GB2531646B - Method for evaluating suppressive action on vascular hyper-permeability - Google Patents
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Description
DESCRIPTION
Title of Invention
METHOD FOR EVALUATING SUPPRESSIVE ACTION ON
VASCULAR HYPER-PERMEABILITY
Technical Field [0001] The present invention relates to a method for evaluating a suppressive action on vascular hyper-permeability.
Background Art [0002] A type-I allergy reaction occurs in the following mechanisms.
If an allergen invades the body and binds to IgE on the cell surface of a mast cell, the mast cell releases physiologically active substances such as histamine. This induces vascular expansion and vascular hyper-permeability, thereby causing symptoms such as edema and pruritus.
[0003] The vascular hyper-permeability is a phenomenon that once vascular endothelial cells respond to histamine, an intercellular gap widens; and then vascular permeability is promoted. This phenomenon has been evaluated by taking a two-dimensional image of vascular endothelial cells and then by measuring an expansion of an intercellular gap (Non Patent Literatures 1 and 2). Because antihistamine agents have a suppressive action on vascular hyper-permeability caused by histamine stimulation, an evaluation system for measuring suppression of widening an intercellular gap is available for a drug efficacy evaluation of the antihistamine agents. Citation List
Non Patent Literature [0004] Non Patent Literature 1: Wysolmerski et al., The American Journal of Pathology, 132(1), 28-37 (1988).
Non Patent Literature 2: Killackey et al., The American journal of pathology, 122(1), 50-61 (1986).
Summary of Invention
Technical Problem [0005] Unfortunately, no expansion of an intercellular gap can be observed until about 30 minutes have passed after histamine stimulation, and consequently, it is difficult to perform a quick measurement.
[0006] In addition, cell adhesion is high in some vascular endothelial cells and thus no expansion of an intercellular gap may occur, and hence, the present inventors have found a possibility that no accurate evaluation can be conducted.
[0007] The purpose of the present invention is to provide a method for evaluating a suppressive action on vascular hyper-permeability, in which a quick and accurate measurement is possible.
Solution to Problem [0008] The present inventors are the first to find that when vascular endothelial cells after histamine stimulation are three-dimensionally observed, the morphology of the vascular endothelial cells is changed immediately after the histamine stimulation. Specifically, the vascular endothelial cells have been found to exhibit the following morphological changes: the apical height of the cell increases immediately after the histamine stimulation, and then, the adhesion area of the cell decreases after a certain period. Further, the present inventors have found that even if the adhesion of the vascular endothelial cell is high and no change in the adhesion area is eventually observed, the apical height of the cell increases immediately after the histamine stimulation. The present inventors have considered that by utilizing a change in the apical height of the cell on the basis of the above findings, the suppressive action on the vascular hyper-permeability can be quickly and accurately evaluated, and completed the present invention.
[0009] Specifically, an aspect of the present invention provides a method for evaluating a suppressive action of a test substance on vascular hyper-permeability by measuring a morphological change of a vascular endothelial cell, the method comprising: a step of contacting the test substance with the vascular endothelial cell; a step of contacting an agent for promoting vascular permeability of the vascular endothelial cell with the vascular endothelial cell; and a step of measuring an apical height of the vascular endothelial cell, wherein the apical height of the vascular endothelial cell is used as an index for the suppressive action of the test substance on vascular hyper-permeability of the vascular endothelial cell; and wherein the apical height of the vascular endothelial cell is the distance from the cell bottom attached to a culture vessel to the highest height point of the cell surface not attached to the culture vessel.
[0010] The above index can be used to determine that the test substance suppresses the vascular hyper-permeability of the vascular endothelial cell when the apical height of the vascular endothelial cell in the presence of the test substance is lower than the apical height of the vascular endothelial cell in the absence of the test substance.
[0011] It is preferable that the above method further comprises a step of measuring an adhesion area of the vascular endothelial cell, wherein the apical height and the adhesion area of the vascular endothelial cell are used as indexes for the suppressive action of the test substance on vascular hyper-permeability of the vascular endothelial cell. Using the adhesion area in addition to the apical height as indexes makes it possible to perform a more accurate evaluation. In this case, the above indexes can be used to determine that the test substance suppresses the vascular hyper-permeability of the vascular endothelial cell when the apical height of the vascular endothelial cell in the presence of the test substance is lower than the apical height of the vascular endothelial cell in the absence of the test substance, and the adhesion area of the vascular endothelial cell in the presence of the test substance is larger than the adhesion area of the vascular endothelial cell in the absence of the test substance.
[0012] In the above methods, it is preferable that the agent for promoting vascular permeability of the vascular endothelial cell is histamine. Use of an intrinsic ligand histamine enables a response similar as an actual in vivo response to be observed.
[0013] In the above methods, it is preferable to measure the apical height and/or the adhesion area of the vascular endothelial cell by using a quantitative phase microscope. Use of the quantitative phase microscope makes it possible to accurately and conveniently measure the apical height and/or the adhesion area.
Advantageous Effects of Invention [0014] In the method for evaluating a suppressive action on vascular hyper-permeability according to the present invention, a quick and accurate measurement is possible. Use of this evaluation method makes it possible to perform a drug efficacy evaluation of antihistamine agents. In addition, use of this evaluation method makes it possible to screen for antihistamine agents.
Brief Description of Drawings [0015]
Figure 1 is a schematic view to illustrate a histamine stimulation-induced morphological change in a vascular endothelial cell, (a) The cell before the histamine stimulation; (b) the cell immediately after the histamine stimulation; (c) the cell at about 10 min after the histamine stimulation; (d) the cell at about 15 min after the histamine stimulation.
Figure 2 is pictures showing morphological changes in vascular endothelial cells when histamine stimulation was performed without pretreatment, (a) After 270 sec; (b) after 402 sec; (c) after 1002 sec; and (d) after 1170 sec. The histamine stimulation was performed at 300 sec.
Figure 3 is pictures showing morphological changes in vascular endothelial cells when histamine stimulation was performed after pretreatment with levocetirizine. (a) After 270 sec; (b) after 402 sec; (c) after 1002 sec; and (d) after 1170 sec. The histamine stimulation was performed at 300 sec.
Figure 4 is a graph showing a morphological change in a single vascular endothelial cell when histamine stimulation was performed without pretreatment. The histamine stimulation was performed at 300 sec.
Figure 5 is a graph showing a morphological change in a single vascular endothelial cell when histamine stimulation was performed after pretreatment with levocetirizine. The histamine stimulation was performed at 300 sec.
Figure 6 is graphs showing histamine stimulation-induced changes in the apical height and the adhesion area of a vascular endothelial cell, (a) Without pretreatment; and (b) with pretreatment with levocetirizine. The histamine stimulation was performed at 300 sec.
Figure 7 is graphs showing changes in the apical height and the adhesion area of a vascular endothelial cell when histamine stimulation was performed without pretreatment, (a) A cell which had weak adhesion and was easy to detach; and (b) a cell which had high adhesion and was hard to detach. The histamine stimulation was performed at 300 sec.
Figure 8 is pictures showing morphological changes in vascular endothelial cells when histamine stimulation was performed after pretreatment with dextrocetirizine. (a) After 270 sec; and (b) after 1170 sec. The histamine stimulation was performed at 300 sec.
Figure 9 is pictures showing morphological changes in vascular endothelial cells when histamine stimulation was performed after pretreatment with a cetirizine racemate, (a) After 270 sec; and (b) after 1170 sec. The histamine stimulation was performed at 300 sec.
Figure 10 is graphs showing histamine stimulation-induced changes in the apical heights of vascular endothelial cells. The ordinate represents a change in the apical height that has been normalized using the apical height at the time of the histamine stimulation. The abscissa represents an elapsed time (sec) when the time of the histamine stimulation is set to 0 sec. (a) Without pretreatment; (b) with pretreatment with levocetirizine; (c) with pretreatment with dextrocetirizine; and (d) with pretreatment with a cetirizine racemate.
Figure 11 is a graph showing histamine stimulation-induced (average) changes in the apical heights of vascular endothelial cells. The ordinate represents a change in the apical height that has been normalized using the apical height at the time of the histamine stimulation. The abscissa represents an elapsed time (sec) when the time of the histamine stimulation is set to 0 sec.
Figure 12 is a graph using a box plot representing the apical height of a vascular endothelial cell at 8 min after histamine stimulation, which has been normalized using the apical height of the vascular endothelial cell at the time of the histamine stimulation.
Description of Embodiments [0016] (Histamine Stimulation-induced Morphological change in Vascular Endothelial Cell) A histamine stimulation-induced morphological change in a vascular endothelial cell is illustrated by referring to Figure 1. Figure 1(a) shows a vascular endothelial cell before histamine stimulation. Figure 1(b) shows the vascular endothelial cell immediately after the histamine stimulation. First, the histamine stimulation causes the apical height of the cell to increase. The adhesion area of the cell is unchanged immediately after the histamine stimulation. Figure 1(c) shows the vascular endothelial cell at about 10 min after the histamine stimulation. The apical height of the cell stays the same. The adhesion area of the cell begins to decrease. Figure 1(d) shows the vascular endothelial cell at about 15 min after the histamine stimulation. The apical height of the cell further increases, the adhesion area of the cell decreases, and the states are then maintained.
[0017] By contrast, when pretreatment is performed with a substance having a suppressive action on vascular hyper-permeability of a vascular endothelial cell, such as an antihistamine agent, and then the vascular endothelial cell is stimulated with histamine, no cell morphological changes occur and the state of Figure 1(a) is maintained. [0018] Suppressive action on vascular hyper-permeability is evaluated on the basis of this difference in the morphological changes.
[0019] (Method for Evaluating Suppressive Action of Test Substance on Vascular Hyper-permeability of Vascular Endothelial Cell)
Any substance may be used as a test substance used to evaluate the suppressive action. Preferably, a substance that should have an Hi receptor antagonism is used as the test substance.
[0020] Vascular endothelial cells derived from mammals such as a mouse, a rat, a cow, and a human may be used as the vascular endothelial cell. It is possible to properly evaluate the drug efficacy of an antihistamine agent when administered to a human, and accordingly, it is preferable to use human-derived vascular endothelial cells. With regard to the human-derived vascular endothelial cells, a dermal microvascular endothelial cell, an umbilical cord vein vascular endothelial cell, and a vena cava vascular endothelial cell, for example, are available. The vascular endothelial cells may be derived from either an adult or a neonate. The vascular endothelial cells can be collected and isolated from animals by using a known method. Also, the cells may be available from cell banks. Processes for culturing vascular endothelial cells are well known to those skilled in the art, and for example, a commercially available medium for vascular endothelial cells may be used to culture the cells in an incubator at 37°C and 5% CO2.
[0021] The vascular endothelial cells are plated on a dish and are cultured until the cells adhere thereto sufficiently. Preferably, the culture period is 24 hours.
[0022] Next, a test substance is added to the medium to contact the test substance with the vascular endothelial cells (hereinafter, also referred to as pretreatment). The contact time is usually from 30 to 60 min. It is preferable that the test substance is added as a solution in which the test substance is dissolved into a suitable solvent. Preferably, the solvent is a medium. When the test substance is difficult to be dissolved into a medium, the test substance may be first dissolved into a solvent into which the test substance can be dissolved, followed by dilution with the medium. Note that, at this time, a negative control may be a sample in which only the test substance-free solvent is added to the medium.
[0023] Next, an agent for promoting vascular permeability of the vascular endothelial cells is added to the medium to contact the agent with the vascular endothelial cells (hereinafter, also referred to as agent stimulation). The agent for promoting vascular permeability of the vascular endothelial cells may be an Ηχ receptor agonist. Examples of the agent include histamine, 2-methylhistamine, and 2-(2-pyridyl)ethylamine. Because histamine is an intrinsic Hi receptor ligand, it is preferable to use histamine.
[0024] After the agent stimulation, the apical height of the vascular endothelial cell is measured. A three-dimensional imaging device may be used to measure the apical height. Examples of the three-dimensional imaging device include a quantitative phase microscope, a low-coherence interference microscope, a digital holography microscope, a tomographic phase microscope, and a scanning probe microscope, and the quantitative phase microscope is preferable.
[0025] The quantitative phase microscope is disclosed in, for example, JP4090244 and JP2010-48619A. The quantitative phase microscope may be used to quantitatively measure the phase of a light field by using a phase shift interference method. By using the quantitative phase microscope, it is possible to obtain a three-dimensional morphology of a cell at a resolution of 1 micron or less, and thereby it is possible to accurately measure the apical height and the adhesion area of the cell. [0026] The quantitative phase microscope may use the phase shift method to calculate a distribution of phase changes given to light that has passed through a measurement target. An optical thickness (OT) refers to an index represented by formula (1) when a phase change given to light that has passed through a measurement target is defined as (p. Here, λ represents the wavelength of the light. ΟΤ = λχφ/2π(1) [0027] The optical thickness is set forth as formula (2) when the refractive index of the measurement target is defined as nl; the refractive index of a medium surrounding the measurement target is defined as nO; and the height of the measurement target is defined as h. Hence, by measuring the optical thickness, a value in proportion to the height of the measurement target can be approximated. OT = h x (nl - nO) (2) [0028] The apical point of a cell refers to the highest height point in a single cell. The height refers to a distance from the cell bottom attached to a culture vessel to the cell surface not attached to the culture vessel. The apical height of a cell may be a real height or an optical thickness.
[0029] A measurement of the apical height of a cell may be carried out at any time point after the agent stimulation. When the agent stimulation is performed without pretreatment with the test substance, the apical height of a cell increases immediately after the stimulation, the height remains substantially the same until about 10 min later, and the height further increases after about 15 min. However, in the case of a vascular endothelial cell having high adhesion, no additional increase in the apical height of the cell after about 15 min may be observed. In addition, in order to increase measurement efficiency, it is preferable to perform a measurement in a short period. Thus, it is preferable that the measurement of the apical height of the cell is carried out within a time range from the agent stimulation initiation to 10 min later. In addition, a more accurate measurement can be conducted if the apical height of the cell is measured in a time zone in which a fluctuation of the apical height of the cell is as small as possible.
Hence, it is preferable that the measurement of the apical height of the cell is carried out within a time range from 3 min after the agent stimulation initiation to 9 min later.
[0030] The suppressive action of a test substance on vascular hyper-permeability of a vascular endothelial cell is evaluated on the basis of the apical height of the cell measured. An index for the above evaluation method may be an index based on the apical height of a single cell or an index based on the average of the apical heights of a plurality of cells. More specifically, the index for the above evaluation method can be used to determine that the test substance suppresses the vascular hyper-permeability of the vascular endothelial cell when the apical height of the vascular endothelial cell in the presence of the test substance is lower than the apical height of the vascular endothelial cell in the absence of the test substance.
[0031] In another embodiment, a measurement of the adhesion area of the vascular endothelial cell is conducted in addition to the measurement of the apical height of the vascular endothelial cell. The evaluation based on the adhesion area of the vascular endothelial cell corresponds to an evaluation based on an expansion of an intercellular gap as is a conventional evaluation method, and a more accurate evaluation is possible than in the case of solely using the apical height of the vascular endothelial cell. The adhesion area of the vascular endothelial cell refers to a bottom area of a cell attached to a culture vessel. A three-dimensional imaging device may be used to measure the apical height as well as the adhesion area of a cell. A measurement of the adhesion area of a cell may be carried out at any time point after the agent stimulation. When the agent stimulation is performed without pretreatment with a test substance, the adhesion area of a cell begins to decrease about 10 min after the stimulation and the adhesion area is leveled off at about 15 min later. Thus, it is preferable that the measurement of the adhesion area of a cell is carried out at 15 min or later after the agent stimulation initiation. In addition, a more accurate measurement can be conducted if the adhesion area of a cell is measured in a time zone in which a fluctuation of the adhesion area of a cell is as small as possible. In addition, in order to increase measurement efficiency, it is preferable to perform a measurement in a short period. Thus, it is preferable that the measurement of the adhesion area of a cell is carried out at from 15 to 20 min after the agent stimulation initiation. [0032] The suppressive action of a test substance on vascular hyper-permeability of a vascular endothelial cell is evaluated on the basis of the apical height and the adhesion area of the cell measured. An index for the above evaluation method may be indexes based on the apical height and the adhesion area of a single cell or indexes based on the average of the apical heights and the average of the adhesion areas of a plurality of cells. More specifically, it is possible to determine that the test substance suppresses the vascular hyper-permeability of the vascular endothelial cell when the apical height of the vascular endothelial cell in the presence of the test substance is lower than the apical height of the vascular endothelial cell in the absence of the test substance, and the adhesion area of the vascular endothelial cell in the presence of the test substance is larger than the adhesion area of the vascular endothelial cell in the absence of the test substance.
[0033] (Method for Evaluating Promoting Action of Test Substance on Vascular Permeability of Vascular Endothelial Cell)
Another embodiment provides a method for evaluating a promoting action of a test substance on vascular permeability of a vascular endothelial cell, the method comprising: a step of contacting the test substance with the vascular endothelial cell; and a step of measuring an apical height of the vascular endothelial cell, wherein the apical height of the vascular endothelial cell is used as an index for the promoting action of the test substance on vascular permeability of the vascular endothelial cell. Use of this evaluation method makes it possible to screen for Hi receptor agonists.
[0034] The above index can be used to determine that the test substance promotes the vascular permeability of the vascular endothelial cell when the apical height of the vascular endothelial cell in the presence of the test substance is higher than the apical height of the vascular endothelial cell in the absence of the test substance.
[0035] It is preferable that the above method further comprises a step of measuring an adhesion area of the vascular endothelial cell, wherein the apical height and the adhesion area of the vascular endothelial cell are used as indexes for the promoting action of the test substance on vascular permeability of the vascular endothelial cell. In this case, the above indexes can be used to determine that the test substance promotes the vascular permeability of the vascular endothelial cell when the apical height of the vascular endothelial cell in the presence of the test substance is higher than the apical height of the vascular endothelial cell in the absence of the test substance, and the adhesion area of the vascular endothelial cell in the presence of the test substance is smaller than the adhesion area of the vascular endothelial cell in the absence of the test substance.
[0036] In the above methods, it is preferable to measure with a quantitative phase microscope the apical height and/or the adhesion area of the vascular endothelial cell.
[0037] Any substance may be used as a test substance used to evaluate the promoting action. Preferably, a substance that should have an Ηχ receptor agonistic action is used as a test substance.
[0038] The vascular endothelial cells used and a culturing process therefor are described above.
[0039] A test substance is added to a medium to contact the test substance with the vascular endothelial cells. This step corresponds to the above agent stimulation step.
[0040] Measurements of the apical height and the adhesion area of the vascular endothelial cell after the agent stimulation are described above.
Examples [0041] Materials and Methods [0042] (1) Vascular Endothelial Cells
Human neonatal skin-derived microvascular endothelial cells (HMVEC) were used as vascular endothelial cells. A culture medium used after subculture until just before measurement was 500 mL of an EBM-2 medium (CC-3156, Lonza Ltd) containing a supplemental factor set for microvascular endothelial cells (2 EGM-2-MV SingleQuots (CC-4147, Lonza Ltd)). After subculture, the cells were incubated for 24 hours at 37°C under a 5% CO2 atmosphere, and the cultured medium was replaced by an EBM-2 medium immediately before pretreatment. An untreated plastic dish was used as a culture dish during the subculture.
[0043] (2) Agent
Histamine was used as an agent for promoting vascular permeability of the vascular endothelial cells. In addition, a cetirizine racemate, levocetirizine (1-cetirizine), and dextrocetirizine (d-cetirizine) were used as a pretreatment reagent for histamine. The cetirizine racemate and levocetirizine are known to have an antihistamine action, that is, a suppressive action on vascular hyper-permeability of vascular endothelial cells, but dextrocetirizine is known to have only a weak antihistamine action. In addition, the ability of levocetirizine to bind to the Hi receptor is known to be about 33 times stronger than that of dextrocetirizine.
[0044] (3) Pretreatment A cetirizine racemate was added to the medium at a final concentration of 2 μΜ, and the cells were allowed to stand in a CO2 incubator at 37°C for 30 min. In the case of levocetirizine or dextrocetirizine, the similar pretreatment as of the cetirizine racemate was carried out except that the final concentration was 1 μΜ.
[0045] (4) Histamine Stimulation
The cells were set in the following device and microscopic observation was initiated. At 300 sec after the observation initiation, histamine was added at a final concentration of 100 μΜ, thereby causing vascular hyper-permeability of the vascular endothelial cells. [0046] (5) Device for Measuring Apical Height and Adhesion Area of
Vascular Endothelial Cell A reflection-type quantitative phase microscope was used in a transmission mode by means of glass bottom reflection to subject a sample to time lapse imaging. Exposure time per interference image = 25 msec. Time interval between phase steps = 100 msec. Phase shift method = To-and-fro 5-point method (a total of 10 points). Waiting time between imaging cycles = 2 sec. Time lapse interval = 3 sec. The observation period was 20 to 30 min, which was a period from the initiation to the end. Its objective lens was a dipping lens with a magnification of 20x (Nikon, CFI Fluor 20xW, NA = 0.5).
[0047] (Example 1) A quantitative phase microscope was used to observe how vascular endothelial cells looked after histamine stimulation with or without pretreatment with levocetirizine. Figure 2 shows how the cells looked in the case of no pretreatment. Figure 3 shows how the cells looked in the case of having pretreatment with levocetirizine. Figures 2 and 3 (a), (b), (c), and (d) show how the cells looked at 270, 402, 1002, and 1170 sec after the observation initiation, respectively. The histamine stimulation was performed at 300 sec after the observation initiation.
[0048] As clear from Figure 2(b), the phenomenon that the apical height of the cell began to increase immediately after the histamine stimulation was observed. In addition, as clear from Figures 2(c) and 2(d), the phenomenon that the apical height of the cell further increased and the adhesion area of the cell began to decrease at about 10 min after the histamine stimulation was observed.
[0049] By contrast, as clear from Figure 3, a change in neither the apical height nor the adhesion area of the cell was recognized even if histamine stimulation was performed in the case of having pretreatment with levocetirizine, an antihistamine agent.
[0050] Cell morphological changes in a single cell are shown in Figures 4 and 5. Figure 4 shows a cell morphological change in the case of no pretreatment, and Figure 5 shows a cell morphological change in the case of having pretreatment with levocetirizine. As clear from Figure 4, the phenomenon that the apical height of the cell increased in the beginning and the adhesion area of the cell then decreased was observed. By contrast, as clear from Figure 5, any cell morphological changes were hardly recognized even if histamine stimulation was performed in the case of having pretreatment with levocetirizine.
[0051] (Example 2)
Like Example 1, a quantitative phase microscope was used to observe how vascular endothelial cells looked after histamine stimulation with or without pretreatment with levocetirizine, and then, the apical height and the adhesion area of the cell were measured. The results are shown in Figures 6(a) and 6(b). Figure 6(a) clearly demonstrates that, in the case of no pretreatment, the apical height of the cell increased immediately after the histamine stimulation and the height further increased at about 10 min later. In addition, in the case of no pretreatment, the adhesion area of the cell was demonstrated to decrease at about 10 min after the histamine stimulation. By contrast, as clear from Figure 6(b), almost no change in the apical height or the adhesion area of the cell was observed even if histamine stimulation was performed in the case of having pretreatment with levocetirizine. [0052] (Example 3)
When the histamine stimulation was performed without pretreatment, it was observed that the vascular endothelial cells exhibited different behaviors. The histamine stimulation was performed without pretreatment, and a quantitative phase microscope was used to observe how vascular endothelial cells looked, and then, the apical height and the adhesion area of the cell were measured using a different vascular endothelial cell. The results are shown in Figures 7(a) and 7(b). Figure 7(a) is identical to Figure 6(a). Figure 7(b) shows that the apical height of the cell increased immediately after the histamine stimulation, but the apical height and the adhesion area exhibited almost no change after that. This is presumed to mean that adhesion, that is, resistance to detachment, varies depending on cells, and it can be interpreted that a cell which has weak adhesion and is easy to detach exhibits a behavior as shown in Figure 7(a), whereas a cell which has high adhesion and is hard to detach exhibits a behavior as shown in Figure 7(b).
[0053] The above means that when a cell used for evaluation is a cell which has high adhesion and is hard to detach, no appropriate evaluation can be conducted using a conventional method in which a change in the adhesion area of the cell is measured. By contrast, it can be appreciated from the results shown in Figure 7 that the method of the present invention including measuring a change in the apical height of a cell makes it possible to conduct an appropriate evaluation even in the case of using such a cell.
[0054] (Example 4) A quantitative phase microscope was used to observe how vascular endothelial cells looked after histamine stimulation with pretreatment with dextrocetirizine or a cetirizine racemate instead of using levocetirizine, and then, the apical height and the adhesion area of the cell were measured. Figures 8(a) and 8(b) show how the cells looked in the case of having pretreatment with dextrocetirizine, and Figures 9(a) and 9(b) show how the cells looked in the case of having pretreatment with a cetirizine racemate. In Figures 8 and 9, (a) and (b) show how the cells looked at 270 and 1170 sec after the observation initiation, respectively. The histamine stimulation was performed at 300 sec after the observation initiation.
[0055] As clear from Figure 8(b), the phenomenon that in the case of having pretreatment with dextrocetirizine, the apical height of the cell increased and the adhesion area of the cell decreased by the histamine stimulation was observed. This observation results are in agreement with the fact that dextrocetirizine has almost no antihistamine action. [0056] As clear from Figure 9(b), almost no change in the apical height or the adhesion area of the cell was observed even if histamine stimulation was performed in the case of having pretreatment with the cetirizine racemate. This result is in agreement with the fact that the cetirizine racemate has an antihistamine action.
[0057] (Example 5)
Figure 10 together shows the results obtained by measuring changes in the apical heights of a plurality of cells (N = 25 to 37) and then by normalizing the apical heights of the cells by using the apical heights of the cells at the time of histamine stimulation. Figure 10(a) shows changes in the apical heights of the cells without pretreatment, Figure 10(b) shows those with pretreatment with levocetirizine, Figure 10(c) shows those with pretreatment with dextrocetirizine, and Figure 10(d) shows those with pretreatment with a cetirizine racemate. The abscissa of Figure 10 represents an elapsed time (sec) when the time of the histamine stimulation is set to 0 sec. Figures 10(a) and 10(c) show that the apical heights of the cells increased after the histamine stimulation, whereas Figures 10(b) and 10(d) shows that increases in the apical heights of the cells after the histamine stimulation were smaller than those in Figures 10(a) and 10(c).
[0058] Figure 11 shows the averages of the changes in the apical heights of the cells in each treatment group. As clear from Figure 11, the apical heights of the cells in the case of having pretreatment with dextrocetirizine increased in a manner similar to the case of no pretreatment; and the apical heights of the cells in the case of having pretreatment with levocetirizine or the cetirizine racemate hardly increased.
[0059] The apical heights of the cells at 8 min after the histamine stimulation were normalized on the basis of the apical heights of the cells at the time of the histamine stimulation, and these results are together shown in a box plot of Figure 12. In addition, Table 1 lists the results of a t-test between the respective treatment groups. As clear from the results shown in Figure 12 and Table 1, it was able to be demonstrated that the apical heights of the cells in the case of having pretreatment with levocetirizine or the cetirizine racemate were significantly lower than those in the case of no treatment or having pretreatment with dextrocetirizine; and there was a correlation between the apical heights of the cells and the antihistamine action (suppressive action on vascular hyper-permeability of vascular endothelial cells). [0060] [Table 1]
Claims (6)
1. A method for evaluating a suppressive action of a test substance on vascular hyper-permeability by measuring a morphological change of a vascular endothelial cell, the method comprising: a step of contacting the test substance with the vascular endothelial cell; a step of contacting an agent for promoting vascular permeability of the vascular endothelial cell with the vascular endothelial cell; and a step of measuring an apical height of the vascular endothelial cell, wherein the apical height of the vascular endothelial cell is used as an index for the suppressive action of the test substance on vascular hyperpermeability of the vascular endothelial cell; and wherein the apical height of the vascular endothelial cell is the distance from the cell bottom attached to a culture vessel to the highest height point of the cell surface not attached to the culture vessel.
2. The method according to claim 1, wherein the index is used to determine that the test substance suppresses the vascular hyper-permeability of the vascular endothelial cell when the apical height of the vascular endothelial cell in the presence of the test substance is lower than the apical height of the vascular endothelial cell in the absence of the test substance.
3. The method according to claim 1 or 2, further comprising a step of measuring an adhesion area of the vascular endothelial cell, wherein the apical height and the adhesion area of the vascular endothelial cell are used as indexes for the suppressive action of the test substance on vascular hyper-permeability of the vascular endothelial cell.
4. The method according to claim 3, wherein the indexes are used to determine that the test substance suppresses the vascular hyper-permeability of the vascular endothelial cell when the apical height of the vascular endothelial cell in the presence of the test substance is lower than the apical height of the vascular endothelial cell in the absence of the test substance, and the adhesion area of the vascular endothelial cell in the presence of the test substance is larger than the adhesion area of the vascular endothelial cell in the absence of the test substance.
5. The method according to any one of claims 1 to 4, wherein the agent for promoting vascular permeability of the vascular endothelial cell is histamine.
6. The method according to any one of claims 1 to 5, wherein the measurement of the apical height and/or the adhesion area of the vascular endothelial cell is carried out using a quantitative phase microscope.
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| PCT/JP2014/054528 WO2014129653A1 (en) | 2013-02-25 | 2014-02-25 | Evaluation method for vascular hyperpermeability regulation action |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2005002093A (en) * | 2003-05-20 | 2005-01-06 | Masako Nozaki | Vasopermeability promotion suppressor |
| JP2006506321A (en) * | 2002-04-11 | 2006-02-23 | チルドレンズ メディカル センター コーポレーション | Methods for inhibiting increased vascular permeability |
| JP2012524058A (en) * | 2009-04-15 | 2012-10-11 | フォンダツィオーネ アイアールシーシーエス イスティトゥト ナツィオナーレ デイ トゥモリ | Use of multikinase inhibitors in the treatment of hypervascular permeability |
| JP2012530784A (en) * | 2009-06-22 | 2012-12-06 | ディエムアイ アクイジション コーポレイション | Methods for treatment of disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2006506321A (en) * | 2002-04-11 | 2006-02-23 | チルドレンズ メディカル センター コーポレーション | Methods for inhibiting increased vascular permeability |
| JP2005002093A (en) * | 2003-05-20 | 2005-01-06 | Masako Nozaki | Vasopermeability promotion suppressor |
| JP2012524058A (en) * | 2009-04-15 | 2012-10-11 | フォンダツィオーネ アイアールシーシーエス イスティトゥト ナツィオナーレ デイ トゥモリ | Use of multikinase inhibitors in the treatment of hypervascular permeability |
| JP2012530784A (en) * | 2009-06-22 | 2012-12-06 | ディエムアイ アクイジション コーポレイション | Methods for treatment of disease |
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