MXPA06013354A - Methods involving micromosal prostaglandin e2 synthase - Google Patents
Methods involving micromosal prostaglandin e2 synthaseInfo
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- MXPA06013354A MXPA06013354A MXPA/A/2006/013354A MXPA06013354A MXPA06013354A MX PA06013354 A MXPA06013354 A MX PA06013354A MX PA06013354 A MXPA06013354 A MX PA06013354A MX PA06013354 A MXPA06013354 A MX PA06013354A
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Abstract
Methods of determining the potency, specificity, and toxicity of microsomal prostaglandinE2 synthase inhibitors are provided that utilize one cell-based assay system.
Description
METHODS TO DETERMINE THE POWER, SPECIFICITY AND TOXICITY OF PROSTAGLANDINE E2 MICROSOMAL SYNTHASE
FIELD OF THE INVENTION The present invention relates to a new and useful method for testing compounds and agents to determine their ability to reduce or inhibit the activity of microsomal prostaglandin E2 synthase (mPGES). Specifically, the present invention relates to assays for measuring the potency, specificity and toxicity of mPGES inhibitors.
DESCRIPTION OF THE RELATED ART Prostaglandins are a class of eicosanoids that play an important role in pain, fever and inflammation. They are synthesized from arachidonic acid and have a ring of five members of carbon atoms that had been part of the arachidonic acid chain. Prostaglandins act locally, that is, close to their synthesis site. Prostaglandin E2 intervenes particularly in the origin of fever, pain and inflammation (Funk, C. Science, 294: 1871-1875 (2001)). It has been shown that PGE2 is an important proinflammatory lipid mediator and inducer of hyperalgesia. The synthesis of PGE2 is catalyzed by two classes of enzymes. The first class of enzymes, cyclooxygenases, generates prostaglandin H2 (PGH2) using an arachidonic acid precursor released from membrane phospholipids. Examples of cyclooxygenases include COX-1 and COX-2. The second class of enzymes, PGE2 synthases, produces PGE2 using a PGH2 precursor. Examples of PGE2 synthases include cytosolic PGE2 synthase (cPGES) and microsomal PGE2 synthase (mPGES) (Murakami, M et al, Progress in Lipid Research 43: 3-35, (2004)). It has been shown that PGE2 is produced by two different routes.
The first route produces PGE2 at the basal level. The constitutively produced PGES is expressed in the cytosol (cPGES) under basal conditions in a wide variety of mammalian cells. Under basal conditions, COX-1 stimulates the production of PGH2 from arachidonic acid, which in turn produces basal levels of PGE2 after activation with cPGES. The second route produces PGE2 after induction by external stimuli such as cytokines and is located in the microsomal compartment of the cell; For this reason it is called mPGES. Upon stimulation by a proinflammatory stimulus such as cytokines, COX-2 reacts with arachidonic acid to produce PGH2, which in turn induces the production of PDGE2 from PGH2 by mPGES. (See figure 1). It is this second "inducible" route that is involved in inflammation and hyperalgesia, while it has been shown that the first "constitutive" route participates little or nothing in this process. (Murakami, M et al., J. Biol. Chem. 275: 32783-32792, (2000) and Tanioka et al, J. Biol. Chem. 275: 32775 -32782 (2000)). It is known that mPGES inhibitors are potentially useful in treating inflammation and pain. An mPGES inhibitor may have undesirable qualities such as lack of specificity and toxicity. For example, in addition to mPGES, there are other prostaglandin synthases, such as prostaglandin 12 synthase (PGIS), which catalyzes the formation of PGI2, a prostaglandin that produces vasation and inhibits platelet aggregation. A non-specific PGES inhibitor can inhibit these other prostaglandins synthases, resulting in undesirable side effects. In addition, mPGES inhibitors can inhibit the level of basal production of PGE2. As PGE2 plays an important role in fever, pain and inflammation, attempts have been made to create assays for compounds that can inhibit their production. In particular, techniques such as high pressure liquid chromatography (HPLC), enzyme-linked immunosorbent assays (ELISA), also known as enzyme immunoassays (EIA) or radioimmunoassays (RIA) have been used to quantify PGE2 production to determine the capacity of a compound or agent to reduce or inhibit the production of PGE2. In the past, the evaluations of potency, specificity and toxicity have been separated in different experiments. In addition, specificity was more feasible in biochemical assays that in many cases could not mimic the complex real conditions existing in cells. It is desirable to design a cell-based assay that measures the potency, specificity and toxicity of mPGES inhibitors in the same experimental sample. This design would shorten the steps that normally require secondary selection and provide more precisely associated information with cell conditions in vivo.
BRIEF COMPENDI OF THE INVENTION The present invention establishes a single cell-based assay to simultaneously assess the potency, specificity and cytotoxicity for mPGES inhibitors. Inhibitors of mPGES can be therapeutic strategies for inflammation and pain. The assay design is suitable for use in any mammalian cell line in which it has been shown that the production of PGE2 is induced by external stimuli. In the present invention, mammalian cells are treated with one or more cytokines, such as TNFa and
IL-1 ß. PGE2 occurs at such high levels that inhibitor assay compounds can be assessed by incubating the test compound with the cells and measuring the reduction of PGE2 in the supernatant. In addition to measuring the potency of a test compound, the present invention provides for specificity and cytotoxicity measurements in the same cell-based assay system. This is designed by evaluating a second reading of the prostanoid 6-keto PGF1a, and evaluating the effect of the test compound on the basal production of PGE2. The above aspects and other aspects, peculiarities and advantages of the present invention will be better understood from the following detailed description considered together with the attached figures.
BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 is a schematic representation of the metabolic routes of arachidonic acid. The abbreviations are the following: arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin H2 (PGH2), prostaglandin 12 (PGI2), 6-keto prostaglandin F1a (PGF1a), prostaglandin l2 synthase (PGIS), cytosolic PGE2 synthase ( cPGES), microsomal PGE2 synthase
(mPGES). FIG 2 is a representative selection system that uses both a single concentration (a single point) and multiple concentrations (8 points in this example) of compound in the mPGES cell-based assay. The test compounds can be rapidly selected using high-throughput screening followed by IC50 measurements in positive hits using multiple concentrations of test compound.
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a cell-based assay for the selection of inhibitors of microsomal PGE2 synthase (mPGES). The assay evaluates the potency, specificity and toxicity of PGES inhibitors as test compounds in living cells. The present invention allows the identification of inhibitory compounds that can be used as oral drugs to treat pain, fever, Alzheimer's disease and many inflammatory diseases including, but not limited to, asthma, allergic rhinitis, arthritis, inflammatory bowel diseases and multiple sclerosis. The present invention relates, in general, to a cell-based assay that is used to measure the potency, specificity and cytotoxicity of mPGES inhibitors as test compounds, in the same cell-based assay. Measurements can be made quickly and can be scaled up to test many different inhibitor compounds simultaneously. There are no particular restrictions regarding the inhibitor compound used. Examples are: low molecular weight chemical molecules, libraries of synthetic compounds of low molecular weight, purified proteins, expression products of gene libraries, synthetic peptide libraries, cell extracts and culture supernatants. Any mammalian cell line can be used in the present invention provided that the mammalian cell line can be induced by one or more cytokines to express mPGES. The greater the induction of PGE2 after the stimulation with cytokines, the greater the window of selection available for measuring the potency, specificity and cytotoxicity of the test compounds. The preferred cell line is A549, a line of human lung epithelial cells of macrocytic cancer that is commercially available. Nevertheless, A549 can be easily substituted by other mammalian cell lines, for example human cell lines such as HEK 293, monkey cell lines such as COS cells and mouse cell lines such as J774. Cell lines that exhibit high levels of PGE2 production upon stimulation by one or more cytokines are then used to measure the potency of the compounds by incubating the test compound with the cells after stimulation with one or more molecules of external stimulus. The external stimulus is selected among proinflammatory cytokines, growth factors and promoters.
The concentration of PGE2 can be easily measured before and after stimulation with one or more cytokines, growth factors, promoters and the like using a specific enzyme immunoassay for
PGE2. The potency of the inhibitor compound is measured by the reduction of the level of PGE2 in the supernatant after its addition. The present invention also contemplates any stimulus compound alone or in combination which, as demonstrated, stimulates the inducible PGE2 pathway upon mixing with the chosen cell type. In a preferred embodiment, cytokines, growth factors or tumor promoters alone or in combination can be used to stimulate the production of PGE2 in the chosen cell to induce PGE2 levels between
100 and 400 times with respect to the basal levels. In a more preferred embodiment, A549 cells are stimulated with TNFa and IL-1β to induce the COX-2 and mPGES-coupled pathway for the production of PGE2 (see Figure 1). In addition to potency, the present invention incorporates the evaluation of specificity and cytotoxicity of the test compounds in the same cell-based system. This was designed by evaluating a second reading of the prostanoid production of 6-keto PGF1a and evaluating the effect of the test compounds on the basal production of PGE2, which is mediated by the route coupled to COX-1 and cPGES. (See figure 1). The base inhibition measures the effect of the compounds on the production at the base level of PGE2, which is mediated by cPGES. The inhibition of the base level is determined by measuring the concentrations of PGE2 in non-induced cells in the absence and presence of the inhibitor compound.
The specific inhibition measures the desired effect of the compound: particularly, the production of PGE2 mediated by mPGES in an induced cell. Specific inhibition is measured by measuring PGE2 concentrations in cells induced in the absence and presence of the inhibitor compound. In this way, base and specific inhibition measurements are indicators of the cytotoxicity and specificity of the test compound. In a preferred embodiment, the A549 cells induced both with
TNFα as with IL1β produce a synergic increase of 400 times in the production of PGE2, which allows a more accurate measurement of inhibition levels (in terms of potency, specificity and toxicity) than previously contemplated. Non-specific inhibition measures other effects that the test compound can have, such as inhibition of enzymes other than mPGES. Nonspecific inhibition is determined by measuring the effect of the compound on the PGI2 pathway or other prostaglandin pathways, which catalyzes the production of PGI2, and finally 6-keto PGF1 a (or other prostaglandins), from the same substrate as mPGES . In a preferred embodiment, the concentration of 6-keto PGF1 a in the cell supernatant is measured after induction in the absence and in the presence of the compound. If it is shown that the compound inhibits the production of 6-keto PGF1 a as well as of PGE2, it can be deduced that the compound is not acting only on mPGES, but also on some previous element responsible for the production of its common precursor, such as PGH2 . Inhibition levels are determined not only by taking measurements in the presence and absence of the inhibitor compound, but also at various concentrations thereof. By taking measurements at various concentrations of inhibitor compound, it is possible to calculate certain properties such as its IC50.
An IC50 is defined as the concentration at which the compound produces a 50% inhibition. (See Figure 2, in which 8 concentrations ("8-points") of the compound are prepared). It is also understood that the assay of the present invention can also be carried out as a single-point assay by evaluating the inhibition of the compound on the production of PGE2 using only a concentration of compound. Both single-point selections and IC50 assays are susceptible to high-throughput screening. (See figure 2). High-throughput screening systems are available in the market (see, for example, Zymark Corp., Hopkington, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif .; Precision Systems, Inc., Natick, Mass., Etc.). These systems typically automate entire procedures including the pipetting of all samples and reagents, fluid distribution, scheduled incubations, and final reading of samples in the detector or appropriate detectors for the assay. These configurable systems provide high performance and fast startup, as well as a high degree of flexibility and customization. The manufacturers of these systems provide detailed protocols for the various high performance tests. Methods for measuring the concentration of PGE2 and 6-keto PGF1 a are well known in the art. Examples include high pressure liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), enzyme-linked immunosorbent assay
(ELISA), also known as enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence polarization (FP) and proximity scintillation assay (SPA). The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the appended figures. Such modifications are intended to be included within the scope of the appended claims. All references cited herein are incorporated by reference in their entirety.
EXAMPLES EXAMPLE 1: TREATMENT OF CELLS WITH CYTOKINES E
INCUBATION WITH TEST AND CONTROL COMPOUNDS In this example, A549 cells were grown in a 96-well flat bottom polystyrene plate at a density of 10 4 cells per well. Then, the plate was incubated overnight at 37 ° C. The next day, a COX-2 inhibitor, NS398, was thawed
(purchased from Cayman Chemical) and mixed well before use at a concentration of 1 mM. Aliquots of TNF-a and IL1-β (R & D Systems) were kept on ice until used. The test compound was thawed and placed in a tabletop mixer at 500 rpm. A series of sterile clustered tubes was arranged according to the following distribution:
A, B and C refer to media containing the TNF-α and IL1-β cytokines at three different concentrations of DMSO (reagents A, B and C) as described below. Reagent A contained cell culture medium at a concentration of 5 ng / ml of TNF-α and 5 ng / ml of IL1-β without DMSO. Reagent B was created by adding DMSO to reagent A at a DMSO concentration of 0.2%. Reagent C was created by adding DMSO to reagent A at a DMSO concentration of 0.3%. To the negative control tubes, 1 ml of culture medium without cytokines was added. To all tubes were added 200 μl of reagent A. To the first column of the pooled tubes was added 315 μl of reagent A. To the second column of the pooled tubes was added 315 μl of reagent B. The first row of the control tubes of NS398 was added 500 μl of reagent C. To the grouped tubes of test compound in rows 2-4 were added 450 μl of reagent C. To the positive control tubes were added 1 ml of reagent C. (See table above.) To prepare a stock solution of the test compound, 2 μl of test compound of a 10 mM stock solution was added to its corresponding tube (final dilution 1: 100) and the The mixture was vortexed.
Dilutions of the test compound were made as follows: 135 μl of the stock solution was taken and 30 μM was added to the tube and 45 μl of the stock solution was taken and 10 μM was added to the tube. Serial dilutions were made to generate concentrations of 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0.003 μM of compound. To prepare a control NS398 stock solution, 2 μl of 1 mM NS398 was added to its corresponding tube (final dilution 1: 100) and the mixture vortexed. Dilutions of NS398 were made as follows: 15 μl of the stock solution was removed and added to the 300 nM NS398 tube and 5 μl of the stock solution was removed and added to the 100 nM NS398 tube. Serial dilutions were made to generate concentrations of 300, 100, 30, 10, 3, 1, 0.3 and 0.1 nM of NS398. After completing all dilutions, the incubated cells were treated as follows. The medium was removed from the cells and rapidly replaced by the diluted compounds and controls. Then, the 96-well plates were incubated at 37 ° C for 16 hours. The plates were centrifuged at 1000 rpm for 5 minutes. 80 μl of the supernatant of each well was collected for assay in the enzyme immunoassay (EIA) or proximity scintillation assay (SPA) described in the following examples. The supernatants were stored at 4 ° C for a period of up to two hours or at -80 ° C for a period of up to three months. For an alternative short-term assay, A549 cells were first treated with TNFa and IL1β and incubated with cytokines at 37 ° C for 16 hours.
The culture medium was replaced with HBSS buffer plus 0.1% BSA and then the compounds diluted in series for a pre-incubation of 30 minutes were added. Subsequently, 10 μM arachidonic acid was added and the cell supernatant was collected after 30 minutes.
EXAMPLE 2: QUANTIFICATION OF THE PROSTAGLANDINE LEVEL: ENZYMATIC IMMUNOASSAY In this example, the concentration of PGE2 and 6-keto PGF1a were measured using an enzyme immunoassay (El A). EIAs for measuring PGE2 and 6-keto PGF1a are available in the market. For example, EIA kits of PGE2 and 6-keto PGF1 a are available in Assay Designs. The PGE2 EIA employs a mouse monoclonal antibody specific for PGE2. Free PGE2 in solution competes with a known amount of PGE2 indicator for binding to a limited amount of the anti-PGE2 antibody. The PGE2 indicator is a conjugate of PGE2 and acetylcholinesterase. The anti-PGE2 antibody is then fixed using a goat anti-mouse Ig antibody. The bound antibody is then developed with Ellman's reagent, which contains the substrate for acetylcholinesterase.
The product produced by this reaction has a yellow color and has a strong absorption at 412 nm. If there is a high concentration of PGE2, the PGE2 will surpass the indicator in the competition and the resulting sample will have a weak absorbance at 412 nm. On the contrary, if there is a low concentration of
PGE2, the indicator will surpass the PGE2 in the competition and the resulting sample will have a strong absorbance at 412 nm. When this test was performed, the absorbance at 412 nm was compared to a pattern of absorbances at known concentrations of PGE2. Similar assays can be performed for 6-keto PGF1 by substituting the monoclonal antibodies and the indicators used by the appropriate ones. This assay can easily be converted into an automatic format by one skilled in the art using a Zymark SciClone Deck, for example. After determining the levels of prostaglandin in cells treated with 8 concentrations of test compounds, an IC50 of the compound was generated by performing a semilogarithmic plot of the prostaglandin level versus the logarithmic scale of the compound concentration. This assay can also be performed as a single-point assay by evaluating the inhibition of the compound on the production of PGE2 using only a concentration of compound.
EXAMPLE 3: QUANTIFICATION OF THE PROSTAGLANDINE LEVEL: SCALED TEST FOR PROXIMITY In this example the concentration of PGE2 in a cell suspension was measured using a scintillation proximity assay ("SPA"). As described in U.S. Patent No. 4,568,649 for example, the SPA measures the concentration of a radiolabelled compound in a test sample. A one-night antibody and bead mixture was prepared by diluting an anti-PGE2 antibody (Cayman Chemical) in a test buffer at a 1: 6.67 dilution. The assay buffer used was TBS with 0.05% Tween 20.
Protein A SPA beads were then added in a volume equal to the mixture. The mixture was combined at 4 ° C overnight. A non-radioactive ("cold") PGE2 standard was diluted in 0.5% FBS medium. Samples of the supernatant to be measured were similarly diluted. An indicator was prepared in such a way that 3-H PGE2 had a total radioactivity of 50 mCi in a total volume of 500 μl, and the reference mean in 5 μl was 4.74 x 10? 5 cpm. The assay was performed by adding 20 μl of assay buffer to each well, 20 μl of cold standard or sample to the appropriate wells, 25 μl of indicator to all wells except the control blank wells, 130 μl of antibody / bead mixture to all wells except non-specific binding wells (NSB) and 130 μl of beads appropriately diluted without antibody to the NSB wells. The plate was stirred for two hours at room temperature. Radioactivity readings were taken using a Wallac 1450 Microbeta Trilux.
Claims (12)
1 .- A method for selecting a compound that specifically inhibits mPGES in mammalian cells that respond to elevated levels of PGE2 upon induction with an external stimulus, comprising the steps of: i) contacting a mammalian cell with said compound, ii) stimulate said mammalian cell with an external stimulus iii) measure the level of PGE2 and 6-keto PGF1a in said mammalian cell, and iv) compare the levels of PGE2 and 6-keto PGF1a in said mammalian cell with the levels of PGE2 and 6-keto PGF1a in a mammalian cell to which no compound was added and in another mammalian cell to which no compound or external stimulus was added; where the potency is determined as the ratio of the PGE2 levels in the presence and in the absence of compound in the cells exposed to the external stimulus and where the specificity and the cytotoxicity are determined as the ratio of the levels of 6-keto PGF1 a in presence and in the absence of compound in the cells exposed to the external stimulus.
2. - The method of claim 1, wherein said external stimulus is selected from one or more proinflammatory cytokines, growth factors and tumor promoters.
3. - The method of claim 2, wherein said cytokines are TNFa and IL1β.
4. - The method of claim 1, wherein said mammalian cell is selected from the group consisting of A549, HEK293, COS and J774.
5. - The method of claim 4, wherein said mammalian cell is A549.
6. - The method of claim 1, wherein said levels of PGE2 and 6-keto PGF1a are measured using HPLC, ELISA, GM-MS, EIA, RIA, FP and SPA.
7. A method for selecting a compound that specifically inhibits mPGES in mammalian cells that respond to high levels of PGE2 upon induction with an external stimulus, comprising the steps of: i) contacting a mammalian cell with said compound at two or more different concentrations of said compound, ii) stimulate said mammalian cell with an external stimulus ni) measure the level of PGE2 and 6-keto PGF1a in said mammalian cell, and iv) compare the levels of PGE2 and 6-keto PGF1 a in said mammalian cell with the levels of PGE2 and 6-keto PGF1a in a mammalian cell to which no compound was added and calculate a percentage of control value, and subsequently an IC50 value based on two or more points of concentration, and in another mammalian cell to which no compound or external stimulus was added; wherein the potency is determined as the IC50 of PGE2 in the presence and absence of compound in said mammalian cells exposed to the external stimulus and where specificity and cytotoxicity are determined as the IC50 of 6-keto PGF1a in the presence and absence of compound in said cells exposed to the external stimulus.
8. - The method of claim 7, wherein said external stimulus is selected from one or more proinflammatory cytokines, growth factors and tumor promoters.
9. The method of claim 8, wherein said cytokines are TNFa and IL1β.
10. - The method of claim 7, wherein said mammalian cell is selected from the group consisting of A549, HEK293, COS and J774.
11. - The method of claim 7, wherein said mammalian cell is A549.
12. The method of claim 7, wherein said levels of PGE2 and 6-keto PGF1a are measured using HPLC, ELISA, GM-MS, EIA, RIA, FP and SPA.
Applications Claiming Priority (1)
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US60/584,459 | 2004-06-30 |
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