of aqueous liquids for injection include physiological saline,
Isotonic solutions containing glucose and other adjuvants are used,
Suitable solubilizing agents, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene)
(50mol) adduct of hydrogenated castor oil)]
And the like. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in a suitable ampoule.
Suppositories used for rectal administration are prepared by mixing the above compound, its derivative or salt with a conventional suppository base. The oral or parenteral pharmaceutical composition,
Conveniently, dosage units may be prepared in dosage units that are compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like. Usually, each dosage unit dosage form is 5 to 500 mg, especially 5 to 100 mg for injection.
mg or another dosage form preferably contains 10 to 250 mg of the above compound, derivative or salt thereof. Each of the above-described compositions may contain other active ingredients as long as the compound and its derivative or salt do not cause an undesirable interaction. Since the preparation thus obtained is safe and has low toxicity, for example, mammals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, human, etc.) Can be administered.

The dose of the compound, its derivative or its salt varies depending on its action, target disease, subject to be administered, administration route and the like. For example, a CD14 antagonist or a CD14 inhibitor compound is used for the purpose of treating septic shock. In the case of oral administration, generally an adult (body weight 60 kg
), The CD14 antagonist or CD14 inhibitor is administered in an amount of about 0.1 to 100 m / day.
g, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When the CD14 antagonist or the CD14 inhibitor is administered parenterally, the single dose varies depending on the administration subject, the target disease, and the like.
When the 14-antagonist or CD14-inhibiting compound is usually administered to an adult (as 60 kg) in the form of an injection, about 0.01 to 30 mg, preferably about 0.1 to 30 mg of the CD14-antagonist or CD14-inhibiting compound is administered per day.
About 1 to 20 mg, more preferably about 0.1 to 10 mg
It is convenient to administer the degree by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. On the other hand, when a CD14 agonist or a CD14 promoting compound is orally administered as an immunostimulant, generally, in an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg, preferably, 100 mg of the CD14 agonist or CD14 promoting compound per day is used. About 1.0-50m
g, more preferably about 1.0-20 mg. The C
The parenteral administration of the D14 agonist or CD14-promoting compound may vary depending on the administration target, the target disease, etc.
When the 14 agonist or CD14 promoting compound is usually administered to an adult (as 60 kg) in the form of an injection, about 0.01 to 30 mg, preferably about 0.1 to 20 mg of the CD14 agonist or CD14 promoting compound per day is administered.
It is convenient to administer about mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

[0025]

The present invention will be further described in detail with reference to the following reference examples and examples, which do not limit the present invention and may be changed without departing from the scope of the present invention.

[Reference Example 1] Preparation of FITC-labeled LPS To 1 mg of Salmonella minnesota Re595 LPS (Sigma) was added 500 µl of triethylamine (Wako Pure Chemical Industries, Ltd.), and the mixture was placed in an ice water bath using a multi ultrasonic cleaner VS-III (Iuchi Seieido). At 15
After sonication for 50 minutes, 50 μl of 100 mM ethylenediaminetetraacetic acid (EDTA) was added, followed by sonication for 5 minutes in an ice water bath. 22.5 μl of 1N hydrochloric acid in the reaction solution after sonication
Was added, and it was confirmed that the pH of the reaction solution was 5 or less. After allowing the mixture to stand at room temperature for 2 minutes, 1N sodium hydroxide was added to neutralize the reaction solution. To this reaction solution, a solution prepared by dissolving 5 mg of FITC isomer I (Dojinsha) in 200 μl of 0.5 M carbonate / bicarbonate buffer (pH 9.5) was added, and the mixture was again subjected to ultrasonic treatment in an ice water bath for 1 minute. After sonication, 1.6% sodium deoxycholate
(DOC; Wako Pure Chemical Industries, Ltd.) 250 μl of the solution was added, the reaction vessel was covered with aluminum foil to shield from light, and the mixture was incubated at 37 ° C. for 18 hours with stirring. After the incubation, distilled water was added to adjust the volume of the reaction solution to 2.5 ml. Sephadex G-2
PD-10 column packed with 5M (Pharmacia Biotech
The free FITC was removed by performing gel filtration using the above method. That is, after the reaction solution was applied, 3 ml of elution buffer was applied to the column, and the fraction eluted when this elution buffer was applied to the column was FITC.
A fraction containing labeled LPS was used. As a buffer for equilibration and elution, Dulbecco's PBS (without Mg ²⁺ and Ca ²⁺ ; Dainippon Pharmaceutical Co., Ltd.) to which DOC was added to a final concentration of 0.16% was used. FITC of the obtained FITC-labeled LPS
When the amount was measured using a fluorometer Fluoroskan II (Labsystems) at an excitation wavelength of 485 nm and a measurement wavelength of 538 nm, LP
36.18 μg of FITC was bound per mg of S. FI
TCisomer I has a molecular weight of 389.4, S. minnesota Re595 L
Since the molecular weight of PS is 3,400, the prepared FIT
The binding ratio between FITC and LPS of the C-labeled LPS was about 1: 3.

[0026]

Reference Example 2 Induction of CD14 Expression in THP-1 Cells As cells, human monocyte THP-1 cells (purchased from Dainippon Pharmaceutical Co., Ltd.) were used. Medium is RPMI medium 1640 with Lg
Inactivated fetal bovine serum (GIBCO) was added to lutamine (RPMI; GIBCO).
At a concentration of 10%, penicillin-streptomycin (G
IBCO) were added as penicillin G sodium and streptomycin sulfate to give concentrations of 100 U / ml and 100 μg / ml, respectively. CD14 expression induction is 3 × 1
0, ^25- cells were added to cells suspended in
Dihydroxyvitamin D ₃ (VD ₃ ; Roussel Uclaf) was added to a final concentration of 100 nM, and the CO ₂ incubator MO was added.
5 using DEL 6200-03 (National appliance company)
This was performed by culturing at 37 ° C. for 3 days in the presence of% CO ₂ .
The cultured cells were suspended in PBS at 10 ⁶ cells / ml, and the FITC-labeled anti-human C was added to the cell suspension at a concentration of 1.6 μg / ml.
D14 monoclonal antibody clone.UCHM1 (Ancell) was added and incubated at 4 ° C. for 1 hour. The cells are washed three times by centrifugation (1,000 rpm, 3 minutes, 20 ° C) using PBS.
Suspend in S and filter tube (FALCON2235 [Becton
Dickinson]). For the obtained cell suspension, the amount of the FITC-labeled anti-CD14 antibody bound to 10,000 cells was measured using a flow cytometer Cyto ACE-300 (JASCO Corporation).
It measured using. FACS Flow (Becton
Dickinson) was used. The obtained average fluorescence intensity (Mean f
The value of luorescence intensity [MFI]) is VD ₃ addition CD1
4 Expression induced THP-1 cells at 114.9, VD ₃ not added THP-
It was 40 per cell. This value is the amount of FITC-labeled anti-CD14 antibody bound to 10,000 cells, ie, CD14.
It is considered to correspond to the expression level. Therefore, the amount of clearly CD14 expression was increased by VD ₃ addition.

[0027]

Example 1 CD14 Expression-Induced THP-1 Cells and F
Screening of CD14 antagonist using ITC-labeled LPS (1) FIT using CD14 expression-induced THP-1 cells
Measurement of C-labeled LPS binding inhibitory activity CD14 expression-induced THP-1 cells were centrifuged three times using PBS
(1,000 rpm, 3 minutes, 20 ° C) After washing, the final concentration of cells is 6.67
× 10 ⁶ cells / ml suspended in PBS, 150 μl each in a multi-filtration system 96-well plate (Millipore
Was dispensed into each well of the company) (final concentration 10 ⁶ cells / well). Test compounds dispensed each well 20 [mu] l, 5% CO ₂ presence 37
Preincubated for 15 minutes at ° C. PBS was used to measure the maximum binding amount, and PBS was used to measure the specific binding amount to mCD14.
Anti-human CD14 monoclonal antibody clon diluted 10 times with
e.MEM18 (SANBIO) was dispensed at 20 μl each. After pre-incubation, FITC-labeled L at a concentration of 0.25 μg / ml
20 μl of PS (final concentration 5 ng / 10 ⁶ cells), 10 μl of human serum (Bio Whittaker) diluted 10 times with PBS was dispensed to each well by 10 μl (final concentration 1%), and in the presence of 5% CO ₂ , Incubated at 37 ° C. for 1 hour. Cells are washed in PBS with a multi-screen filtration system vacuum manifold (Millipo
After washing three times using Re. Co., Ltd., the cells were suspended in 300 μl of PBS and filtered through a filter tube. The amount of FITC-labeled LPS bound to 1,000 cells of the obtained cell suspension was measured using a flow cytometer Cyto ACE-300.
FACS Flow was used as an elution buffer. Obtained MFI
The value was defined as the amount of FITC-labeled LPS bound to the cells, and the activity of each test compound to inhibit the specific binding between mCD14 and FITC-labeled LPS was determined by the above formula (I) based on the measured value. (2) Measurement of TNF-α production inhibitory activity using THP-1 cells THP-1 cells were centrifuged three times using RPMI (1,000 rpm, 3 rpm).
After washing, the cells were suspended in PBS so that the final concentration of the cells was 6.67 × 10 ⁶ cells / ml, and 150 μl was dispensed into each well of a multifiltration system 96-well plate (Millipore). (final concentration of 10 ⁶ cells / well). 20 μl of test compound
Each well was dispensed to each well and pre-incubated at 37 ° C. for 15 minutes in the presence of 5% CO ₂ . 20 μl of RPMI was dispensed into control wells. After preincubation, a concentration of 0.25 μg / ml Es
20 μl (final concentration 5 ng / 10 ⁶ cells) of cherichia coli O111: B4 LPS (Sigma), human serum (Bio W
10 μl (final concentration 1%) was dispensed into each well and incubated at 37 ° C. for 4 hours in the presence of 5% CO ₂ .
After the incubation, the culture supernatant was recovered using a multi-screen filtration system vacuum manifold (Millipore). TNF-α in the obtained culture supernatant
The amount was measured using an ELISA kit for human TNF-α measurement (Genzyme). (3) Cytotoxicity test using THP-1 cells THP-1 cells were centrifuged three times (1,000 rpm, 3 minutes, 20 ° C.) using RPMI supplemented with 10% inactivated fetal bovine serum, and then the final concentration of the cells was determined. Was suspended in the same medium at a concentration of 10 ⁶ cells / ml, and 90 μl was dispensed into each well of a 96-well microwell plate (ICN) (final concentration 9 × 10 ⁴ cells / well). 10 μl of the test compound was dispensed into each well and incubated at 37 ° C. for 24 hours in the presence of 5% CO ₂ . 10 μl of RPMI was dispensed into control wells. After the incubation, the presence or absence of cytotoxicity was examined using a Cell counting kit (Dojinsha). That is, 10 μl of the attached color reagent was dispensed, incubated at 37 ° C. for 3 hours in the presence of 5% CO ₂ , and the absorbance was measured at a measurement wavelength of 450 nm and a reference wavelength of 620 nm.

The binding inhibitory activity of the ten compounds was measured at a concentration of 2 μM using the above method (1). The results shown in Table 1 were obtained. From Table 1, it was confirmed that Compounds 1 to 10 have strong binding inhibitory activity on the binding between LPS and CD14.

Further, the results obtained by measuring the in vitro activities of the three compounds using the above methods (1) to (3) are shown in Table 2. [Table 2] Compound No. FITC-labeled LPS binding inhibition TNF-α production inhibition cytotoxicity IC50 value IC50 value IC50 value A 4.39 μM 4.79 μM> 10 μM B 4.39 μM 10.39 μM> 10 μM C 4.26 μM 9.21 μM> 10 μM From Table 2, It was confirmed that Compounds A to C had a strong binding inhibitory activity on the binding between LPS and CD14 and also had a TNF-α production inhibitory activity, ie, a so-called CD14 antagonist activity.

[0030]

The screening method and the screening kit of the present invention are useful for screening a compound that changes the binding between LPS and CD14, and a compound or a compound that changes the binding between LPS and CD14 that can be obtained by the screening. The derivative is useful as a prophylactic / therapeutic agent for septic shock or an immunostimulant.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 G01N 33/53 Y 33/566 33/566

Claims (29)

[Claims] 1. Labeled lipopolysaccharide and membrane-bound CD1
4. A method for screening a compound that alters the binding between lipopolysaccharide and CD14, comprising using a cell that has expressed No.4. 2. The screening method according to claim 1, wherein the lipopolysaccharide is fluorescein isothiocyanate, biotin, an enzyme or a lipopolysaccharide labeled with a radioisotope. 3. The lipopolysaccharide according to claim 1, wherein the lipopolysaccharide is a rough lipopolysaccharide.
The screening method as described above. 4. The screening method according to claim 1, wherein the lipopolysaccharide is a lipopolysaccharide labeled with fluorescein isothiocyanate. 5. A lipopolysaccharide labeled with fluorescein isothiocyanate having a binding ratio (fluorescein isothiocyanate / lipopolysaccharide) of about 0.03 to about 1.0, and high expression of membrane-bound CD14. The screening method according to claim 1, wherein cells are used. 6. The method according to claim 1, wherein the membrane-bound CD14 per cell is about 100,
6. The screening method according to claim 1, wherein cells expressing at least 000 molecules are used. 7. The method according to claim 1, wherein the cells are 1,25-dihydroxyvitamin D.
The screening method according to claim 1, wherein the cells that are induced expression of membrane-bound CD14 by _3. 8. The screening method according to claim 1, wherein the cells are mononuclear cells or polymorphonuclear cells. 9. The screening method according to claim 1, wherein the cells are monocytes, macrophages or neutrophils. 10. The screening method according to claim 1, wherein the cells are human cells. 11. The screening method according to claim 1, wherein the cells are THP-1. 12. About 1 ng to about 3 cells per about 10 ⁶ cells.
The screening method according to claim 1, wherein 0 ng of the labeled lipopolysaccharide is used. (I) labeled lipopolysaccharide and membrane-bound C
And (ii) labeled lipopolysaccharide and a test compound and membrane-bound CD14.
2. The screening method according to claim 1, wherein the binding between the labeled lipopolysaccharide and the membrane-bound CD14 is measured when the cell is brought into contact with a cell that has expressed. 14. The method according to claim 14, wherein the flow cytometry method is used.
(I) Mean fluorescence intensity (Mean fluor) when the labeled lipopolysaccharide is brought into contact with cells expressing membrane-bound CD14
luminescence intensity (MFI)) value, (ii) MFI value when the labeled lipopolysaccharide and test compound are brought into contact with cells expressing membrane-bound CD14, and (iii)
Labeled lipopolysaccharide and anti-CD14 antibody and membrane-bound C
The MFI value was measured when the cells were brought into contact with cells expressing D14, and the following formula (I) inhibition rate (%) = (1−MFI ¹ / MFI ² ) × 100 (I) (MFI ¹ = MFI when added-MFI when anti-CD14 antibody was added MFI ² = MFI when test compound was not added-anti-CD14
A method for screening a compound that alters the binding between lipopolysaccharide and CD14, wherein the inhibition rate is determined by MFI when an antibody is added. 15. The screening method according to claim 1, wherein the compound that alters the binding between lipopolysaccharide and CD14 is a CD14 antagonist. 16. The screening method according to claim 1, wherein the compound that alters the binding between lipopolysaccharide and CD14 is a CD14 agonist. 17. A CD14 agonist or CD, which is for determining the presence or absence of a lipopolysaccharide-like action of a compound obtained by the screening method according to claim 13 or 14.
14 antagonists, compounds that do not bind to membrane-bound CD14 but act on lipopolysaccharide, CD14 or lipopolysaccharide binding protein to inhibit the binding of lipopolysaccharide to CD14 or compounds that promote the binding of lipopolysaccharide to CD14 Screening method. (18) the lipopolysaccharide-like action is TNF-α, IL-
1, IL-6, IL-8, NO, superoxide,
The screening method according to claim 17, wherein the method is production of a platelet activating factor or an arachidonic acid cascade product. 19. Labeled lipopolysaccharide and membrane-bound CD
A screening kit for a compound that alters the binding between lipopolysaccharide and CD14, comprising a cell in which 14 has been expressed. [20] a compound or a derivative thereof which alters the binding property between lipopolysaccharide and CD14, which can be obtained by the screening method according to [1]; 21. A lipopolysaccharide, CD14 or a lipopolysaccharide-binding protein which does not bind to a CD14 agonist, a CD14 antagonist, or a membrane-bound CD14 obtainable by the screening method according to claim 17. A compound that inhibits the binding of lipopolysaccharide to a compound or a derivative thereof that promotes the binding between CD14 and lipopolysaccharide. (22) A prophylactic / therapeutic agent for septic shock, comprising a compound capable of changing the binding property between lipopolysaccharide and CD14 or a derivative thereof obtainable by the screening method according to (1). 23. The agent for preventing or treating septic shock according to claim 22, wherein the compound is a CD14 antagonist. 24. The septic according to claim 22, wherein the compound does not bind to membrane-bound CD14 but acts on lipopolysaccharide, CD14 or lipopolysaccharide binding protein to inhibit the binding of lipopolysaccharide to CD14. Shock prevention
Therapeutic agent. 25. An immunostimulator comprising a compound capable of changing the binding property between lipopolysaccharide and CD14 or a derivative thereof obtainable by the screening method according to claim 1. 26. The immunostimulant according to claim 25, wherein the compound is a CD14 agonist. 27. The immunostimulant according to claim 25, wherein the compound promotes the binding between lipopolysaccharide and CD14. (A) (i) contacting a labeled lipopolysaccharide with cells expressing membrane-bound CD14;
(Ii) contacting the cells expressing the membrane-bound CD14 with the labeled lipopolysaccharide and the test compound,
Measuring the binding property between the labeled lipopolysaccharide and membrane-bound CD14, or (B) using flow cytometry to contact (i) the cell that expressed membrane-bound CD14 with the labeled lipopolysaccharide Average fluorescence intensity (Mean f
luorescence intensity (MFI)) value, (ii) MFI value when the labeled lipopolysaccharide and test compound are brought into contact with cells expressing membrane-bound CD14, and (ii)
i) MFI when contacting labeled lipopolysaccharide and anti-CD14 antibody with cells expressing membrane-bound CD14
The values were measured and the following formula: Inhibition rate (%) = (1−MFI ¹ / MFI ² ) × 100 (I) (MFI ¹ = MFI when test compound was added−MFI when anti-CD14 antibody was added MFI MFI ² = MFI without test compound added-anti-CD14
The compound that alters the binding between lipopolysaccharide and CD14 is selected by determining the inhibition rate by MFI when an antibody is added, and then TNF-α, I
L-1, IL-6, IL-8, NO, superoxide, platelet activating factor, or arachidonic acid cascade product production-inducing activity was measured, and (a) a compound having the production-inducing activity was identified as a CD14 agonist or lipopolysaccharide. Selected as a compound that promotes the binding of
(B) as a compound that does not bind to the CD14 antagonist or membrane-bound CD14 but acts on lipopolysaccharide, CD14 or lipopolysaccharide binding protein to inhibit the binding between lipopolysaccharide and CD14 A compound that promotes the binding between CD14 agonist, lipopolysaccharide and CD14,
A method for screening a compound that does not bind to a CD14 antagonist or membrane-bound CD14 but acts on lipopolysaccharide, CD14 or a lipopolysaccharide binding protein to inhibit the binding of lipopolysaccharide to CD14. 29. A compound library which can be produced by a combinatorial chemistry technique.
CD selected using the screening method described in Item 8
14 agonist, compound promoting binding of lipopolysaccharide to CD14, CD14 antagonist, membrane-bound C
A compound or derivative thereof that does not bind to D14 but acts on lipopolysaccharide, CD14 or lipopolysaccharide-binding protein to inhibit the binding between lipopolysaccharide and CD14.