WO2004081230A1 - Method of evaluating activity of function factor reflecting physiologically active structural function and utilization of the same - Google Patents

Abstract

A method of evaluating activity in vitro which is consistent with activity expression in vivo and utilization of the method in drug discovery. Namely, a method of evaluating activity reflecting a physiologically active structural function and utilization of this method in drug discovery, for example, the production of an inhibitor using an inhibitory activity indicator in a test using the method. More specifically speaking, a method of evaluating the activity of IVA-cytosolic phospholipase A (2) reflecting its physiologically active structural function; utilization of this method in drug discovery, for example, the production of an inhibitor using an inhibitory activity indicator in a test using the method; utilization of a difference in sensitivity in a method of evaluating activity reflecting a physiologically/artificially false active structural function in the production of a physiologically active cPLA (2)-selective inhibitor; and so on.

Description

 Specification

 Method for evaluating activity of functional factor reflecting physiologically active structural function and use thereof

Technical field

 The present invention relates to an activity evaluation method reflecting physiologically active structural functions, an inhibitor of an activity indicator in a test using the method, use of the method for creating an activator, and activity in a test using the method. Inhibitors to be evaluated and activators, evaluation methods using the difference in sensitivity in each activity evaluation method reflecting physiological / artificial pseudo-active structure function as an index, physiologically active form of the index Use of structure-function selective inhibitors and activators for the creation of physiologically active structure-function selective inhibitors and activators evaluated by their indicators, and use of those inhibitors and activators More specifically, the present invention relates to the above-mentioned method or the like in which the functional factor is VA—cytoplasmic phospholipase A (2) (hereinafter abbreviated as cPLA (2)).

Background art

 Phospholipase A (2) (hereinafter abbreviated as PLA (2)) preferentially hydrolyzes fatty acids bound to the sn-2 position of the glycerol skeleton of daricerophospholipid, a major constituent of biological membranes A generic term for enzymes. ―

 In recent years, more than 19 new isozymes have been discovered in PLA (2) one after another, and it has been reported that each isozyme has different characteristics and shares functions (Six, DA & Dennis, Ε. Biochimica et Biophysica Acta 1488: 1-19 (2000)., Balsinde J., Winstead MV, & Dennis EA FEBS Letters 531: 2-6. (2002)). Among these, c PLA (2) showed high specificity for arachidonic acid-linked phospholipids, increased activity in inflammatory diseases, and found that gene-deficient mice (Uozumi, N. et al., Mure 390: 619-22 (1997)., Bonventre, JV et al., Nature 390: 622-25 (1997).) Is expected as a high drug discovery target.

c PLA (2) is abundantly expressed in the cytoplasm, and its hydrolysis activity is much weaker than that of the physiologically active enzyme due to the interaction with the phospholipid membrane substrate depending on the increase in ^{Ca 2+} concentration. Fortunately, purification and isolation were successful. Thereafter previously c PL A (2) is to understand the complex activity control mechanism two prism, non-dependent on the C a ²⁺ concentration increased by utilizing a biochemical technique and information, including genetic handling techniques Cell lines and cell test systems have been constructed, and screening for inhibitors has been energetically carried out.However, artificial false activities with low similarity to the functional structure of such physiologically active enzymes are Activity against enzymes in an evaluation system using an evaluation system that reflects sex type enzyme activity and in WO pharmacology The association with activity was low.

 The present inventors, using U933 cells, a fluorescently labeled phospholipid 1,2-bis- (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-5 -indecene-3-undecanoyl)-A glycero-3- phosphocholine (hereinafter abbreviated as bis-BODIPY-PC, Molecular Probes) as an index for screening a PLA (2) inhibitor using a cell-based assay. An evaluation method to be performed has been found (WO 01/072723, WO 03/000668, Japanese Patent Application No. 2001-307301). However, although this method is an evaluation method superior to the conventional test method, there is a difference in sensitivity of the compound, and the evaluation method is not sufficiently correlated with the in′ΤΌ anti-inflammatory activity and includes noise. Disclosure of the invention

The present inventors have conducted intensive studies for the purpose of solving the above-mentioned problems, and as a result, the present inventors have found that U937 cell Hydrolysis of bis-BODIPY-PC substrate added to cells is independent of elevated C a2 ⁺ concentration and is related to physiological conditions such as production of inflammatory lipid mediators in cells c PLA (2) Was found to be dependent on the activation state. C a ^{2 +} In Many cell culture conditions used in the evaluation method, etc. depending on the concentration increase is not observed highly inflammatory lipid Mede Isseki One class production promoting conventional contrast, bis- BODIPY- The hydrolysis activity of the PC substrate was also found to be low.

 The present inventor has proposed an IV A—cytoplasmic phospholipase A (2) inhibitory activity and an artificial indicator that reflects this physiologically active structural function and an indicator of VA—cytoplasmic phospholipase A (2) inhibitory activity. The present inventors have found that a method for evaluating VA-cytoplasmic phospholipase A (2) inhibitory activity has a good correlation with i'fl n'TO pharmacological activity, thereby completing the present invention.

 That is, the present invention is shown in the following 1. to 4.

 1. (a) reflecting physiologically active structural function] VA—a step of obtaining an inhibitory activity index in the method for evaluating the activity of cytoplasmic phospholipase A (2),

 (b) Reflects artificial pseudo-active structural functions with low similarity to physiologically active structural functions] VA—Inhibition activity index in the method for evaluating the activity of cytoplasmic phospholipase A (2) The step of obtaining

(c) obtaining a selectivity index calculated by comparing (a) and (b),

A physiologically active form] VA—a method for evaluating cytosolic phospholipase A (2) activity.

2. (a) reflecting the physiologically active structural function] VA—a step of obtaining an inhibitory activity index in the method for evaluating the activity of cytoplasmic phospholipase A (2), (b) Reflects artificial pseudo-active structural function with low similarity to physiologically active structural function] VA—obtains an inhibitory activity index in the method for evaluating the activity of cytoplasmic phospholipase A (2) Process,

(c) obtaining a selectivity index calculated by comparing (a) and (b),

2. The evaluation method according to claim 1, wherein the ratio of the selectivity index in the evaluation method characterized by including 10 is 10 times or more.

3. In the step (a), the production of PGE ₂ and / or LTC ₄ and / or PAF is enhanced 10 times or more, and the production of Z or active oxygen is enhanced 10 times or more, and / or the activity 3. The evaluation method according to claim 1 or 2, wherein a human leukemia cell line U937 differentiated cultured cell exhibiting a morphological reaction similar to that of pharmacokinetics is used.

4. The activity evaluation method according to any one of claims 1 to 3, wherein in the step (b), cells having an increase in lipid mediator production of less than 10 times are used.

 Here, the term "functional factor" as used herein means a substance which is involved in any physiological reaction by forming a complex with a biological component alone or with the same or different species. Includes proteins, nucleic acids, enzymes, receptors, transposers, rafts, etc.

 The term “structural function” means a physiological reaction involving a functional factor, and includes a catalytic reaction, interaction such as binding, movement, and the like.

 The term “physiological activation” refers to enhancement of the function of a functional factor caused by various stimuli in a physiological phenomenon of an organism, and necessarily involves a change in the functional structure.

 The term “functional molecular probe” refers to a molecule that is used as a probe to recognize a specific functional factor and / or its function.

 The term “artificial activation” refers to the enhancement of the function of a functional factor caused by a specific physiologically active reagent in an i / zW test system or the like, and is referred to as “artificial false activation”. The term means "artificial activation" that has low homology to functional structural changes associated with physiological activation and low relevance to various stimuli in biological physiological phenomena.

The term “inhibition activity index” means an index of the inhibitory activity strength of a compound in an iTi70 test system or the like, and specifically, a value such as a 50% inhibitory concentration (IC ₅₀ ) is used.

 The term “selectivity index” refers to the difference in the sensitivity of the inhibitory activity of the compound in each of the compared activity evaluation methods, and is specifically calculated as the ratio of the “inhibitory activity index” in each / '72 W0 test system. Is done.

As a method for evaluating cPLA (2) activity, any of the conventionally used measurement techniques described below is used, but as a test system that reflects `` physiological activation '' used in the present specification, reaction This refers to measurement under conditions where a "physiologically activated" state is identified, such as enhanced arachidonic acid cascade and enhanced production of PGs and LTs in a Cel-based Assay system that simulates the above. In addition, since c PLA (2) has been accumulated in various physiological reactions from various physiological studies, knowledge that it is acting as a Trigger has been accumulated. It is considered a test system that reflects “physiological activation”.

The activity of PLA (2) has long been measured using radioisotopes, chromophore or iluorophore-labeled lipids as substrates, or using a Thio-based test method, etc. (Dennis, EA ed., 'Methods in E Hana logy', 197 (1991). c) The hydrolysis activity of PLA (2) can also be determined by separating the substrate and the hydrolyzate by an appropriate method after the reaction, if necessary, and measuring the radioactivity, absorbance or fluorescence intensity based on the label on the substrate. You. The substrate phospholipid or arachidonic acid ester is often labeled with a radioisotope such as [ ³ H] or [ ¹⁴ C], which has high measurement sensitivity, or 4, 4_dif luoro-5, 7-dimet. Labeled with fluorophore such as yl-4-bora-3a, 4a-diaza-s ^ indecene-3-undecanoyl (hereinafter abbreviated as BODIPY ⁹ ) or 7-hydroxy-coumariii (Huang, Z Et al., Analytical Biochemistry, 222: 110-05 (1994).

 The arachidonic acid releasing activity by the non-phosphorylating enzyme is considered as a typical example of an artificial pseudo-active enzyme activity evaluation method having low similarity to the physiologically active functional structure.

BEST MODE FOR CARRYING OUT THE INVENTION

 Next, the present invention will be described in more detail.

 In the present specification, in order for a cPLA (2) inhibitor to exhibit i / z Fira anti-inflammatory activity, it is important that the inhibitory activity against an enzyme having a physiologically active structure is important. Based on the finding that it is more advantageous that the selectivity of the inhibitory activity in the evaluation system that reflects the physiologically active enzyme activity is higher than the inhibitory activity in the evaluation system that reflects the enzymatic activity Active C

Describes methods or techniques for screening, detecting and / or characterizing novel compounds that inhibit PLA (2), and compounds or drugs newly discovered by such methods have anti-inflammatory activity above the cellular level Will be described.

As a method for measuring a cell function accompanied by a physiological increase in the activity of the enzyme, there is a method for measuring an increase in the production of an inflammatory lipid mediator. The reported measurement of the physiological response in a known cell line was used to determine the characteristics of the cell used in the physiological / artificial pseudo-active cPLA (2) activity evaluation of the present invention. used. Physiologically active cPLA (2) In vivo pharmacological test methods with physiologically enhanced activity include: An inflammatory disease state model induced by an appropriate inflammatory stimulus is exemplified. However, models induced by arachidonic acid, a hydrolysis product of the enzyme, are not suitable as examples. The test method is used for the purpose of verifying the in-activity evaluation method, which is the subject of the present invention, with the in-n'ra activity expression. The method also includes evaluating the pharmacological activity of the PL A (2) inhibitor and Z or the anti-inflammatory compound by the test method.

 Examples of the cell-free system for evaluating the activity based on an artificial pseudo-active functional structure having low similarity to the physiologically active functional structure of the present invention include a non-phosphorylated form in an expression system such as Escherichia coli. There is a method of evaluating the hydrolysis activity of phospholipid substrates and the like in the micelle state of Triton X-100 using the prepared recombinant enzyme.

 The hydrolysis activity of cPLA (2) in a cell line is quantitatively analyzed by several measurement methods as described above, but for the purpose of measuring the physiologically active enzyme activity defined in the present invention. First, it is necessary to use cells whose enzymes have been physiologically activated. The cell line used in the test is preferably a cell line that is appropriately controlled so as to simulate the physiological phenomenon reported to be involved in the activation of cPLA (2). Measurement of status is also an indicator of the pharmacological activity associated with activation of cPLA (2) in the cell line.

 First, c PLA (2) is an enzyme at the rate-limiting step of arachidonic acid cascade. It is considered a physiologically activated state. That is, in cells in which the enzyme has not been physiologically activated, LT is not involved in the activation of the downstream arachidonic acid cascade, and the production of LTs is enhanced by COX-2 depending on the production of PGs. I can't. In contrast, in cells in which the enzyme has been physiologically activated, at least the downstream enzyme is properly coupled and the production of lipid mediators is enhanced, and the state is the production of appropriate PGs and LTs It can be evaluated by measuring the amount and the enhancement of the activity of an enzyme such as COX-2. By using cells in which the production of these lipids is markedly enhanced, it is possible to construct a cell test system that strongly reflects the activity of physiologically activated enzymes. By using cells in which no progression is observed, it is possible to construct a cell test system that has low similarity to physiologically active structural functions and reflects the activity of artificial pseudo-activated enzymes. It is possible.

The amount (or concentration) of fatty acid (or concentration) released and released (Leaked) by a non-physiological stimulus such as C a ²⁺ — ionophore in a test solution in which appropriate cells are suspended, directly or as an appropriate derivative Separation and quantitative analysis by HP LC or GC enables phospholipid hydrolysis by artificially activated enzymes Gender is measured. In particular, a test method using cells in which the enzyme is not subjected to phosphorylation evaluates the activity based on an artificial pseudo-active functional structure having low similarity to the physiologically active functional structure of the present invention. This is considered a typical example. On the other hand, when cells that reflect the hydrolysis activity of the phosphorylated enzyme are used, the activity of the physiologically active enzyme is not reflected in principle, but it is evaluated by the method of the present invention. When the activity of the physiologically active cPLA (2) selective inhibitor can be selectively evaluated with high sensitivity, the activity based on the physiologically active functional structure of the present invention is estimated. It is similar to the evaluation method suitable for

Enzyme activity evaluated by a method of measuring the release of radioactivity in response to stimulation from cells in which arachidonic acid previously labeled with [ ³ H] or [ ¹⁴ C] has been incorporated into the cells is determined by the physiological activity of the cells used. In order to reflect the sum of the hydrolytic activities of the various activated enzymes present in the cell, the enzyme activities in both the non-phosphorylated state and the phosphorylated state were evaluated. It is also possible to compare the sensitivity of the compounds to the inhibitory activity between the two. That is, the test method using cells in which the enzyme does not undergo phosphorylation evaluates the activity based on an artificial pseudo-active functional structure having low similarity to the physiologically active functional structure of the present invention. This is an example. On the other hand, an evaluation method that reflects physiologically active enzyme activity using cells under appropriately controlled physiological conditions, such as coupling to enhanced lipid mediator production, is based on the physiologically active function of the present invention. This is an example of evaluating the activity based on the structure. However, some enzymes are subject to physiological activity, and the enzyme activity in this test method reflects the sum of the hydrolysis activities of various activated states present in the cells. Therefore, caution is required in interpreting the inhibitory activity of a compound.

The measuring method using a functional molecular probe substrate in which the compatibility as a substrate differs depending on the activity of the enzyme, particularly such that it is selectively hydrolyzed to a physiologically active enzyme, is described in the present invention. It is also desirable in principle as an activity evaluation system that reflects the physiologically active structure and function of E. coli. Human monoblast-like lymphoma cell line U933 is a cell line that was originally reported to be used for the isolation of this enzyme, and it is Phorbol 12-Myristate 13-Acetate (hereinafter abbreviated as TPA). ), Activated vitamin D ₃ , pyruvamine A derivative, dimethyl sulfoxide (hereinafter abbreviated as DMSO), 2 -0- Dibutyry 1 adenosine-3 ', 5'- (eye 1 ic)- By treatment with various kinds of differentiation inducing substances such as mono-phosphate (hereinafter abbreviated as dBc AMP), cell differentiation into monocyte, macrophage or granulocyte system was observed, It is often used for the study of nervous system and arteriosclerosis.

Chemical analysis or immunoassay can be used for quantitative analysis of lipid mediators. In any measurement method, extraction, concentration, purification, and induction from biological samples, cells, or culture supernatants to be analyzed If necessary, the sample containing the target lipid mediator is prepared in a state suitable for separation and analysis. In a chemical analysis method in which quantification is performed by an appropriate detection method after separation by thin-layer chromatography, HP LC or GC, multiple lipid mediators can be separated and quantified from the same preparation sample. When measuring a specific lipid mediator, an immunoassay such as radioimmunoassenzym immunoassay has high detection sensitivity and is suitable for high-speed measurement of multiple sample samples. Example

 Hereinafter, the present invention will be described with reference to examples. These examples are not intended or intended to limit the scope of the disclosure.

 First, an anti-inflammatory pharmacological test is described as a method for evaluating the pharmacological activity of cPLA (2) inhibitors.

Pharmacological test example 1

 Mouse pinna edema induced by TP A

 Carlson, RP et al. (Agents and Actions, 17 (2): 197-204 (1985).) And Chang, J et al. (European Journal of Pharmacologic 142: 197-205 (1987).) went. That is, TPA 5 fig / 20 L dissolved in ethanol was applied to the front and back of the right auricle of an ICR male mouse (6 to 7 weeks old), and after 7 hours, the thickness of a fixed region of both auricles was digitized. The measurement was performed three times using a matic micrometer, and the average value of each measurement was calculated. The thickness of the untreated left auricle was reduced from the thickness of the right auricle to which TPA was applied, resulting in pinna edema. The anti-inflammatory activity was evaluated by applying a 0.1¾ Tween 80 / acetone solution of the test compound to the front and back of the right auricle 30 minutes after the application of TPA. As a positive control, 0.1% Tween 80 / acetone solution of Dexamethasone-2-acetate (hereinafter abbreviated as DEX-Ac) or Indomethacin was administered in the same manner as the test compound. Table 5 shows the results.

Pharmacological test example 2

Ear edema in mice induced by arachidonic acid

The method was performed with reference to the method of Chang, J et al. (European Journal of Phanmcology, 142: 197-205 (1987)). That is, 5 mg 20 L of arachidonic acid dissolved in acetone was applied to the front and back of the right auricle of an ICR male mouse (6 to 7 weeks old). The average was calculated by measuring three times using a micrometer. The thickness of the untreated left pinna was reduced from the thickness of the right pinna to which arachidonic acid was applied, resulting in pinna edema. A 0.1% Tween 80 / acetone solution of the test compound was added 30 minutes before the application of arachidonic acid and 15 minutes after the right auricle. The anti-inflammatory activity was evaluated by applying to the front and back of the sample. As a positive control, a 0.1 l Tween 80 / aceton solution of Indometliaciii was administered in the same manner as the test compound. In the pharmacology test induced by arachidonic acid, a PLA (2) hydrolysis reaction product, the physiologically active c PLA (2) inhibitor of the present invention showed anti-inflammatory activity by local administration. Did not. Table 5 shows the results.

 Next, the quantitative analysis measurement method used for the in i activity evaluation method is described.

Quantitative analysis test example 1

Quantification of PGE ₉ and LTC _± in cell culture

By centrifuging the culture solution containing various types of fractionated cells and the floating cells in the flask, adherent cells cultured in a 96-well plate should not be detached. The mixture was removed with care, and washed three times with Hanks' Balanced Salt Solution (hereinafter abbreviated as HBSS) used as a reaction buffer-0.1% bovine serum albumin (hereinafter abbreviated as BSA). The washed cells are suspended in a reaction buffer at 2 x 10 ⁶ ce 11 s / mL for suspension cells, and a 96-well plate is used so that the final reaction solution volume is 0.2 mL. The adherent cells were prepared at 0.2 mL reaction buffer / well. If necessary, TPA was added to the reaction solution to a final concentration of 10 nM, and then cultured at 37 for 1 hour to activate. Indomethacin was used as a positive control compound as an inhibitor of COX-1 and COX-2, NS-398 as a selective inhibitor of COX-2, and AA-861 as an inhibitor of 5-LOX. Formation reaction of PG £ ₂ Oyopishi Ding Ji _4, 50 M Arakidon C a ^{2 +} acid or appropriate concentration - ionophore A23187 was started by adding Stimulator such (hereinafter abbreviated as A23187), 37 The culture was continued at ° C, and terminated by ice cooling. The cells were pelleted by centrifugation at 4 V, and the concentrations of PGE ₂ and LTC _{4 in} the supernatant were determined using the enzyme immunoassay kit (Cayman Chemical), either directly or after appropriate dilution in the reaction buffer. Quantified. Quantitative analysis test example 2

PL A (2) activity in cell lines by fluorescence method

PLA (2) activity was measured by quantifying the fluorescent substance generated by carohydrolysis of bis-BODIPY-PC using appropriate cells. The cells were collected by centrifugation, washed with a reaction buffer (Dulbecco's Phosphate buffered saline (hereinafter abbreviated as PBS) -2.2 mM glucose-2.5 nM MBSA) 2-3 times, and suspended. If necessary, TPA was added to the reaction buffer to a final concentration of 10 nM, and then cultured at 37 ° C for an additional hour to activate. (Rzigalinski, BA & Rosenthal, MD, Biochimica et BiophysicaActa 1223: 219-225 (1994); and Goncliar, MV, Biochemical and Biophysical Research Co., imkation, 249: 829-832 (1998). The substrate ribosome suspension is prepared by mixing bis-BODIPY-PC and phosphatidylserine in a form-form solution so as to have a molecular number ratio of 1: 9, and evaporating the organic solvent under a stream of nitrogen to dryness. The reaction buffer was added to 100 μg / mL, and the mixture was sonicated for 1 hour under ice-cooling from light.

The test was performed by adding the 6x10 ⁶ cells / mL suspension described above to a 96-well microplate (Corning 3603), adding the substrate ribosome suspension to 25 wells, and mixing with the reaction solution in a total of 75 iLZ wells. The cells were cultured at 37 with shading for 30 minutes. 1.5 M A2 3 187 was added to the above reaction solution as needed. For the reaction, 0.01 ^ Ethylene Glycol Bis (j8-aminoethyl ether) -N, N, N, eir && cei \ c Acid (hereinafter abbreviated as EGTA) Dispense 100 L / well of methanol solution and mix. And stopped. Fluorescence intensity based on the hydrolysis product of the enzyme is measured using S PECTRA FLUOR PLUS (TECAN), and the fluorescence intensity at 535 nm (Relative Fluorescence Unit, hereinafter abbreviated as RFU) with 485 nm excitation light is measured by upward photometry. did. In each test, the reaction zone containing no cells was set as the B 1 ank zone, and the average value of the fluorescence intensities was used as the B 1 ank value.The PL A (2) activity of each test zone was calculated for each well. It was determined by subtracting the B 1 ank value from the fluorescence intensity.

Quantitative analysis 3

[Release of ³ H3 arachidonic acid from cultured cell lines

Arachidonic acid to each well of a variety of differentiated flask with cells and cultured or 24-well play Bok, [5, 6, 8, 9, 11, 12, 14, 15 - ³ transliteration - (PerkinElmei \ 7844 GBa / mmoL 3.7 cut off M) so as to have a 10 per ¹⁰⁶ cells 3. 7KBq, continued overnight culture was added to the evening day before the final day of culture, it was incorporated radioactive fatty acid in equilibrium. However, for cells that have been activated and differentiated such as TPA, replace them with a culture solution containing no activator after the activation and differentiation treatment, add radioactive fatty acids, and culture overnight. Continued. Centrifuge the culture of cells that have taken up [ ³ H] arachidonic acid overnight, and centrifuge the suspended cells in the flask so that the adherent cells cultured in the 24-well plate are not removed. The mixture was removed with care, and washed three times with HBSS-0.1% BSA used as a reaction buffer. The washed cells are dispensed in a volume of 5 × 10 ⁵ ce 11 sZ tube, in the case of floating cells, and in a final reaction solution volume of 0.5 mL / tube.In the case of adherent cells, the final reaction solution volume is 0. Dispense the reaction buffer so that the 5 mL Was. If necessary, TPA was added to each reaction solution to a final concentration of 1 OnM, and then cultured at 37 for 1 hour to activate. The [ ³ H] arachidonic acid release reaction was started by adding an appropriate concentration of A23187 and stopped by cooling on ice. For suspended cells, centrifuge at 4 to pellet the cells. For adherent cells, take 200 from 500 L of the supernatant and pay attention to the liquid scintillation counter ( Hereinafter, abbreviated as LSC). 200 L of the lysate 6 obtained by adding 300 L of a 2% aqueous solution of TrUonX-100 to 300 L of the residual solution containing cells was mixed, and the radioactivity was measured by LSC. From these measured values of radioactivity, the "free radioactivity" and "total radioactivity incorporated" for each treatment were calculated, and the free activity was calculated as "free radioactivity" It was calculated as the ratio of free radioactivity (%).

 Next, a test example of the culture of U937 cells for controlling the physiologically active state is described. Cell culture test example 1:

U937 cell culture and differentiation induction

The cells were cultured under the conditions of 37 ° C. and 5% CO ₂ . Each test was performed while confirming that the cells exhibited stable proliferation and differentiation reactivity during the period of 3 to 14 weeks after freeze-thawing.

[I] Subcultured, undifferentiated cells

U937 cells are subcultured every 3 to 4 days in RPMI 1640 culture medium supplemented with 10% inactivated fetal bovine serum (hereinafter abbreviated as i FBS) in RPMI 1640 culture medium. Was done. The non-differentiated cells TP A very low Katsuta although differentiated gently reaction proceeded even the production of PGE ₂ by LTC ₄ production and C OX 2 for processing.

 [Π] DM SO differentiated cells 1

When the undifferentiated cells of [I] were cultured for 48 hours under the same culture conditions as in [I] under the conditions of the final concentration of 0.16M DMS O in the culture solution, no clear morphological change was observed and remarkable adhesion was observed. Although not shown, the growth rate was reduced, and the cells were stimulated with A23187 to promote the activity of hydrolyzing arachidonic acid from phospholipids into cells with enhanced activity. The minute I human cells low reactivity enhancement for TPA activation process, the production of P GE ₂ by hyperactivity and C OX 2 of LTC ₄ production was also very low.

 [m] DM SO differentiated cells—2

After culturing the undifferentiated cells of [I] for at least 1 day in a Tissue Culture Treated flask in RPMI 1640 ± 咅 nutrient solution supplemented with 10% fetal calf serum (hereinafter abbreviated as FBS, Sigma F2442) Cell density under culture conditions of 0.16M DMS〇, cell density 2 ~ 8xl0 ⁵ ce 11 When culturing was continued for 96 to 144 hours while controlling to s / mL, the growth rate decreased, and the cells were differentiated into adherent Mactophage cells. The differentiated cells causes morphological change in the very strong cell adhesion react rapidly with respect to TPA treatment, very high or the like enhancement of PGE ₂ production by LTC ₄ production and COX 2, preferred characteristics as an inflammatory cell model showed that.

[IV] TP A cell

[I] non-differentiated cells with 10% 83 added? 1 After pre-incubation for at least 1 day using a Tissue Culture Treated flask in 1640 culture medium, the cell density was adjusted to 2.5 × 10 ⁵ ce 11 sZmL under the conditions of 10 nM TPA in the culture medium. After dispensing 0.2mL / well into a 96-well microplate or 1.OmLZ hole into 24-well microplate and continuing culture for 48-72 hours, it was dissociated into macrophage cells showing strong adhesion. However, enhancement of PGE ₂ production was inferior to that of [、], and no enhancement of LTC ₄ production was observed. When lipopolysaccharide and inflammatory cytokine were added to the differentiated cells and the culture was further continued, remarkable activity was observed.

[V] dBcAMP differentiated cells

After pre-incubation of the undifferentiated cells of [I] in RPMI 1640 culture medium supplemented with 10% FBS using Tissue Culture Treated flask for at least one day, the cell density was 2.5 X under the conditions of final concentration ImM dBcAMP in the culture medium. Dispense the solution prepared to give 10 ⁵ ce 11 s ZmL into a 96-well microplate with 0.2 mL / well in each well or into a 24-well microplate with 1.OmL no-well and continue culturing for 48 to 72 hours. The cells differentiated into granulocytic cells exhibiting somewhat strong adhesion. Next, test examples that characterize the method for evaluating cPLA (2) activity according to the present invention will be described.

PLA (2) activity in cell lines based on fluorescent substrate hydrolysis

The above cell culture test example 1 [I]-[! Q] were collected by centrifugation, and PL A (2) activity was measured according to the method of Quantitative Analysis Test Example 2. For activation, 10 nM TPA was added as necessary before the measurement, and the culture was continued for 1 hour. For the purpose of observing the effect of the increase in the concentration of C a2 ⁺ , a group to which A23187 was added and a group to which no A23187 was added were set so that the final concentration was 1 M in the reaction solution. For example, cells under various differentiation activity conditions exhibited hydrolysis activity as shown in Table 1. Quantitative analysis using activated TPA-treated differentiated cells with activated Macguchi phage-like morphological characteristics and highly enhanced PGE ₂ and LTC ₄ production under Tangling conditions [ΠΙ] Cell-based Assay systems 11 and 12 whose c PLA (2) activity was measured by the method of analytical test example 2 are suitable as a method for measuring the physiologically active c PLA (2) activity of the present invention. Indicated. The method of Quantitative Analysis Test Example 2 CPL A (2) activity was not affected by A23187 treatment (increased Ca ²⁺ concentration), but was dependent on cell differentiation and activation status.

Table 1

* TPA¾ttft: + (1 hour MTPA activation treatment 1 hour),-(no TPA activation treatment) "A 23187 Addition in reaction solution: + (1! 丄 Add MA 23 187), 1 (No addition of A 23 187 ) *** PGE ₂ / LTC ₄ production: — «10 nmole / hole), Soil (10 15 nmole / hole), + (15 30 nmole / hole), ++ (30 100 nmoleoleZ hole), + + + (> 100 nmoleZ hole)

PL. A (2) activity indicated by the release of [ ³ H] arachidonic acid from cultured cell lines

The above cell culture test example 1 [I] [! The unbranched I spoon and differentiated cells V], collected by centrifugation, respectively, indices for radioactivity ^[3 Eta] Arakidon acid liberated in a final concentration of 3 Μ Α23187 15 minutes after the addition according to the method of quantitative analysis Test Example 3 And the PL A (2) activity was measured. Activation was performed by adding 10 nM TPA as necessary and culturing for 1 hour before measurement. For example, cells under various differentiation activity conditions exhibited hydrolysis activity as shown in Table 2. The activity measurement by Quantitative Analysis Test Example 3 was carried out in the Cell-based Assay System 12-2 using cells obtained by activating TPA-treated differentiated cells under the same differentiation conditions [m] as described above. It was shown that the method of the present invention can be used as a method for measuring physiologically active PLA (2) activity. In addition, the cell-based Assay system 6-2, which uses cells that have not been activated by TPA to differentiate cells under differentiating conditions [Π], which has been widely used, has low similarity to the physiologically active form of the present invention. However, it was shown to be suitable as a measurement method reflecting the activity of artificially activated PLA (2). Table 2

* TP A activated: + (10 nMTPA activation treatment 1 hour), - (TP A Non activation) "PGE ₂ ZLTC ₄ Production: A« 10 nmole / well) and (. 10 to 15 nmole / well) , + (15 '30 nmole / well), ++ (30-100 nmole / well), +++ (> 100 nmole / well) Test compound and in F'TO anti-inflammatory activity

In order to examine the selectivity of the physiologically active type artificially active type c PLA (2) inhibitory activity, which is compatible with the in v o anti-inflammatory activity according to the present invention, WO 01 / Three typical compounds were selected from the compounds listed in 072723 and WO 03/000668. Known PLA (2) inhibitors and anti-inflammatory active compounds were also purchased or synthesized. Table 3 shows these compounds. The possible compounds were evaluated for their anti-inflammatory activity by the test method of Pharmacological Test Example 1. Further, the pharmacological activity was evaluated by the test method of pharmacological test example 2 as necessary, in order to confirm that it was not in Fiw anti-inflammatory activity based on the effect of the downstream arachidonic acid cascade on the enzyme amount. Table 4 shows the results of the In Wra pharmacological test.

Table 3—

第 ΰ 2¾^— 2

Chapter ΰ 2¾ ^— 2

第 4表

Table 4

 * N.D .: PL A (2) inhibitory activity in cultured cell lines by untested compounds

The test compound was dissolved in DMSO and diluted with DMSO or each reaction buffer so that the final concentration of DMSO in the reaction solution in each test was 0.1% or less. The compound solution was added 10 to 15 minutes before the start of the reaction in each test using the cultured cells, and the cells were cultured at 37 ° C. As a positive control Arachidonyl Trif luoromethyl Ketone (hereinafter, abbreviated as AACOC F ₃₎ was used. For comparison, Bromoenol lactone (hereinafter abbreviated as BEL), an inhibitor of V_i PLA (2), was used. The activity of the inhibitor was calculated by the following formula as a comparison with the control group to which no inhibitor was added (hereinafter abbreviated as a control group).

 Inhibition rate () = (control plot data-inhibitor treatment plot data / control plot data) x 100 Perform multiple concentration treatment tests for each inhibitor and use the logarithmic concentration to determine the inhibition rate at each concentration.

(%) Was plotted on a graph to determine a 50% inhibitory concentration (IC ₅₀ ) value.

As a system for evaluating the physiologically active cPLA (2) inhibition according to the present invention, each of the conjugates was evaluated using the above-described Celt based Assay system 12 for quantitatively measuring the hydrolytic activity of the fluorescent substrate. The inhibitory activity was examined. As an artificial false active cPLA (2) inhibition evaluation system with low similarity to conventional physiologically active forms, the above-mentioned [ ³ H] arachidonic acid free radioactivity is quantitatively measured. Cell based Assay system 6-2 was performed. For the purpose of comparison with these, the cell culture conditions were the same as for the Cell-based Assay System 12 and the Cell-based Assay System 12-2, which quantitatively measures the free radioactivity of [ ³ H] arachidonic acid, was also examined. Added debate. In each test system calculates the 50% inhibitory concentration IC _5Q a (FIM), in any physiological to inhibitors sensitivity in a test system that reflects the enzyme activity, test system that reflects the artificial fake enzymatic activity The ratio of inhibitor sensitivity was calculated and used as the selectivity index.

 For example, PLA (2) P, which has anti-inflammatory activity in pharmacological test example 1 or has been reported for in Wra anti-inflammatory activity, is listed in Table 5 Thus, it was shown that the PLA (2) inhibitory activity differs depending on the state of the cells used and the test method. That is, the inhibitory activity of the compound in the conventional activity evaluation method reflecting the artificial enzyme activity was not related to its in′TO anti-inflammatory activity. Activity that reflects physiologically active enzyme activity The inhibitory activity in the evaluation method is improved compared to the conventional method, but it also includes noise, and by calculating the selectivity between different evaluation methods, it is more advantageous to calculate in It has been shown that anti-inflammatory active compounds can be screened in vivo.

Table 5

 ΦΝ. D .: Not tested Industrial applicability

 Based on the above test results, the heterocyclic derivative compound exhibiting anti-inflammatory activity shows a sensitivity difference of 10 times or more in an evaluation method reflecting physiologically artificial pseudo active cPLA (2) activity, The evaluation method of the present invention is an excellent evaluation method that reflects cPL A (2) activity. Although the present invention has examined cPLA (2) activity in relation to anti-inflammation, it can be applied to an evaluation method for various symptoms caused by cPLA (2).

Claims

The scope of the claims  1. (a) reflecting physiologically active structural function] VA—a step of obtaining an inhibitory activity index in the method for evaluating the activity of cytoplasmic phospholipase A (2),  (b) a step of obtaining an inhibitory activity index in the method for evaluating the activity of IVA-cytoplasmic phospholipase A (2) that has low similarity to physiologically active structural functions and reflects artificial pseudo-active structural functions , (c) obtaining a selectivity index calculated by comparing (a) and (b), A physiologically active form] VA—a method for evaluating cytosolic phospholipase A (2) activity.  2. (a) reflecting the physiologically active structural function] V A—a step of obtaining an inhibitory activity index in the method for evaluating the activity of cytoplasmic phospholipase A (2),  (b) a step of obtaining an inhibitory activity index in the method for evaluating the activity of 1VA-cytoplasmic phospholipase A (2), which has low similarity to physiologically active structural functions and reflects artificial pseudo-active structural functions , (c) obtaining a selectivity index calculated by comparing (a) and (b), 2. The evaluation method according to claim 1, wherein the ratio of the selectivity index in the evaluation method characterized by including 10 is 10 times or more. 3. In the step (a), the production of PGE ₂ and / or LTC ₄ and / or PAF is enhanced 10 times or more, and / or the production of active oxygen is enhanced 10 times or more, and z or 3. The evaluation method according to claim 1 or 2, wherein a human leukemia cell line U937 differentiated cultured cell exhibiting an activated ligament macrophage-like morphological reaction is used. 4. The activity evaluation method according to any one of claims 1 to 3, wherein in the step (b), cells having an increase in lipid mediator production of less than 10 times are used.