U.S. patent application number 15/611316 was filed with the patent office on 2017-09-21 for method for evaluation of drug efficacy of a medicine having a therapeutic or preventive effect against a disease related to el activity and a method for screening an inhibitor of el activity.
This patent application is currently assigned to SHIONOGI & CO., LTD.. The applicant listed for this patent is SHIONOGI & CO., LTD.. Invention is credited to Takashi ONO, Shuhei SHIGAKI, Atsuko YAMAMOTO.
Application Number | 20170268034 15/611316 |
Document ID | / |
Family ID | 50627543 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170268034 |
Kind Code |
A1 |
ONO; Takashi ; et
al. |
September 21, 2017 |
METHOD FOR EVALUATION OF DRUG EFFICACY OF A MEDICINE HAVING A
THERAPEUTIC OR PREVENTIVE EFFECT AGAINST A DISEASE RELATED TO EL
ACTIVITY AND A METHOD FOR SCREENING AN INHIBITOR OF EL ACTIVITY
Abstract
The present invention is related to a method for evaluation of
drug efficacy of a medicine having a therapeutic or preventive
effect against a disease related to EL activity wherein
phosphatidylinositol or lysophosphatidylinositol is used as an
indicator. The present invention is also related to a method for
screening an inhibitor of EL activity using phosphatidylinositol
and a kit for use in the method.
Inventors: |
ONO; Takashi; (Toyonaka-shi,
JP) ; YAMAMOTO; Atsuko; (Osaka, JP) ; SHIGAKI;
Shuhei; (Toyonaka-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHIONOGI & CO., LTD. |
Osaka |
|
JP |
|
|
Assignee: |
SHIONOGI & CO., LTD.
Osaka
JP
|
Family ID: |
50627543 |
Appl. No.: |
15/611316 |
Filed: |
June 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14437941 |
Apr 23, 2015 |
9695462 |
|
|
PCT/JP2013/079814 |
Nov 5, 2013 |
|
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15611316 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/92 20130101;
G01N 2500/20 20130101; C12Q 1/44 20130101; G01N 2500/04 20130101;
G01N 2405/06 20130101; G01N 2560/00 20130101; G01N 2500/00
20130101; G01N 2333/92 20130101 |
International
Class: |
C12Q 1/44 20060101
C12Q001/44; G01N 33/92 20060101 G01N033/92 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 5, 2012 |
JP |
2012-243324 |
Claims
1. A method for treating a patient having a disease related to
endothelial lipase (EL) activity, said method comprising:
administering an effective amount of a test substance as an EL
inhibitor to the diagnosed patient, thereby increasing the high
density lipoprotein cholesterol (HDL-c) in said patient; wherein
said patient has been diagnosed with said disease by comparing a
concentration of phosphatidylinositol or lysophosphatidylinositol
in a sample from said patient after the administration of the test
substance with a concentration of phosphatidylinositol or
lysophosphatidylinositol in the sample before the administration of
the test substance; and wherein the test substance has increased
the concentration of phosphatidylinositol or decreased the
concentration of lysophosphatidylinositol in the sample obtained
from the patient after the administration of the test substance
relative to the concentration of phosphatidylinositol or
lysophosphatidylinositol in the sample before the administration of
the test substance.
2. The method of claim 1, wherein said sample is blood, serum or
plasma.
3. The method of claim 1, wherein the test substance is an anti-EL
neutralizing antibody.
4. A method for treating a patient having a coronary artery disease
related to endothelial lipase (EL) activity, said method
comprising: administering an effective amount of a test substance
as an EL inhibitor to the diagnosed patient, thereby increasing the
high density lipoprotein cholesterol (HDL-c) in said patient;
wherein said patient has been diagnosed with said disease by
comparing a concentration of phosphatidylinositol or
lysophosphatidylinositol in a sample from said patient after the
administration of the test substance with a concentration of
phosphatidylinositol or lysophosphatidylinositol in the sample
before the administration of the test substance; wherein the test
substance has increased the concentration of phosphatidylinositol
or decreased the concentration of lysophosphatidylinositol in the
sample obtained from the patient after the administration of the
test substance relative to the concentration of
phosphatidylinositol or lysophosphatidylinositol in the sample
before the administration of the test substance; wherein said
coronary artery disease is dyslipidemia, hyperlipidemia,
arteriosclerosis, atherosclerosis, hypercholesterolemia,
hypertriglyceridemia, diabetes, obesity or syndrome X.
5. The method of claim 4, wherein said sample is blood, serum or
plasma.
6. The method of claim 4, wherein the test substance is an anti-EL
neutralizing antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a 37 C.F.R. .sctn.1.53(b) divisional of
U.S. application Ser. No. 14/437,941 filed on Apr. 23, 2015, which
is the National Phase of PCT International Application No.
PCT/JP2013/079814, filed on Nov. 5, 2013, which claims priority
under 35 U.S.C. .sctn.119(a) to Patent Application No. 2012-243324,
filed in Japan on Nov. 5, 2012, all of which are hereby expressly
incorporated by reference into the present application.
TECHNICAL FIELD
[0002] The present invention relates to a method for measurement of
the activity of vascular endothelial lipase (hereinafter, referred
to EL), wherein phosphatidylinositol or lysophosphatidylinositol is
used as an indicator. More specifically, the present invention
relates to a method for evaluation of drug efficacy of a medicine
having a therapeutic or preventive effect against a disease related
to EL activity and a method for screening an inhibitor of EL
activity.
BACKGROUND ART
[0003] EL is phospholipase belonging to triglyceride lipase
(hereinafter, referred to TGL) family (Non-patent document 1).
Human EL consists of 500 amino acid residues (NCBI Accession No.
NP_006024.1) and mouse EL also consists of 500 amino acid
residues.
[0004] Lipoprotein lipase (hereinafter, referred to LPL) and
hepatic lipase (hereinafter, referred to HL) are included in TGL
family.
[0005] The analysis of EL knockout mice and EL transgenic mice
revealed the involvement of EL in HDL cholesterol (hereinafter,
referred to HDL-c) metabolism due to its strong phospholipase
activity for HDL, and EL is noted as a determinant of HDL levels
(Non-Patent Document 2). The negative correlation between coronary
artery disease (hereinafter, referred to CAD) and plasma HDL-c
levels has been known for a long time. HDL-c has been believed to
show anti-atherogenic effect via anti-oxidant, anti-inflammatory
and reverse cholesterol transport action and low HDL-c
cholesteremia has been recognized as one of the risk factors for
CAD. Therefore, EL inhibitor can be a therapeutic agent against
CAD, and the increase in HDL-c levels and the decrease of
atherosclerotic lesion area have actually been reported in the
animal model of disease using EL-knockout mice (Non-Patent Document
3). These findings exhibit utility of a specific inhibitor for EL
as a therapeutic agent against lipid metabolism disorder and
atherosclerosis.
[0006] In evaluation of drug efficacy of EL inhibitor, it is
important to grasp the degree of inhibition of EL in blood by
administered drug in order to understand the potency of drug
efficacy. However, because other enzymes having lipase activity
similar to EL exist in blood, the finding of assay method which can
detect EL activity specifically is a problem to be solved for
selecting a promising inhibitor.
[0007] The lipase having higher activity than EL exists in blood
under the general assay condition well known (Non-Patent Document
4). Thereby, as EL activity can not be measured accurately, it is
difficult to accurately determine inhibition rate of administered
EL inhibitor in blood. When blood is collected to measure EL
activity in non-clinical study, it is necessary to administer a
large amount of heparin intravenously in order to release and
collect EL from blood vessel wall, but intravenous injection of a
large amount of heparin to a subject is not allowed in clinical
trial. Therefore, the marker as an indicator of EL activity is
essential for clinical trial, whereas there has never been any
report about the presence of maker for EL activity. [0008]
Non-patent document 1: Nature Genetics., 1999, vol. 21(4), p.
424-428 [0009] Non-patent document 2: TCM., 2004, vol. 14(5), p.
202-206 [0010] Non-patent document 3: The Journal of Biological
Chemistry., 2004, vol. 279(43), p. 45085-45092 [0011] Non-patent
document 4: The Journal of Clinical Investigation, 2003, vol.
111(3), p. 347-355
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0012] An objective of the present invention relates to measure EL
activity specifically in sample collected from non-clinical and
clinical specimens. In principle, it is impossible to administer a
large amount of heparin intravenously in clinical trial. Therefore,
an objective of the present invention is to measure EL activity
specifically without administering a large amount of heparin to a
subject.
[0013] The present inventors have contemplated that the exploring
substrate specific for EL from phospholipids in plasma lipoprotein
might be the best strategy for solving the problem of the
improvement in accuracy and selectivity of assay for EL activity
and the identification of marker of EL activity. The present
inventors found that phosphatidylinositol can be used as a specific
substrate for EL by intensive studies, and achieved the present
invention.
[0014] To be more specific, the present invention provides below
[1] to [10]:
[0015] [1] A method for measurement of EL activity, wherein
phosphatidylinositol or lysophosphatidylinositol is used as an
indicator.
[0016] [2] A method for evaluation of drug efficacy of a medicine
having a therapeutic or preventive effect against a disease related
to EL activity, comprising [0017] (1) a step for comparing a
concentration of phosphatidylinositol or lysophosphatidylinositol
in a sample obtained from a mammal after the administration of a
test substance with a concentration of phosphatidylinositol or
lysophosphatidylinositol in a sample obtained from the mammal
before the administration of the test substance, and [0018] (2) a
step for evaluating the test substance as a medicine having a
therapeutic or preventive effect against a disease related to EL
activity when the concentration of phosphatidylinositol or
lysophosphatidylinositol in the sample obtained from the mammal
after the administration of the test substance is increased
relative to the concentration of phosphatidylinositol or
lysophosphatidylinositol in the sample obtained from the mammal
before the administration of the test substance.
[0019] [3] A method for evaluation of drug efficacy of a medicine
having a therapeutic or preventive effect against a disease related
to EL activity, comprising [0020] (1) a step for comparing a
concentration of phosphatidylinositol in a sample obtained from a
mammal after the administration of a test substance with a
concentration of phosphatidylinositol in a sample obtained from the
mammal before the administration of the test substance, and [0021]
(2) a step for evaluating the test substance as a medicine having a
therapeutic or preventive effect against a disease related to EL
activity when the concentration of phosphatidylinositol in the
sample obtained from the mammal after the administration of the
test substance is increased relative to the concentration of
phosphatidylinositol in the sample obtained from the mammal before
the administration of the test substance.
[0022] [4] A method for evaluation of drug efficacy of a medicine
having a therapeutic or preventive effect against a disease related
to EL activity, comprising [0023] (1) a step for comparing a
concentration of lysophosphatidylinositol in a sample obtained from
a mammal after the administration of a test substance with a
concentration of lysophosphatidylinositol in a sample obtained from
the mammal before the administration of the test substance, and
[0024] (2) a step for evaluating the test substance as a medicine
having a therapeutic or preventive effect against a disease related
to EL activity when the concentration of lysophosphatidylinositol
in the sample obtained from the mammal after the administration of
the test substance is increased relative to the concentration of
lysophosphatidylinositol in the sample obtained from the mammal
before the administration of the test substance.
[0025] [5] The method described in [2]-[4], wherein the sample is
blood, serum or plasma.
[0026] [6] A method for screening an inhibitor of EL activity,
comprising [0027] (1) a step for contacting phosphatidylinositol, a
test substance and EL, [0028] (2) a step for comparing a
concentration of phosphatidylinositol when the test substance is
contacted with a concentration of phosphatidylinositol when the
test substance is not contacted and [0029] (3) a step for selecting
the test substance as an inhibitor of EL activity
[0030] when the concentration of phosphatidylinositol when the test
substance is contacted is increased relative to the concentration
of phosphatidylinositol when the test substance is not
contacted.
[0031] [7] The method described in [6], wherein
phosphatidylinositol is phosphatidylinositol-liposomes,
phosphatidylinositol-HDL, phosphatidylinositol-reconstituted HDL or
phosphatidylinositol-detergent mixed micelles.
[0032] [8] A method for screening an inhibitor of EL activity,
comprising [0033] (1) a step for contacting phosphatidylinositol, a
test substance and EL, [0034] (2) a step for comparing a
concentration of lysophosphatidylinositol when the test substance
is contacted with a concentration of lysophosphatidylinositol when
the test substance is not contacted, and [0035] (3) a step for
selecting the test substance as an inhibitor of EL activity [0036]
when the concentration of lysophosphatidylinositol when the test
substance is contacted is decreased relative to the concentration
of lysophosphatidylinositol when the test substance is not
contacted.
[0037] [9] The method described in [8], wherein
phosphatidylinositol is phosphatidylinositol-liposomes,
phosphatidylinositol-HDL, phosphatidylinositol-reconstituted HDL or
phosphatidylinositol-detergent mixed micelles.
[0038] [10] A kit for use in the method according to [6]-[9]
Effect of the Invention
[0039] A method for evaluation of drug efficacy of a medicine
having a therapeutic or preventive effect against a disease related
to EL activity, wherein a concentration of phosphatidylinositol or
lysophosphatidylinositol is used as an indicator, is provided by
the present invention. A method for screening an inhibitor of EL
activity using phosphatidylinositol and a kit for use in the method
is also provided by the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 shows the change in phosphatidylcholine (hereinafter,
also called PC) levels, when mouse preheparin plasma is incubated
with or without EL. The horizontal axis and the vertical axis
indicate molecular species of phosphatidylcholine and
phosphatidylcholine levels, respectively.
[0041] FIG. 2 shows the change in phosphatidylethanolamine
(hereinafter, also called PE) levels, when mouse preheparin plasma
is incubated with or without EL. The horizontal axis and the
vertical axis indicate molecular species of
phosphatidylethanolamine and phosphatidylethanolamine levels,
respectively.
[0042] FIG. 3 shows the change in phosphatidylinositol
(hereinafter, also called PI) levels, when mouse preheparin plasma
is incubated with or without EL. The horizontal axis and the
vertical axis indicate molecular species of phosphatidylinositol
and phosphatidylinositol levels, respectively.
[0043] FIG. 4 shows mouse EL activity when
phosphatidylinositol-induced HDL (hereinafter, also called PI-HDL)
or HDL is used as a substrate. The horizontal axis and the vertical
axis indicate the substrate used and the amount of non-esterified
free fatty acid (hereinafter, also called NEFA), respectively.
[0044] FIG. 5 shows the inhibitory effect of orlistat, which is
known inhibitor of lipase, on mouse EL when PI-HDL is used as a
substrate. The horizontal axis and the vertical axis indicate the
concentration of orlistat and the inhibition rate,
respectively.
[0045] FIG. 6 shows EL activity and lipase activity other than EL
(hereinafter, referred to non-EL activity) in postheparin plasma of
EL transgenic mice when PI-HDL or HDL is used as a substrate. The
horizontal axis and the vertical axis indicate the substrate used
and the amount of NEFA, respectively.
[0046] FIG. 7 shows the result converted the data shown in FIG. 6
into the ratio of EL activity to non-EL activity for PI-HDL or HDL,
respectively The horizontal axis and the vertical axis indicate the
substrate used and the ratio of EL activity to non-EL activity,
respectively.
[0047] FIG. 8 shows phosphatidylinositol levels in preheparin
plasma of wild-type mice (C57BL/6J strain) and EL-KO mice. The
horizontal axis and the vertical axis indicate molecular species of
phosphatidylinositol and phosphatidylinositol levels,
respectively.
[0048] FIG. 9 shows phosphatidylethanolamine levels in preheparin
plasma of wild-type mice and EL-KO mice. The horizontal axis and
the vertical axis indicate molecular species of
phosphatidylethanolamine and phosphatidylethanolamine levels,
respectively.
[0049] FIG. 10 shows the ratio of HDL cholesterol levels in
preheparin plasma collected with time after intravenous injection
of anti-EL neutralizing antibody to cynomolgus monkey to those
before administration. The horizontal axis and the vertical axis
indicate the sampling time of preheparin plasma after
administration of anti-EL antibody and the ratio to the
concentration before administration of anti-EL neutralizing
antibody.
[0050] FIG. 11 shows the ratio of phosphatidylinositol levels in
preheparin plasma collected with time after intravenous injection
of anti-EL neutralizing antibody to cynomolgus monkey to those
before administration. The horizontal axis and the vertical axis
indicate the sampling time of preheparin plasma after
administration of anti-EL antibody and the ratio to the
concentration before administration of anti-EL neutralizing
antibody.
[0051] FIG. 12 shows the ratio of lysophosphatidylinositol
(hereinafter, also called LPI) levels in preheparin plasma
collected with time after intravenous injection of anti-EL
neutralizing antibody to cynomolgus monkey to those before
administration. The horizontal axis and the vertical axis indicate
the sampling time of preheparin plasma after administration of
anti-EL antibody and the ratio to the concentration before
administration of anti-EL neutralizing antibody.
MODE FOR CARRYING OUT THE INVENTION
[0052] A term in the present invention, unless specifically
referred to, is used as the meaning commonly used in the art.
[0053] In one embodiment, the present invention provides a method
for measurement of EL activity, wherein phosphatidylinositol or
lysophosphatidylinositol is used as an indicator. The method
includes a method for evaluation of drug efficacy of a medicine
having a therapeutic or preventive effect against a disease related
to EL activity, wherein phosphatidylinositol or
lysophosphatidylinositol is used as an indicator, and a method for
screening an inhibitor of EL activity, wherein phosphatidylinositol
or lysophosphatidylinositol is used as an indicator.
[0054] Phosphatidylinositol is one of the acidic phospholipids with
inositol in polar head group and is sometimes abbreviated as PI.
Many kinds of molecular species consisting of the differences in
combination of fatty acids at the sn-1 and sn-2 positions exist.
For example, from molecular weight of phosphatidylinositol
estimated from the results of analysis by mass spectrometry, if the
predicted sum of the carbon number of fatty acid esterified at the
sn-1 and sn-2 positions are 38 and the predicted sum of the double
bond are 4, respectively, it is shown as 38:4.
[0055] Phosphatidylcholine is also called lecithin and sometimes
abbreviated as PC. Many kinds of molecular species consisting of
the differences in combination of fatty acids at the sn-1 and sn-2
positions exist. For example, from molecular weight of
phosphatidylcholine estimated from the results of analysis by mass
spectrometry, if the predicted sum of the carbon number of fatty
acid esterified at the sn-1 and sn-2 positions are 36 and the
predicted sum of the double bond are 3, respectively, it is shown
as 36:3.
[0056] Phosphatidylethanolamine is sometimes abbreviated as PE.
Many kinds of molecular species consisting of the differences in
combination of fatty acids at the sn-1 and sn-2 positions exist.
For example, from molecular weight of phosphatidylethanolamine
estimated from the results of analysis by mass spectrometry, if the
predicted sum of the carbon number of fatty acid esterified at the
sn-1 and sn-2 positions are 40 and the predicted sum of the double
bond are 6, respectively, it is shown as 40:6.
[0057] EL means vascular endothelial lipase which is a kind of
phospholipase belonging to TGL family. The GenBank Accession
numbers of human, rabbit and mouse EL are Accession No.
NP_006024.1, NP_001182567.1 and NP_034850.3, respectively. In the
present specification, EL includes not only the proteins consisting
of amino acid sequence registered in said GenBank Accession numbers
but also the proteins having the same
[0058] amino acid sequences as those described above except that
one amino acid or some amino acids is deleted, substituted or
added. However, these are limited to proteins having phospholipase
activity equal to EL registered in said GenBank Accession
numbers.
[0059] A disease related to EL activity means dyslipidemia,
hyperlipidemia, arteriosclerosis, atherosclerosis,
hypercholesterolemia, hypertriglyceridemia, diabetes, obesity,
syndrome X and the like.
[0060] In another embodiment, the present invention provides a
method for evaluation of drug efficacy of a medicine having a
therapeutic or preventive effect against a disease related to EL
activity, comprising
[0061] (1) a step for comparing a concentration of
phosphatidylinositol or lysophosphatidylinositol in a sample
obtained from an mammal after the administration of a test
substance with a concentration of phosphatidylinositol or
lysophosphatidylinositol in a sample obtained from the mammal
before the administration of the test substance, and
[0062] (2) a step for evaluating the test substance as a medicine
having a therapeutic or preventive effect against a disease related
to EL activity when the concentration of phosphatidylinositol or
lysophosphatidylinositol in the sample obtained from the mammal
after the administration of the test substance is altered relative
to the concentration of phosphatidylinositol or
lysophosphatidylinositol in the sample obtained from the mammal
before the administration of the test substance.
[0063] A disease related to EL activity means dyslipidemia,
hyperlipidemia, arteriosclerosis, atherosclerosis,
hypercholesterolemia, hypertriglyceridemia, diabetes, obesity,
syndrome X and the like.
[0064] In the step (1) of the above method, the test substances
include any known and novel compounds, and examples thereof include
nucleic acids, carbohydrates, lipids, proteins, peptides, organic
low molecular compounds, compound library prepared using the
technology of combinatorial chemistry, random peptide library
prepared by solid phase synthesis or phage display method or
natural products derived from microorganisms, animals and plants,
and marine organism.
[0065] In the step (1) of the above method, for example, the
quantification of phosphatidylinositol levels in the sample can be
performed by mass spectrometry, for example, with reference to
Analytical Letters, 2006, vol. 39, p. 957-972.
[0066] On the other hand, the quantification of
lysophosphatidylinositol levels in the sample can be performed by
TLC development with reference to unexamined patent application
2010-63470 or mass spectrometry with reference to Chromatographia.,
2010, vol. 72(7/8), p. 659-664.
[0067] In the step (1) of the above method, any species of mammal
is permissible. And if a sample is obtained from a mammal, any
sample is permissible, but blood, serum or plasma is preferred.
[0068] In the step (2) of the above method, a concentration of
phosphatidylinositol or lysophosphatidylinositol in a sample
obtained from a mammal after the administration of a test substance
is compared with a concentration of phosphatidylinositol or
lysophosphatidylinositol in a sample obtained from the mammal
before the administration of the test substance. The comparison of
a concentration of phosphatidylinositol or lysophosphatidylinositol
is preferably carried out on the basis of the presence or absence
of a significant difference. A concentration of
phosphatidylinositol or lysophosphatidylinositol in the sample
obtained from the mammal before the administration of the test
substance may be acceptable both a concentration of
phosphatidylinositol or lysophosphatidylinositol measured in
advance and at the same time against measurement after the
administration of the test substance, but a concentration of
phosphatidylinositol or lysophosphatidylinositol measured at the
same time is preferable in terms of accuracy of the experiment or
reproducibility of the experiment.
[0069] In the step (1) and (2) of the above method, it is never
necessary to release EL into blood. Therefore, the present
invention has the advantage that the present evaluation method do
not need the intravenous injection of heparin in collecting blood
from a subject and is also applicable for a subject in clinical
trial.
[0070] In another embodiment, the present invention provides a
method for screening an inhibitor of EL activity, comprising
[0071] (1) a step for contacting phosphatidylinositol, a test
substance and EL,
[0072] (2) a step for comparing a concentration of
phosphatidylinositol when the test substance is contacted with a
concentration of phosphatidylinositol when the test substance is
not contacted, and
[0073] (3) a step for selecting the test substance as an inhibitor
of EL activity
[0074] when the concentration of phosphatidylinositol when the test
substance is contacted is increased relative to the concentration
of phosphatidylinositol when the test substance is not
contacted.
[0075] Phosphatidylinositol used in the step (1) of the above
method is acceptable for any forms as long as phosphatidylinositol
is contained, even if phosphatidylinositol is
phosphatidylinositol-liposomes, phosphatidylinositol-HDL,
phosphatidylinositol-reconstituted HDL or
phosphatidylinositol-detergent mixed micelles.
[0076] Phosphatidylinositol-liposomes mean dispersion in solution
of phosphatidylinositol or the mixture of phosphatidylinositol and
lipid, prepared by the mechanical process like the sonication. The
lipids used in the said mixture include, for example, lipids
contained in blood. Phosphatidylinositol-liposomes can be prepared
appropriately with reference to The journal of Lipids Research,
1976, vol. 17(3), p. 239-247.
[0077] Phosphatidylinositol-HDL means phosphatidylinositol-induced
artificial HDL prepared by mixing phosphatidylinositol with HDL.
The HDL in the said phosphatidylinositol-induced artificial HDL
includes, for example, HDL or its components, namely, HDL2 and
HDL3.
[0078] Phosphatidylinositol-reconstituted HDL means the
reconstituted HDL prepared by mixing phosphatidylinositol or the
mixture of phosphatidylinositol and lipids with apoproteins. The
lipids in the said mixture include, for example, lipids contained
in blood. The apoproteins used for the preparation of
phosphatidylinositol-reconstituted HDL include, for example,
apoproteins contained in blood. Phosphatidylinositol-reconstituted
HDL can be prepared appropriately with reference to The Journal of
Biological Chemistry., 1982, vol. 257(8), p. 4535-4540.
[0079] Phosphatidylinositol-detergent mixed micelles mean the mixed
micelles prepared by solubilizing phosphatidylinositol or the
mixture of phosphatidylinositol and lipid by detergent. The lipids
in the said mixture include, for example, lipids contained in
blood. The detergents used for the preparation of
phosphatidylinositol-detergent mixed micelles include, for example,
ionic and non-ionic detergents. Phosphatidylinositol-detergent
mixed micelles can be prepared appropriately with reference to The
Journal Biological Chemistry, 1988, vol. 263(12), p. 5724-5731.
[0080] The test substances which are subjected to the screening
methods include any known and novel compounds, and examples thereof
include nucleic acids, carbohydrates, lipids, proteins, peptides,
organic low molecular compounds, the compound library prepared
using the technology of combinatorial chemistry, random peptide
library prepared by solid phase synthesis and phage display method
and the natural products derived from microorganisms, animals and
plants, and marine organism.
[0081] In the step (2) of the above method, for example, the
quantification of phosphatidylinositol levels in the sample can be
performed by mass spectrometry, for example, with reference to
Analytical Letters, 2006, vol. 39, p. 957-972 or can be performed
by measuring NEFA levels produced from phosphatidylinositol of
substrate.
[0082] In the step (3) of the above method, a concentration of
phosphatidylinositol when the test substance is contacted is
compared with a concentration of phosphatidylinositol when the test
substance is not contacted. The comparison of a concentration of
phosphatidylinositol is preferably carried out on the basis of the
presence or absence of a significant difference. A concentration of
phosphatidylinositol when the test substance is not contacted may
be acceptable both a concentration of phosphatidylinositol measured
in advance and at the same time against measurement of a
concentration of phosphatidylinositol when the test substance is
contacted, but a concentration of phosphatidylinositol measured at
the same time is preferable in terms of accuracy of the experiment
or reproducibility.
[0083] An inhibitor of EL activity discovered by the screening has
a therapeutic or preventive effect against a disease related to EL
activity, namely has a therapeutic or preventive effect against
dyslipidemia, hyperlipidemia, arteriosclerosis, atherosclerosis,
hypercholesterolemia, hypertriglyceridemia, diabetes, obesity,
syndrome X and the like.
[0084] In another embodiment, the present invention provides a
method for screening an inhibitor of EL activity, comprising
[0085] (1) a step for contacting phosphatidylinositol, a test
substance and EL,
[0086] (2) a step for comparing a concentration of
lysophosphatidylinositol when the test substance is contacted with
a concentration of lysophosphatidylinositol when the test substance
is not contacted, and
[0087] (3) a step for selecting the test substance as an inhibitor
of EL activity
[0088] when the concentration of lysophosphatidylinositol when the
test substance is contacted is decreased relative to the
concentration of lysophosphatidylinositol when the test substance
is not contacted.
[0089] In the step (2) of the above method, the concentration of
lysophosphatidylinositol which is a lipolytic product of
phosphatidylinositol is measured. The quantification of
lysophosphatidylinositol levels in the sample can be performed by
TLC development with reference to unexamined patent application
2010-63470 or mass spectrometry with reference to Chromatographia.,
2010, vol. 72(7/8), p. 659-664.
[0090] The present invention is described below in more detail by
the way of examples. However, the present invention is not limited
to the following examples.
Example 1
[0091] Verification of Substrate Specificity of EL for
Phospholipids in Mouse Plasma Lipoproteins
[0092] The pcDNA-DEST40 (Invitrogen Corp.) cloned DNA encoding
mouse EL was transfected into Freestyle 293F cells and the cells
were cultured at 37.degree. C. for 2 days in an atmosphere of 8%
CO.sub.2. The cells were collected by centrifugation of the culture
medium, suspended in Freestyle 293 Expression Medium (Invitrogen
Corp.) containing 20 U/ml heparin and incubated at 37.degree. C.
for 45 min. After that, the supernatant obtained by removing the
cells by centrifugation was used as mouse EL enzyme solution. The
reaction mixture consisting of preheparin plasma of C57BL/6J mice
and mouse EL enzyme solution(9:1, v/v) was incubated at 37.degree.
C. for 2 hr and added EDTA (final concentration of 10 mM) to
terminate the reaction. After that, various phospholipids in
reaction solution were quantified by mass spectrometry with
reference to Analytical Letters 2006., vol. 39, p. 957-972. From
the comparison of the change in each phospholipid levels between
incubation without and with EL addition, the phospholipids showing
specific change for EL addition were explored (FIG. 1-3). As a
result, the actions of lipase other than EL for phosphatidylcholine
and phosphatidylethanolamine were found, whereas it became clear
that EL alone showed the activity for phosphatidylinositol.
Example 2
[0093] Measurement of Mouse EL Activity and Evaluation of an
Inhibitor of EL Activity Using Phosphatidylinositol-HDL as a
Substrate
[0094] Phosphatidylinositol (Avanti Polar Lipids Inc.) in
chloroform was collected the necessary amount, which was completely
evaporated the solvent to dryness under nitrogen. After addition of
PBS, phosphatidylinositol-HDL induced phosphatidylinositol (2.5
mg/ml) into HDL (5 mg/ml) was prepared by mixing
phosphatidylinositol vesicle prepared by sonication on ice with
human HDL (Athens Research & Technology, Inc.) and incubating
at 4.degree. C. for 24 hr. The pcDNA-DEST40 (Invitrogen Corp.)
cloned DNA encoding mouse EL was transfected into Freestyle 293F
cells and the cells were cultured at 37.degree. C. for 2 days in an
atmosphere of 8% CO.sub.2. The cells were collected by
centrifugation of the culture medium, suspended in Freestyle 293
Expression Medium (Invitrogen Corp.) containing 20 U/ml heparin and
incubated at 37.degree. C. for 45 min. After that, the supernatant
obtained by removing the cells by centrifugation was used as mouse
EL enzyme solution. Phosphatidylinositol-HDL or HDL was added to
reaction solution including 20 mM Tris-HCl buffer (pH 7.5), 0.5%
bovine serum albumin, 4 mM calcium chloride and 150 mM sodium
chloride to achieve a final concentration of 2 mg/ml and finally,
mouse EL enzyme solution was added (total volume of 10 .mu.l).
After reaction at 37.degree. C. for 2 hr, the amount of NEFA
produced from substrate by EL was measured with NEFA C-test Wako
(Wako Pure Chemical Industries, Ltd.) and the amount of NEFA
(absorbance) was assumed an index of enzyme activity. The enzyme
activity was calculated by subtracting the background value
equivalent to the amount of endogenous NEFA without mouse EL enzyme
solution from the value measured in enzyme reaction of EL (FIG. 4).
As a result, mouse EL exhibited about 2-fold higher activity when
using phosphatidylinositol-HDL as a substrate than when using HDL
as a substrate, suggesting that phosphatidylinositol-HDL was higher
sensitivity substrate for EL than HDL. Furthermore, when the
inhibitory activity of a well-known lipase inhibitor, orlistat
against mouse EL was examined using phosphatidylinositol-HDL as a
substrate, a dose-dependent inhibition was observed, demonstrating
that phosphatidylinositol-HDL can be applied to evaluation of EL
inhibitor (FIG. 5).
Example 3
[0095] Measurement of Endogenous EL Activity in Plasma of
EL-Transgenic Mice
[0096] Phosphatidylinositol (Avanti Polar Lipids Inc.) in
chloroform was collected the necessary amount, which was completely
evaporated the solvent to dryness under nitrogen. After addition of
PBS, phosphatidylinositol-HDL induced phosphatidylinositol (2.5
mg/ml) into HDL (5 mg/ml) was prepared by mixing
phosphatidylinositol vesicle prepared by sonication on ice with
human HDL (Athens Research & Technology, Inc.) and incubating
at 4.degree. C. for 24 hr. The saline (Otsuka pharmaceutical Co.,
Ltd.) contained 50 KU/Kg heparin (Sigma Aldrich, Co.) was injected
into tail vein of EL-transgenic mice. After 30 min, the blood
obtained by drawing from the abdominal vena cava was centrifuged
and plasma containing endogenous EL by collecting the supernatant
was obtained. Phosphatidylinositol-HDL or HDL was added to reaction
solution including 100 mM Tris-HCl buffer, 5 mM calcium chloride
and 150 mM sodium chloride to achieve a final concentration of 1.5
mg/ml, and finally, plasma which was treated with an excessive
amount of anti-mouse EL neutralizing antibody or normal antibody
was added (total volume of 10 .mu.l). After reaction at 37.degree.
C. for 60 min, the concentration of NEFA produced by enzyme
reaction was measured with NEFA C-test Wako (Wako Pure Chemical
Industries, Ltd.) and the amount of NEFA (absorbance) was assumed
an index of enzyme activity. The enzyme activity was calculated by
subtracting the background value equivalent to the amount of
endogenous NEFA in blood from the measured value in enzyme reaction
(FIG. 6). The enzyme activity with anti-mouse EL neutralizing
antibody was defined as non-EL activity. The EL activity was
obtained by subtracting the enzyme activity with anti-mouse EL
neutralizing antibody from the enzyme activity with normal
antibody. As a result, the EL activity in plasma showed more
apparent activity for phosphatidylinositol-HDL than HDL and it was
revealed that the relative ratio of EL activity to non-EL activity
(EL activity/non-EL activity) using phosphatidylinositol-HDL as a
substrate was 3.5-fold higher than that using HDL as a substrate
(FIG. 6, 7).
Example 4
[0097] Comparison of Phosphatidylinositol and
Phosphatidylethanolamine Levels in Plasma Between Wild-Type Mice
and EL Knockout Mice
[0098] Phosphatidylinositol and phosphatidylethanolamine levels in
preheparin plasma collected from C57BL/6J strain, wild-type mice
and EL knockout mice were quantified by mass spectrometry with
reference to Analytical Letters 2006., vol. 39, p. 957-972 (FIGS. 8
and 9). As a result, phosphatidylinositol levels in plasma of EL
knockout mice showed 1.8-fold higher than that of wild-type mice,
whereas phosphatidylethanolamine levels of EL knockout mice were
only 1.1-fold higher than that of wild-type mice. These results
demonstrated that phosphatidylinositol levels express endogenous EL
activity highly sensitively.
Example 5
[0099] Change in HDL Cholesterol, Phosphatidylinositol and
Lysophosphatidylinositol Levels in Blood after Administration of
Anti-EL Neutralizing Antibody to Cynomolgus Monkey
[0100] HDL cholesterol, phosphatidylinositol and
lysophosphatidylinositol levels in preheparin plasma collected with
time after administration of anti-EL neutralizing antibody to
cynomolgus monkey were measured. HDL cholesterol was quantified by
automated analyzer (FIG. 10). Phosphatidylinositol and
lysophosphatidylinositol were quantified by mass spectrometry and
the total amount of each molecular species was calculated (FIG. 11,
12). As a result, HDL cholesterol, phosphatidylinositol and
lysophosphatidylinositol increased with time after administration
of anti-EL neutralizing antibody and the increasing rate at 7 hr
after administration of anti-EL neutralizing antibody showed 1.6
times in HDL cholesterol, 2.4 times in phosphatidylinositol and 2.6
times in lysophosphatidylinositol relative to each initial value
before administration of antibody.
Example 6
[0101] Measurement of EL Activity Using
Phosphatidylinositol-Liposomes, Phosphatidylinositol-Reconstituted
HDL and Phosphatidylinositol-Detergent Mixed Micelles as a
Substrate
[0102] Phosphatidylinositol-liposomes are prepared appropriately
with reference to The Journal Lipid Research, 1976, vol. 17(3), p.
239-247 and phosphatidylinositol-reconstituted HDL is prepared
appropriately with reference to The Journal of Biological
Chemistry, 1982, vol. 257(8), p. 4535-4540.
[0103] On the other hand, the measurement of EL activity using
phosphatidylinositol-liposomes or
phosphatidylinositol-reconstituted HDL as a substrate is performed
with reference to Biochemistry, 2003, vol. 42(46), p.
13778-13785.
[0104] On the other hand, phosphatidylinositol-detergent mixed
micelles are prepared appropriately with reference to The Journal
of Biological Chemistry, 1988, vol. 263(12), p. 5724-5731.
Furthermore, EL activity is measured using
phosphatidylinositol-detergent mixed micelles as a substrate with
reference to ibid.
* * * * *