The invention relates to methods for identifying macrolides having properties for reducing inflammation and associated sequalae (e.g., fibrosis).
Macrolide antibiotics having 14- or 15-member lactone rings are known to have anti-inflammatory properties in patients with chronic airway inflammation. There is need in the art for methods of identifying other macrolides with anti-inflammatory properties. Such macrolides may be useful for treating airway or other inflammation and associated conditions (e.g., cystic fibrosis).
The invention provides at least the following embodiments:
1. A method of screening test macrolides to identify potential anti-inflammatory agents, comprising:
1) contacting a first biological sample with a test macrolide; and
2) detecting, as an alteration, a first level of expression or activity of a target molecule involved in inflammation in the first biological sample relative to a second level of expression or activity of the target molecule involved in inflammation in a second biological sample that has not been contacted with the test macrolide.
2. The method of embodiment 1, wherein a test macrolide that alters the first level of alteration of expression or activity of the target molecule involved in inflammation relative to the second level of alteration of expression or activity of the target molecule involved in inflammation identifies the test macrolide as a potential anti-inflammatory agent.
3. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the level of de novo arachidonic acid biosynthesis.
4. The method of embodiment 3, wherein a macrolide compound that increases or decreases the level of de novo arachidonic acid biosynthesis is selected.
5. The method of embodiment 4, wherein the level of de novo arachidonic acid biosynthesis is detected by measuring incorporation of labeled arachidonic acid into phospholipids.
6. The method of embodiment 4, wherein the level of de novo arachidonic acid biosynthesis is detected by measuring incorporation of label into an intermediate in an arachidonic acid biosynthesis pathway.
7. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the level of extracellular matrix decomposition.
8. The method of embodiment 7, wherein a macrolide compound that decreases the level of extracellular matrix decomposition is selected.
9. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the leucocyte infiltration number.
10. The method of embodiment 9, wherein a macrolide compound that decreases the leucocyte infiltration number is selected.
11. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the level of expression of a gene involved in arachidonic acid biosynthesis or activation.
12. The method of embodiment 11, wherein a macrolide compound that increases or decreases the level of expression of the gene is selected.
13. The method of embodiment 11, wherein the gene is at least one type selected from the group consisting of A5 fatty acid desaturase (FADS1), A6 fatty acid desaturase (FADS2), fatty acid elongase, arachidonyl CoA synthetase, cyclooxygenase 2, prostaglandin E receptor EP4, cytosolic phospholipase A2, fatty acid desaturase, prostaglandin E synthase, prostaglandin E receptor EP1, prostaglandin 12 receptor, and secretory phospholipase A2 receptor 1.
14. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the level of expression of a gene involved in extracellular matrix decomposition.
15. The method of embodiment 14, wherein a macrolide compound that increases the level of expression of the gene is selected.
16. The method of embodiment 15, wherein the gene is at least one type selected from the group consisting of serine proteinase inhibitor, clade B (ovalbumin), and member 3 (SERPINB3).
17. The method of embodiment 14, wherein a macrolide compound that decreases the level of expression of the gene is selected.
18. The method of embodiment 17, wherein the gene is at least one type selected from the group consisting of matrix metalloproteinase 12 (MMP12), matrix metalloproteinase 19 (MMP19), matrix metalloproteinase 9 (MMP9), cathepsin C, and cathepsin K.
19. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the level of expression of a gene involved in leucocyte infiltration.
20. The method of embodiment 19, wherein a macrolide compound that decreases the level of expression of the gene is selected.
21. The method of embodiment 19, wherein the gene is at least one type selected from the group consisting of small inducible cytokine subfamily A (Cys-Cys) member 18 (CCL18), small inducible cytokine subfamily B (Cys-X-Cys) member 10 (CXCL10), small inducible cytokine subfamily B (Cys-X-Cys) member 11 (CXCL11), and small inducible cytokine subfamily B (Cys-X-Cys) member 9 (CXCL9).
22. The method of any one of embodiments 11, 14 and 19, wherein expression of the gene is determined by detecting mRNA, protein or protein synthesis.
23. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the activity of an enzyme involved in arachidonic acid biosynthesis.
24. The method of embodiment 23, wherein a macrolide compound that increases or decreases the activity of the enzyme is selected.
25. The method of embodiment 24, wherein the enzyme is at least one type selected from the group consisting of A5 fatty acid desaturase (FADS1), A6 fatty acid desaturase (FADS2), fatty acid elongase, and arachidonyl CoA synthetase.
26. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the activity of an enzyme involved in extracellular matrix decomposition.
27. The method of embodiment 26, wherein a macrolide compound that increases the activity of the enzyme is selected.
28. The method of embodiment 27, wherein the enzyme is at least one type selected from the group consisting of serine proteinase inhibitor, clade B (ovalbumin), and member 3 (SERPINB3).
29. The method of embodiment 26, wherein a macrolide compound that decreases the activity of the enzyme is selected.
30. The method of embodiment 29, wherein the enzyme is at least one type selected from the group consisting of matrix metalloproteinase 12 (MMP12), matrix metalloproteinase 19 (MMP19), matrix metalloproteinase 9 (MMP9), cathepsin C, and cathepsin K.
31. The method of embodiment 1, wherein the alteration of the target molecule in 2) above is an alteration in the activity of a chemokine involved in leucocyte infiltration.
32. The method of embodiment 31, wherein a macrolide compound that decreases the activity of the chemokine is selected.
33. The method of embodiment 32, wherein the chemokine is at least one type selected from the group consisting of small inducible cytokine subfamily A (Cys-Cys) member 18 (CCL18), small inducible cytokine subfamily B (Cys-X-Cys) member 10 (CXCL10), small inducible cytokine subfamily B (Cys-X-Cys) member 11 (CXCL11), and small inducible cytokine subfamily B (Cys-X-Cys) member 9 (CXCL9).
34. The method of embodiment 1, wherein the biological sample comprises cells.
35. The method of embodiment 1, wherein the biological sample comprises a cellular extract.
36. The method of embodiment 1, wherein the contacting in 1) above is carried out in vitro.
37. The method of embodiment 1, wherein the contacting in 1) above is carried out in VIVO.
38. The method of embodiment 1, wherein the contacting in 1) above is carried out in a cell culture.
39. The method of embodiment 38, wherein the cell culture comprises an epithelial cell line.
In embodiments below, the screening method for a macrolide compound having anti-inflammatory action includes the following steps.
1) contacting a first biological sample with a test macrolide; and
2) detecting, as an alteration, a first level of expression or activity of a target molecule involved in inflammation in the first biological sample relative to a second level of expression or activity of the target molecule involved in inflammation in a second biological sample that has not been contacted with the test macrolide.
In this screening method, a test macrolide that alters the first level of alteration of expression or activity of the target molecule involved in inflammation relative to the second level of alteration of expression or activity of the target molecule involved in inflammation may be identified as the test macrolide as a potential anti-inflammatory agent.
Specific examples of the alteration of the target molecule in the above-mentioned step 2) include
(i) that related to arachidonic acid biosynthesis,
(ii) that related to extracellular matrix decomposition, and
(iii) that related to leucocyte infiltration.
Specific examples of (i) above include an alteration in the level of de novo arachidonic acid biosynthesis, an alteration in the level of expression of a gene involved in arachidonic acid biosynthesis or activity, and an alteration in the level of activity of an enzyme involved in arachidonic acid synthesis.
Specific examples of (ii) above include an alteration in the level of extracellular matrix decomposition, an alteration in the level of expression of a gene involved in extracellular matrix decomposition, and an alteration in the activity of an enzyme involved in extracellular matrix decomposition.
Specific examples of (iii) above include an alteration in the leucocyte infiltration number, an alteration in the level of expression of a gene involved in leucocyte infiltration, and an alteration in the activity of a chemokine involved in leucocyte infiltration.
Furthermore, in accordance with the screening method of this embodiment, it becomes possible to carry out screening for a macrolide compound useful for, for example, an inflammatory disease of the lung.
Examples of the inflammatory disease of the lung include pulmonary emphysema (COPD), bronchitis, pulmonary fibrosis, acute lung injury, viral pneumonia, bacterial pneumonia, and acute respiratory disease syndrome (ARDS).
In such lung diseases, examples of compounds involved in the inflammation include those described in Categories 1 to 3 below.
Category 1: In inflammatory disease of the lung, decomposition of extracellular matrix forming lung tissue is important, and a compound that controls (decreases) expression or activity thereof can become a therapeutic drug. Category 1 relates to such compounds, and specific examples thereof include serine or cystein proteinase inhibitor (SERPINB3), which is present in the living body. In particular, SERPINB3 localizes in the lung, inhibits the activity of a cathepsin, which is activated by an inflammatory reaction, and suppresses the inflammatory action. As described in Examples below, clarithromycin (CAM) has an effect of enhancing the expression of a SERPINB3 gene.
Category 2: In inflammatory disease of the lung, decomposition of extracellular matrix forming lung tissue is important, and a compound that controls (decreases) expression or activity thereof can become a therapeutic drug. Category 2 relates to such compounds, and specific examples thereof include MMP12, MMP9, MMP19, cathepsin K, and cathepsin C. These compounds are proteinases that are important in lung inflammation. As described in Examples below, CAM suppresses the expression of these proteinase genes, which are induced during inflammatory stimulation.
Category 3: In inflammatory disease of the lung, infiltration of leucocytes (T cells, macrophages, neutrophils, etc.) into lung tissue is important in making sickness chronic and more virulent. Category 3 is a group of chemokines that promote leucocyte infiltration. As described in Examples below, CAM suppresses expression of the chemokine gene, which is induced during inflammatory stimulation. Because of this, it is useful as a therapeutic drug for suppressing leucocyte infiltration.
In the screening method of this embodiment, detecting an alteration, due to contact with a macrolide compound, in the level of expression or the activity of a target molecule involved in inflammation in the lung enables the action of the macrolide compound on the lung inflammatory action to be reliably evaluated and a macrolide compound that alters the level of expression or the activity of the target molecule to be identified. Because of this, it is possible to select a macrolide compound having an anti-inflammatory action as a material that is useful as a therapeutic drug.
Furthermore, with regard to the above-mentioned (i), using microarray-based expression analysis, we have shown that macrolide treatment of lung epithelial cell lines alters expression of multiple components in the pathway leading to arachidonate, prostaglandin, and leukotriene production. Delta 5 and delta 6 desaturases are responsible for generation of arachidonate from linoleic acid in the diet. Arachidonate is then incorporated into phospholipids. Upon stimulation (e.g., by interleukin 1), arachidonate is cleaved from phospholipid by phospholipase A2 (PLA2) and then undergoes further reactions to generate bioactive lipids, i.e., prostaglandins and leukotrienes. Delta 5 desaturase (FADS1) is consistently upregulated in A549 epithelial cells treated with immune-active, but not inactive, macrolides. In addition to FADS1, active macrolides specifically alter expression of genes encoding PLA2, PGE synthase, and PGE2 receptors. The pathway leading from arachidonate to generation of prostaglandins is highly validated as a therapeutic target for anti-inflammatory drugs, such as aspirin, inhibiting cyclo-oxygenases (COX1 and COX2).
Macrolides have been used clinically, and have demonstrated anti-inflammatory activity with few serious side effects, suggesting that other macrolides identified on the basis of arachidonic acid inhibition will also be safe.
Screening Methods
The invention provides methods of identifying macrolides having anti-inflammatory activity. Methods of the invention can be used to develop macrolides with improved activity and increased efficacy to suppress inflammation, or alternatively to identify new structures with desirable properties.
Examples of the first and second biological samples used in screening include cells and cell extracts.
Contacting these biological samples with a macrolide compound may be carried out in vitro or may be carried out in vivo. It may be carried out in a cell culture. Examples of the cell culture here include an epithelial cell line such as A549 and a macrophage cell line such as THP-1.
The case of the above-mentioned (i), that is, the case in which the target molecule is arachidonic acid or a material involved in arachidonic acid biosynthesis, is explained below as an example, and when carrying out measurement with respect to the above-mentioned other target molecules, screening can be carried out in accordance with the method as in the case of (i).
In the case of the above-mentioned (i) related to arachidonic acid biosynthesis, specifically, methods of the embodiment involve assaying whether a test macrolide alters biosynthesis of arachidonic acid, alters the expression or activities of enzymes involved in the biosynthesis or activation of arachidonic acid (e.g., arachidonic acid CoA synthetase, delta-6 desaturase, fatty acid elongase, and delta-5 desaturase, AA-related genes in Tables 2 and 3), or alters the expression of genes identified as predictive for the anti-inflammatory macrolide effect (Appendix E). Assays in which arachidonic acid or gene expression or enzymatic activity is detected can be carried out after contacting a cell with a test macrolide. Any cell line that secretes prostaglandin or produces arachidonic acid can be used. Epithelial and macrophages lines (e.g., A549 or THP-1) are suitable for this purpose; other cell lines can be identified by carrying out assays such as those described in the specific Examples, below. Alternatively, screening assays can be carried out after adding a test macrolide to an in vitro cell system (e.g., a cellular extract or liver microsome preparation) or an in vitro enzyme assay system.
Furthermore, in the case of the above-mentioned (ii) related to extracellular matrix decomposition, the following can be selected as macrolide compounds.
Those that decrease the level of extracellular matrix decomposition;
those that increase the level of expression of a gene involved in extracellular matrix decomposition, such as serine proteinase inhibitor, clade B (ovalbumin), and member 3 (SERPINB3) (Appendix F);
those that decrease the level of expression of a gene involved in extracellular matrix decomposition, such as matrix metalloproteinase 12 (MMP12), matrix metalloproteinase 19 (MMP19), matrix metalloproteinase 9 (MMP9), cathepsin C, and cathepsin K (Appendix F);
those that increase the activity of an enzyme involved in extracellular matrix decomposition, such as serine proteinase inhibitor, clade B (ovalbumin), and member 3 (SERPINB3); or
those that decrease the activity of an enzyme involved in extracellular matrix decomposition, such as matrix metalloproteinase 12 (MMP12), matrix metalloproteinase 19 (MMP19), matrix metalloproteinase 9 (MMP9), cathepsin C, and cathepsin K.
In the case of the above-mentioned (iii) related to leucocyte infiltration, the following can be selected as macrolide compounds.
Those that decrease the leucocyte infiltration number;
with regard to chemokines such as small inducible cytokine subfamily A (Cys-Cys) member 18 (CCL18), small inducible cytokine subfamily B (Cys-X-Cys) member 10 (CXCL10), small inducible cytokine subfamily B (Cys-X-Cys) member 11 (CXCL11), and small inducible cytokine subfamily B (Cys-X-Cys) member 9 (CXCL9), those that decrease the level of expression of a gene involved in leucocyte infiltration (Appendix F); or
with regard to chemokines such as small inducible cytokine subfamily A (Cys-Cys) member 18 (CCL18), small inducible cytokine subfamily B (Cys-X-Cys) member 10 (CXCL10), small inducible cytokine subfamily B (Cys-X-Cys) member 11 (CXCL11), and small inducible cytokine subfamily B (Cys-X-Cys) member 9 (CXCL9), those that decrease the activity of a chemokine involved in leucocyte infiltration.
Test Macrolides
“Test macrolides” according to the invention include any macrolide desired to be tested for potential anti-inflammatory activity. Test macrolides may or may not be known to have anti-bacterial or other activities. Macrolides known to have anti-inflammatory activity can be screened in methods of the invention, for example to screen such macrolides for relative potency. Screening methods of the invention also can be used to assess relative potency after modification of test macrolides.
Preferred test macrolides have 14- or 15-member lactone rings and can be natural products or semi-synthetic derivatives thereof. Representatives of macrolides having 14-member lactone rings are the erythromycins, produced from Streptomyces erythreus, such as erythromycin A, erythromycin B, erythromycin C, erythromycin D, erythromycin E, erythromycin estolate, erythronolid, and clarithromycin. Other examples of macrolide antibiotics with 14-member lactone rings include megalomycin and its derivatives, picromycin, narbomycin, oleandomycin, triacetyl-oleandomycin; and the neutral compounds laukamycin, kujimycin A, albocyclin, and cineromycin B. Examples of macrolides having 15-member lactone rings include azalides, such as azithromycin.
Test macrolides can be synthesized using methods well known in the art. Methods of synthesizing macrolides are disclosed, for example, in U.S. Pat. Nos. 4,921,978; 4,933,474; 4,963,582; 5,023,348; 5,034,542; 5,116,968; and 6,100,239.
Typically, a test macrolide identified as a potential anti-inflammatory agent either (1) increases or decreases biosynthesis of arachidonic acid intermediates or products by at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200% or more, (2) alters (i.e., increases or decreases) expression of a gene in the arachidonic acid biosynthesis pathway by at least 1.3-fold as determined by methods described in the specific examples, below, or (3) alters (i.e., increases or decreases) enzymatic activity of an enzyme in the arachidonic acid biosynthesis pathway by at least 10, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200% or more.
Assays for Decreased Arachidonic Acid Production
Quantitative in vitro or in vivo assays can be used to determine whether arachidonic acid production decreases in response to a test macrolide, e.g., by measuring a decrease in radiolabeled arachidonic acid or measuring an increase or decrease of radiolabeled precursors, as is known in the art. Free arachidonic acid can be measured, for example, as described in U.S. Pat. No. 5,663,053. Alternatively, liver microsomes can be incubated in the presence of radiolabeled linoleic acid or other precursor, and the production of labeled arachidonic acid can be detected, as is known in the art. Alternatively, incorporation of label into phospholipids containing arachidonic acid could be measured. See, e.g., Obukowicz et al., Biochem. Pharmacol. 55, 1045-58, 1998; Obukowicz et al., J. Pharm. Exptl. Ther. 287, 157-66, 1998. This type of assay can readily be adapted to high throughput screening (see below).
Determining Gene Expression
Either mRNA or protein can be detected to determine gene expression. mRNA can be detected a variety of methods well known in the art, including, but not limited to, RNA dot blots, slot blots, Northern blots, RT-PCR (including TaqMan RT-PCR), SAGE (serial analysis of gene expression), and electrochemical detection of nucleic acid hybridization (e.g., U.S. Pat. No. 6,361,951). See also US 20030096782; Liang & Pardee, Science 257, 967, 1992; Higuchi et al., BioTechnology 10, 413-17, 1992, and Higuchi et al., BioTechnology 11, 1026-30, 1993; Holland et al., Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991; Heid et al., Genome Res. 6, 986-94, 1996; and Gibson et al., Genome Res. 6, 995-1001, 1996. Alternatively, mRNA products can be identified by contacting RNA with one or more oligonucleotide or cDNA probes unique to the FADS1, FADS2, or fatty acid (elongase) gene, either in solution or immobilized on a solid support, such as a membrane or a gene expression “chip.” Such chips can be purchased commercially (e.g., from Affymetrix or Motorola) or can be custom made.
Protein can be detected using a variety of techniques known to the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, protein synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into protein products.
Measuring Individual Enzyme Activity
Various assays are available to measure the activities of delta-6 desaturase (FADS2), fatty acid elongase, delta-5 desaturase (FADS1), and arachidonyl CoA synthetase. For delta-6 desaturase, these include, but are not limited to, assays described in U.S. Pat. No. 6,492,108 and references disclosed therein. For fatty acid elongase, assays include those described in Chang et al., J. Nutr. 122, 2074-80, 1992; U.S. Pat. No. 6,307,128; and US 20030104596. For FADS1 enzyme activity, assays include those described in de Gomez Dumm et al., Lipids 18, 781-88, 1983; Leikin & Brenner, Biochim. Biophys. Acta 963, 311-19, 1988; Chanussot et al., Diabetolgia 32, 786-91, 1989; Venkatesan et al., Biomed. Chromatography 4, 234-38, 1990; Maniongul et al., Lipids 28, 291-97, 1993; Su & Brenna, Anal. Biochem. 261, 43-50, 1998; Leonard et al., Biochem. J. 347, 719-24, 2000; Nishida et al., J. Pineal Res. 32, 26-33, 2002. Obukowicz et al., J. Pharm. Exptl. Ther. 287, 157-66, 1998, describes quantitative in vitro or in vivo assays for both delta-6 and delta-5 desaturase activities. See also Obukowicz et al., Biochem. Pharmacol. 55, 1045-58, 1998. Arachidonyl CoA synthetase activity can be measured as described, for example, in Bhat & Block, Am. J. Physiol. 262, L606-13, 1992; Beaumelle & Vial, Biochim. Biophys. Acta 958, 1-9, 1988; or Gerritsen & Perry, Biochim. Biophys. Acta 1045, 174-79, 1990.
Measuring Level of Extracellular Matrix Decomposition
An inflammatory substance (e.g. lipopolysaccharide, inflammatory cytokine, tobacco smoke, hydrochloric acid, anticancer agent) is pulmonarily or systemically administered to an animal. After administration of the inflammatory substance, lung lavage fluid or serum of an animal to which administration of the inflammatory substance has been carried out or has not been carried out is prepared. The amount of desmosine that is solubilized accompanying extracellular matrix decomposition is measured by the ELISA method (Laurent P et. al. J Immunol Methods. 1988 Feb. 24; 107(1): 1-11.)
Alternatively, the amount of solubilized hydroxyproline is measured in accordance with a standard method.
Measuring Leucocyte Infiltration Number
An inflammatory substance (e.g. lipopolysaccharide, inflammatory cytokine, tobacco smoke, hydrochloric acid, anticancer agent) is pulmonarily or systemically administered to an animal. After administration of the inflammatory substance, lung lavage fluid is recovered from a mouse to which administration of the inflammatory substance has been carried out or has not been carried out, and by counting the leucocyte infiltration number, the leucocyte infiltration number accompanying the inflammation can be measured.
Furthermore, after administration of the inflammatory substance, a lung pathological specimen is prepared from a mouse to which administration of the inflammatory substance has been carried out or has not been carried out, and by counting the number of cells that are stained by an antibody that recognizes leucocytes (e.g. MAC-1), the leucocyte infiltration number accompanying the inflammation can be measured.
Measuring Chemokine Activity
An inflammatory substance (e.g. lipopolysaccharide, inflammatory cytokine, tobacco smoke, chemical substance, anticancer agent) is pulmonarily or systemically administered to an animal. After administration of the inflammatory substance, a lung lavage fluid and a soluble fraction of lung lysate are prepared from an animal to which administration of the inflammatory substance has been carried out or has not been carried out. Alternatively, an inflammatory substance is added to cultured cells, and the cell supernatant is prepared to give a chemokine-containing liquid.
This chemokine-containing liquid is used to measure the activity of chemokine toward leucocytes by a Boyden chamber assay. Alternatively, the chemokine-containing liquid is contacted with highly chemokine receptor-expressing cells, and a transient increase in the intracellular Ca concentration is detected to give an activity.
High Throughput Screening
Using high throughput screening, many discrete test macrolides can be tested in parallel so that large numbers of test macrolides can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format. Alternatively, “free format assays,” or assays that have no physical barrier between samples, can be used. See, e.g., Jayawickreme et al., Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18, 1994. Another high throughput screening method is described in Beutel et al., U.S. Pat. No. 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.
All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.
Identification of Differentially Expressed Genes
Microarray experiments were carried out to identify genes that are differentially expressed in response to various immune stimuli in the presence or absence of macrolides. The data for each cell line (A549 and THP-1) are presented in Appendices A and B. For each cell line, the data for all of the hybridizations are represented together in the same table. For each hybridization, there are two columns: the weighted mean ratio (wtd mean ratio) and the minimum fold change (min fold change). The “min fold change” represents the minimum change in expression that meets the 95% confidence threshold. It can be estimated from the log ratio and standard deviation, assuming that the uncertainty in the ratio is normally distributed. Note that the minimum fold change can never be less than one (hence the minimum function). See also Cheng Li and Wing Hung Wong, “Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application,” Genome Biology 2001 2(8):research0032.1-0032.11 (published 3 Aug. 2001).
The analysis method to generate these tables was similar for A549 and THP-1, cells. All of the significance threshold criteria were set for the “min fold change” column, which ensured that the resulting genes would be differentially expressed by greater than the threshold value with 95% confidence (p value <0.05). The lowest significance threshold used was 1.3-fold, because it has been demonstrated that a 1.25-fold threshold has a false positive rate of less than 1 in 1000. It is important to note that the fold change threshold is an absolute number, and the direction of change (increased or decreased expression) must be determined by the looking at the weighted mean ratio. The significance threshold criteria are included in parentheses in each “min fold change” column. The weighted mean ratios representing significant differentials (95% confidence, p value <0.05) are in bold. The “Spot IDs” correspond to publicly available cDNA sequences for the region of the gene represented at that spot on the microarray chip.
For the A549 and THP-1 cell lines (Appendix A and B, respectively), the first table for each cell line (“total differentials”) represents all of the genes that show differential expression in at least one of the hybridizations. The second table for A549 and THP-1 cells (“drug effects, sum ≧4.5” and “drug effects, sum ≧5”, respectively) has an additional final column, “sum of min fold change.” This column represents the sum of the minimum fold changes for all of the groups except the stimulus alone. This column was used to rank the genes in order of the greatest global drug effect, independent of whether the drug used was Clarithromycin or FMA9045 and independent of stimulus effect. A sum min fold change of greater than or equal to 4.5 indicates that at least one of the four groups (Clari alone, FMA9045 alone, Clari+stimulus, FMA+stimulus) had a minimum fold change of at least 1.5.
For A549 cells, the tables of Appendix A include genes that met one or more of the following criteria: greater than 1.5-fold change for IL-1β stimulation and greater than 1.3-fold change for each of the drug groups. As noted above, a sum min fold change ≧4.5 was used as the threshold for ranking the most highly regulated drug effects. In both tables in Appendix A, the genes that meet this criterion (sum ≧4.5) are in bold, and those genes that are bold and italics are the most highly regulated genes and have a min fold change ≧5.0.
For THP-1 cells, the tables of Appendix B include genes that met one or more of the following criteria: greater than two-fold change for LPS stimulation, greater than 1.5-fold change for clarithromycin alone and for clarithromycin+/−LPS, and greater than 1.3-fold change for FMA9045 alone and for FMA9045+/−LPS. For THP-1 cells, a sum min fold change ≧5.0 was used as the threshold for ranking the most highly regulated drug effects. In both tables in Appendix B, the genes that meet this criterion (sum ≧5) are in bold and in italics. A larger sum min fold change threshold was used for THP-1 cells, because the macrolide response in this cell line was more robust than that in A549 cells.
Results Overview
The global transcription response to the various culture conditions is shown in Table 1, below. The min fold change threshold (noted next to each condition) was used to determine the total number of significant differentially expressed genes (95% confidence).
The results of the microarray experiments in the A549 cell line reveal that a large number of genes (>175) are regulated in response to IL-1β stimulation. Similarly, in the THP-1 macrophage line, a large group of genes (>200) are regulated in response to LPS stimulation. While these numbers of differentially expressed genes are similar, it is important to note that the min fold change threshold was set lower for the IL-1, stimulus response in A549 cells (1.5), as compared to a threshold of 2.0 for the LPS stimulus response in THP-1 cells. This is because the transcriptional response to stimulus was larger in THP-1 cells than in A549 cells.
In cells treated with clarithromycin alone, a similar number of genes were differentially expressed in each cell line (117 genes in A549 cells and 127 genes in THP-1 cells). In cells treated with stimulus (IL-1β or LPS)+clarithromycin, the number of differentially expressed genes also was similar (127 genes in A549 and 120 genes in THP-1). As with the stimulus alone condition, however, the min fold change threshold was set lower for the A549 cells for both the clarithromycin alone and the stimulus+/−clarithromycin conditions (1.3 for A549 versus 1.5 for THP-1). Again, this is because the global transcription response was not as robust in the A549 cells as it was in the THP-1 cells.
In both A549 and THP-1 cells, the response to FMA9045 treatment was much weaker than the response to Clarithromycin. In cells treated with FMA9045 alone, a similar number of genes were differentially expressed in both cell lines (20 genes in A549 cells and 22 genes in THP-1 cells). In response to stimulus (IL-1β or LPS)+FMA9045, 16 genes were differentially expressed in A549 cells, and 25 genes were differentially expressed in THP-1 cells. In contrast to the stimulus alone and the Clarithromycin conditions, the min fold change threshold was set at 1.3 for both A549 and THP-1 cells, because the overall response to FMA9045 was relatively weak.
Functions Associated with Macrolide Target Genes
Genes that were differentially expressed in one or more of the macrolide treatment groups were categorized into several groups, as shown in Tables 2 and 3. A selection of representative genes is included under each category, with the full number of differentially expressed genes in each category in parentheses next to the category name. Similar patterns were apparent in both the A549 and THP-1 cell lines, although the particular genes in each category were often different. The macrolide-regulated genes considered for these tables are from the “drug effects” tables in Appendices A and B. As discussed in Example 1, genes in these tables were ranked in order of their sum min fold change, and only the most highly regulated genes that were above a certain threshold were included. For THP-1 cells, genes that had a sum min fold change above 5.0 were included, whereas for A549 cells, genes that had a sum min fold change above 4.5 were included.
In THP-1 cells (Table 2), the largest number of responsive genes were those encoding the cytokines and chemokines (27 total). Interestingly, a large number of genes fell into the global category of tissue/matrix remodeling (30 total). Genes in this category included many protease-related genes (matrix metalloproteinases, thrombin-related genes), as well as structural/matrix genes (fibronectin and collagen) and EGF-related genes. Importantly, the expression of the matrix metalloproteinases was (in most cases) decreased in the presence of stimulus plus macrolide (compared to stimulus alone), while the expression of the metalloproteinase inhibitor TIMP2 was increased in the same treatment group, suggesting coordinate regulation. The signaling cascade of phospholipase A2 to prostaglandin E-related genes (arachidonic acid-related signaling) was another notable set of differentially expressed genes. In this case, PTGS2 and PLA2G4A showed increased expression in response to stimulus and then an additional increase in expression in response to stimulus plus macrolide.
In A549 cells (Table 3), the global transcriptional response to the various treatment conditions was not as robust as that seen in THP-1 cells, but the differentially expressed genes were in similar categories to those seen in THP-1 cells. Similar to THP-1 cells, a large number of genes were in the global category of tissue/matrix remodeling (26 total). There was quite a bit of overlap in the differentially expressed tissue/matrix remodeling-related genes between A549 and THP-1 cells, including several protease-related genes (MMP7, TIMP2, THBS1), and multiple EGF ligands (AREG, EREG, NRG). Also similar to THP-1 cells, genes involved in arachidonic acid signaling were differentially expressed in A549 cells (including FADS1, PTGES, PTGER1, and PLA2G4B), although the specific genes did not overlap with the differentially expressed genes in THP-1 cells. Similar to the observation in THP-1 cells, these arachidonic acid-related genes were up-regulated in response to macrolide. Up-regulation of gene expression is a common signaling feedback response following inhibition of enzyme(s) activity in that signaling pathway.
Macrolide-dependent changes in gene expression in THP-1 and A549 cell lines fall into several functional categories which are relevant to airway inflammation, including tissue/matrix remodeling and arachidonic acid metabolism. Previous microarray results suggested a number of possible target signaling pathways, including thrombin/PAR-1, TGFβ, bradykinin B2 receptor, PGE2, and arachidonic acid (AA) biosynthesis/metabolism. Experiments were then designed to determine which of these pathways was most likely to be the actual signaling pathway targeted by macrolides. In addition, a microarray experiment was carried out with THP-1 macrophages to examine the effect of macrolides on a larger number of genes on multiple microarray slides. The microarray data demonstrated that thrombin, TGFβ, and PGE2 signaling pathways were not likely the direct targets of macrolide activity, although TGF-β may be indirectly affected.
The data from the initial microarray experiments suggested that either the PGE2 signaling pathway or the AA pathway might be a target of macrolides. The PGE2 signaling pathway is downstream of AA metabolism, and cells respond to PGE2 stimulation by changes in cAMP or Ca levels.
A number of genes involved in PGE2 biosynthesis from AA (PLA2, PGE synthase/PTGES), as well as the PGE receptor (PGER) EP1 and EP4 subtypes, were differentially expressed in response to macrolides, suggesting the PGE2 pathway as a possible macrolide target. However, in preliminary experiments, neither A549 nor THP-1 cells showed PGE-dependent changes in cAMP levels. This data suggested that it was unlikely that macrolides were inhibiting PGER-dependent signaling if the cells were not responsive to PGE2. An additional experiment demonstrated, however, that macrolide treatment caused a small, but reproducible, decrease in PGE2 production, in response to IL-1β stimulation. This indicates that macrolides have a regulatory effect on the AA pathway.
The AA pathway can be split into AA biosynthesis and AA metabolism. In the biosynthetic portion of the pathway, AA is synthesized from lipid in the diet and incorporated into phospholipids. AA metabolism involves AA release from phospholipids, usually in response to immune stimulation, and subsequent generation of a variety of lipid products, including prostaglandins and leukotrienes.
FADS1 is a key enzyme in the biosynthesis of AA (
Prostaglandins (including PGE2) and leukotrienes are inflammatory modulators generated from AA. This pathway is attractive as a target of macrolide anti-inflammatory activity, because interference with this pathway is well validated therapeutically by inhibitors of COX1 and 2. In addition to prostaglandins and leukotrienes, other lipid products generated from AA include prostacyclins (from COX enzymes), thromboxanes (from COX), and HETEs (from 12- and 15-lipoxygenases).
To better understand the ability of macrolides to regulate the AA signaling pathway, a microarray experiment was carried in A549 cells to compare the transcriptional response of macrolides to that of several inhibitors of the AA pathway (
A549 cells were pre-incubated overnight (17 hours) with macrolides (10 μM FMA-9045, 50 μM clarithromycin, 50 μM roxithromycin, 50 μM josamycin) and 15 μM triacsin C, and pre-incubated 2 hours with 100 nM indomethacin and 100 nM dexamethasone. All cells then were stimulated for 4 hours with IL-1β, because the activation of PLA2 and downstream signaling events generally requires an inflammatory stimulus. The various treatment conditions are shown below in Table 4. Following macrolide and inhibitor pre-treatment and IL-1 stimulation, the cells were lysed, total RNA was collected, and the poly-A+ mRNA fraction was purified. Competitive hybridizations to the microarray were performed as shown in Table 5. The resulting microarray data (Appendix C) were analyzed using the methods described in the Examples above.
Results
The tables in Appendix C represent the effects of the arachidonic acid pathway inhibitors (“AA pathway inhibitors”) and the effects of the macrolides (“IL1+macrolide effects”). The first table (“AA pathway inhibitors”) represents genes that show differential expression (>1.3-fold) in response to at least one of the inhibitors. The second table (“all macrolides”) represents genes that show differential expression (>1.3-fold) in response to at least one of the macrolides. In both tables in Appendix C, the genes that meet this criterion (≧1.3 min fold change) are indicated in bold.
The global transcriptional response to the various culture conditions is shown in Table 6. The min fold change threshold was used to determine the total number of significant differentially expressed genes (95% confidence).
The main focus of the analysis was to look at the similarities between the response to AA pathway inhibitors and the response to macrolides. It was anticipated that there would be an overlap in the transcriptional responses if the macrolides and the AA pathway inhibitors were acting on the same pathway. In contrast, if macrolides act on a pathway other than the AA pathway, the response to macrolides and the response to the inhibitors should be dissimilar. Additionally, an inhibitor that acts further upstream in a signaling pathway might be expected to affect more genes than an inhibitor acting lower in the pathway.
Arachidonic Acid Biosynthetic Pathway Inhibitors
A small number of genes responded to indomethacin treatment (37), while a larger number of genes responded to triacsin C treatment (228). This is consistent with the idea that inhibitors acting upstream in a pathway will influence transcription of more genes than inhibitors of final steps in a pathway. Triacsin interferes with the biosynthetic portion of the AA pathway by inhibiting AA incorporation into phospholipids. Triascin may also affect other steps in the AA pathway due to its structural similarity to AA, but this has not been demonstrated. Indomethacin inhibits AA metabolism induced by various stimuli by blocking COX enzyme activity, and thus PGE2 production. Dexamethasone affects multiple steps in the AA pathway as well as other unrelated pathways. The number of genes responding to dexamethasone was quite large (341), as predicted based on the multiple transcriptional targets of glucocorticoid receptors.
Many genes were differentially regulated in response to more than one of the AA pathway inhibitors. The overlap of the responses is shown in a Venn diagram, in which numbers represent genes similarly regulated by the corresponding treatments (
Looking at overlap between pairs of inhibitors, the indomethacin-dependent response is almost entirely a subset of the triacsin C response: 37 genes were differentially expressed in response to indomethacin, and 36 of these 37 genes were differentially expressed in response to both triacsin C and indomethacin. This is not unexpected, considering the known mechanism of action of these inhibitors. Indomethacin inhibits COX1 and COX2, and therefore acts downstream of triacsin C on the AA biosynthetic pathway (
In contrast to the strong overlap between triacsin C and indometbacin, the overlap between the response to dexamethasone and the response to triacsin C and indomethacin is smaller. Some overlap was expected, because dexamethasone does affect the AA pathway. However, dexamethasone is a more general inhibitor, affecting pathways other than the AA pathway, so there are also many other genes affected by dexamethasone presumably through other pathways.
Quantitative analysis was done to determine how well the responses to the different AA pathway inhibitors correlated. The correlation values were assigned a p-value to provide statistical confidence to the correlation value. Correlation between experiments, across all spots showing good detection, was assessed by cosine vector angle to generate the data shown in Tables 7-9. A correlation coefficient close to one indicates strong correlation, whereas a value near zero indicates no correlation. P-values for each correlation coefficient were estimated based on a Fisher permutation test. In this test, each spot's expression ratios were randomly re-ordered across experiments and the degree of correlation between experiments was then reassessed. This was done repeatedly. The p-value is the fraction of these random reorderings that result in a larger cosine vector angle than was observed for the unpermuted data. This method of assessing significance was the most conservative of several that were tested. Using Pearson correlation coefficient instead of cosine vector angle also gave equivalent results.
This analysis further supports the conclusions drawn from the amount of gene overlap between the different responses. The dexamethasone response is not strongly correlated with the response to either triacsin C (0.196, p-value 0.1) or indomethacin (0.266, p-value 0.009), reflecting its additional transcriptional targets outside the AA pathway. In contrast, the AA pathway inhibitors triacsin C and indomethacin show a significant correlation (0.485, p-value <0.001), consistent with their shared effect on PGE2 biosynthesis. A related analysis, which only used the subset of genes differentially expressed in response to triacsin C for the correlation assessment, showed an even stronger correlation between triacsin C and indomethacin (0.621, p-value 0.1), while the correlation with the dexamethasone response was still low and was not significant (0.281, p-value 1.0).
Overlap Between Macrolide Responses
The genes differentially expressed in the presence of clarithromycin, FMA-9045, and roxithromycin overlapped moderately (
In this experiment, the transcriptional response to the active macrolides (clarithromycin and roxithromycin) also overlapped with the transcriptional response to josamycin (see Venn diagram,
As with the AA pathway inhibitors, the correlation analysis of the different macrolides (Table 8) reinforces the observed overlapping gene response. All the macrolides are well correlated with one another, and these correlation coefficients are statistically highly significant (p-values <0.001).
Overlap Between Pathway Inhibitors and Macrolide Responses
An experiment was carried out to address whether macrolides affect the arachidonic acid pathway. The similarity or dissimilarity of macrolide and AA pathway inhibitor transcriptional profiles should reflect the degree to which they have similar effects on the pathway. The results support the hypothesis that macrolides inhibit the AA pathway.
The transcriptional response to clarithromycin and roxithromycin overlapped significantly with the response to triacsin C and indomethacin (
The quantitative analysis of the correlations between the response to active macrolides and the response to AA pathway inhibitors (Table 9) demonstrates that responses to all 3 active macrolides are significantly correlated with those to both triacsin C and indomethacin. Responses to clarithromycin and roxithromycin are most highly correlated with the triacsin C response. The effects of macrolides on epithelial cells are most similar to those of triacsin C and indomethacin, inhibitors targeting the AA pathway, supporting the hypothesis that the AA pathway is a likely macrolide target.
A similar analysis was performed using only the subset of genes differentially expressed in response to clarithromycin for the correlation assessment. The subset analysis demonstrates that clarithromycin-induced responses are significantly correlated with triacsin C (0.916, p-value 0.001). The correlation of clarithromycin with indomethacin for this subset of genes was less significant (0.848, p-value 0.07), and little correlation was observed with dexamethasone (0.430, p-value 1.0).
Effect of Triacsin C on Macrolide Response
If it is true that macrolides act on the AA pathway, then chemically inhibiting the pathway is expected to block the effects of macrolides on AA-related gene expression. For example, if the macrolide target is FADS1, then inhibition of AA biosynthesis with triascin should result in a similar gene expression profile with or without the addition of macrolide. This is true because FADS1 is upstream of the step blocked by triascin. The ability of macrolides to alter gene expression in the presence of triascin was tested directly by competitive hybridization of IL1+Triacsin vs. IL1+Triacsin+Clari/Roxi (Table 7, mRNA from culture conditions 8 v. 10 or 11). It is important to remember that results of direct competitive hybridizations are a ratio of gene expression changes from one group versus changes from the other group—i.e., a 10-fold increase in gene X in both IL1+Triacsin and IL1+Triacsin+Clari would result in no observed change in the gene expression ratio (ratio=1) when those groups were compared in a direct competitive hybridization.
There were two possible outcomes from these two final hybridizations. Outcome #1 would be that triacsin C and macrolides act on different signaling pathways. In this case, the AA pathway inhibitor and macrolides would have different effects on transcription and the transcriptional response should resemble the response to macrolide alone, because the IL1+Triacsin in both groups would effectively cancel out the response to triacsin.
Outcome #1: IL1+Triacsin/IL1+Triacsin+ML=ML
A second possibility, Outcome #2, would be that the AA pathway inhibitor (triacsin C) and macrolides are acting on the same signaling pathway. In this case, the transcriptional response to IL1+Triacsin+macrolide would resemble the response to IL1+Triacsin alone, because the macrolide effect would be blocked by or overlap the triacsin effect. The overall observed response to the direct competitive hybridization in Outcome #2 would be no transcriptional changes, since the ratio of IL1+Triacsin vs. IL1+Triacsin+macrolide (resembling triacsin alone) would be 1.
In the hybridization of IL1+Triacsin vs. IL1+Triacsin+Clari, there are only 4 differentially expressed genes across the 2 conditions, in contrast to 54 differentially expressed genes in the comparison of IL1 alone to IL1+clarithromycin (Table 7, culture conditions 2 vs. 4). In a similar direct competitive hybridization between IL1+Triacsin and IL1+Triacsin+Roxi, there were only 7 differentially expressed genes (Table 7, conditions 8 v. 10), compared to 65 differentially expressed genes in response to roxithromycin in the absence of triascin (conditions 2 v. 6).
The results of these hybridization experiments are compelling. The macrolide effect is blocked by, or is overlapping, the triacsin C effect. The minimal change in gene expression in these direct competitive hybridizations corresponds to outcome #2. These data imply that triacsin C blocks the macrolide effect or that the two effects are nearly identical, and strongly support the hypothesis that the AA pathway is a target of macrolides. These results further suggest that the molecular target is likely to be upstream of triacsin C in the biosynthetic pathway, which is comprised of just four possible target enzymes and includes FADS1.
FADS1 Antisense and Inhibitor Microarray Experiment
FADS1, also known as Δ5 desaturase, is the enzyme responsible for conversion of dihomo-gamma-linolenic acid (DGLA) to AA. The related gene FADS2 is A6 desaturase, and is responsible for an earlier step in the biosynthesis of AA—the conversion of linoleic acid (from the diet) to gamma-linolenic acid (GLA). The function of a third related gene, FADS3, is unclear, however it is believed to be more closely related to FADS2 than FADS1.
Experiments in which A549 cells are exposed to a compound reported to be a specific inhibitor of FADS1 activity reduced expression of PGE2 production, as determined by competitive enzyme immunoassay (
To specifically address whether macrolides were specifically targeting the FADS1 enzyme of the arachidonic acid biosynthesis pathway, a microarray experiment was carried out in A549 cells to compare the transcriptional response of macrolides to that of an inhibitor reported to be specific for FADS1 (CP-74006) and to the response of FADS1 mRNA knock-down by antisense oligonucleotides.
A549 cells were transfected with 2 different pairs of FADS1 antisense/reverse control oligonucleotides and incubated for 48 hours. Cells then were pre-incubated overnight (17 hours) with macrolides (50 μM clarithromycin, 50 μM roxithromycin), and 10 μM CP-74006. All cells then were stimulated for 4 hours with IL-1β, because the activation of PLA2 and downstream signaling events generally requires an inflammatory stimulus. The various treatment conditions are shown below in Table 10. Following macrolide and inhibitor pre-treatment and IL-1β stimulation, the cells were lysed, total RNA was collected, and the poly-A+ mRNA fraction was purified. Competitive hybridizations to the microarray were performed as shown in Table 11. The resulting microarray data was analyzed using the methods described in the Examples above. The transcriptional response to CP74006 (FADS1 inhibitor) is in Appendix D.
The table in Appendix D represents the effects of the FADS1 inhibitor in the absence and presence of macrolides, and represents genes that show differential expression (>1.3-fold) in response to at least one of the conditions. The genes that meet this criterion (≧1.3 min fold change) are indicated in bold.
Quantitative analysis was done to determine how well the response to the FADS1 inhibitor correlated with the macrolide response and with the responses to the other inhibitors of the arachidonic acid pathway (triacsin C and indomethacin). The macrolide and arachidonic acid pathway inhibitor data used for the correlations are in Appendix C and the FADS1 inhibitor data are in Appendix D. The correlation analysis was done using the methods described in the Examples above.
The analysis indicates a strong correlation between the FADS1 inhibitor and both arachidonic acid inhibitors triacsin and indomethacin. Using the subset of spots differentially expressed in response to triacsin and indomethacin, the correlation between the FADS1 inhibitor and both triacsin and indomethacin was greater than 0.9 (p-value <0.001). This result was anticipated, since all three of the above inhibitors target the arachidonic acid pathway and therefore should have similar effects on cells.
Significantly, the correlation analysis also demonstrates a strong correlation between the FADS1 inhibitor and the active macrolides. Using a subset of spots differentially expressed in response to clarithromycin and roxithromycin, the correlations between the FADS1 inhibitor and clarithromycin was 0.9 (p-value 0.3), between the FADS1 inhibitor and roxithromycin was greater than 0.9 (p-value 0.03), and between the FADS1 inhibitor and FMA was greater than 0.9 (p-value 0.003). These data suggest that the FADS1 inhibitor and the macrolides are acting on the same pathway.
The correlation analyses demonstrate that the responses to the anti-inflammatory macrolides are highly correlated with the responses to three different inhibitors of the arachidonic acid pathway (triacsin C, indomethacin, and FADS1 inhibitor). These data, in conjunction with the ability of macrolides to inhibit production of PGE2, a product of arachidonic acid activation, strongly support the hypothesis that macrolides are targeting the arachidonic acid pathway.
In this example, the usefulness of a macrolide compound for inflammatory disease of the lung was evaluated.
Clarithromycin, lactose, corn starch, crystalline cellulose, and carmellose calcium were uniformly mixed. To this was added a 10% hydroxypropyl cellulose aqueous solution. After blending the mixture, the granules were dried and sieved with a 30M-screen to make uniform granules. Magnesium stearate was added to the granules and it was compressed to form tablets.
The pharmacological action of clarithromycin (improvement to a pulmonary emphysema condition) will be next explained by way of test examples.
Test Example: Pharmacological effect in model mice with tobacco smoke-induced pulmonary emphysema
The model mice with tobacco smoke-induced pulmonary emphysema mimicking human pulmonary emphysema were constructed according to the method as described in Hautamaki, R. D. et al., Science 1997, 277: 2002-2004 or Shapiro, S. D. et al., Am. J. Pathol (2003) 163: 2329-2335. Specifically, C57black/6 female mice of 12-weeks old were exposed to tobacco smoke at two cigarettes per day, 6 days per week for 6 months.
Administration of clarithromycin was carried out orally at the rate of 25, 50 or 100 mg/body weight twice daily (morning and evening) during the tobacco smoke exposure period. The plasma AUC0-8 of clarithromycin obtained when 50 mg/kg was singly administered to the mice was 2.677 μg·hr/ml while the plasma AUC0-8 of clarithromycin obtained when 200 mg of clarithromycin was singly administered to a normal human subject was 8.98 μg·hr/ml. The former concentration was substantially lower than the latter one and was an adequate concentration to be used clinically.
The index of the improvement to the pulmonary emphysema condition followed the mothod as described in Hautamaki, R. D. et al., ibid. or Shapiro, S. D. et al., ibid.
Hematoxylin eosin-stained pathological specimens were prepared from the model mice and ten visual fields under a microscope were randomly selected, where the average sizes of pulmonary alveoli were measured. An increase in the average size of pulmonary alveolus is considered as an index of the destruction of pulmonary alveoli, i.e., the pulmonary emphysema condition.
Table 12 shows the improvement factor of pulmonary emphysema in the model mice with tobacco smoke induced pulmonary emphysema. Six months' smoking (i.e., exposure to tobacco smoke) increased the pulmonary emphysema rate by 20% relative to the non-smoking group (control). The pulmonary emphysema was improved by 47% in the group administered with clarithromycin (25 mg/kg) and by a maximum of up to 87% in the group administered with clarithromycin (50 mg/kg). These results indicate that clarithromysin is effective against pulmonary emphysema (or pulmonary emphysema condition).
(mean ± SE)
The cell counts of inflammatory cells (microphages and neutrophiles) in the bronchoalveolar lavage fluids after six months' exposure were measured to provide an index of the inflammatory condition in the model mice with tobacco smoke-induced pulmonary emphysema. The results are shown in
The cell counts of macrophages and neutrophiles increased as the pulmonary emphysema progressed. These cells produce the proteases that are involved in pulmonary emphysema. These cells increase exacerbate the pulmonary emphysema. Administration of clarithromycin reduced the cell counts of macrophages and neutrophiles in a dose-dependant manner. This result indicates that clarithromycin inhibits inflammation reaction and suppresses the progression of pulmonary alveoli destruction.
It has been reported that as pulmonary emphysema progresses, pulmonary functions deteriorate. It has also been known that the pulmonary compliance, which is an index of pulmonary functions, increases in model mice with tobacco smoke-induced pulmonary emphysema. Thus, the pulmonary compliance was measured in the model mice with tobacco smoke-induced pulmonary emphysema after six months' exposure. The result is shown in
As the pulmonary emphysema progressed, the pulmonary compliance (the index of pulmonary functions) was increasing in the model mice with tobacco smoke-induced pulmonary emphysema; however, the pulmonary compliance recovered upon the administration of clarithromycin. This result was in accord with the improvement of the pulmonary emphysema ratio as well as with the decrease in the number of the inflammatory cells, and it indicates that the clarithromycin administration is useful for the treatment of pulmonary emphysema (or pulmonary emphysema condition).
In this example, among the genes shown in the above-mentioned Appendixes A to E, the influence of a macrolide compound on the level of expression of a gene of a material related to inflammatory disease of the lung was examined.
The influence of a macrolide compound on the level of expression of the gene of the material related to inflammatory disease of the lung is summarized in Appendix F. In Appendix F, ‘+/−Clari, wtd means’ denotes gene induction (suppression) when clarithromycin is added to THP-1 cells, and the larger the figure, the more the gene is expressed. Furthermore, ‘+LPS+/−Clari, wtd means’ denotes gene induction (suppression) when clarithromycin is added to LSP-stimulated THP-1 cells, and the smaller the figure, the more the gene expression is suppressed.
From Appendix F, it can be seen that clarithromycin acts effectively on the expression of genes of Categories 1 to 3.
The present invention may have the following modes.
1. A method of screening test macrolides to identify potential anti-inflammatory agents, comprising the steps of:
2. The method of embodiment 1 wherein the test macrolide increases the first level of de novo arachidonic acid biosynthesis.
3. The method of embodiment 1 wherein the test macrolide decreases the first level of de novo arachidonic acid biosynthesis.
4. The method of embodiment 1 wherein the biological sample comprises cells.
5. The method of embodiment 1 wherein the biological sample comprises a cellular extract.
6. The method of embodiment 1 wherein the biological sample is in an in vivo system.
7. The method of embodiment 1 wherein the biological sample is in an in vitro system.
8. The method of embodiment 1 wherein the in vitro system is a cell culture.
9. The method of embodiment 1 wherein the first level of de novo arachidonic acid biosynthesis is detected by measuring incorporation of labeled arachidonic acid into phospholipids.
10. The method of embodiment 1 wherein incorporation of label into an intermediate in an arachidonic acid biosynthesis pathway is measured.
11. A method of screening macrolides to identify potential anti-inflammatory agents, comprising the steps of:
12. The method of embodiment 41 wherein the test macrolide increases the first level of expression.
13. The method of embodiment 11 wherein the test macrolide decreases the first level of expression.
14. The method of embodiment 11 wherein the step of contacting is in vitro.
15. The method of embodiment 11 wherein the step of contacting is in vivo.
16. The method of embodiment 15 wherein the step of contacting is in a cell culture.
17. The method of embodiment 16 wherein the cell culture comprises an epithelial cell line.
18. The method of embodiment 17 wherein the epithelial cell line is a macrophage cell line.
19. The method of embodiment 17 wherein the epithelial cell line is A549.
20. The method of embodiment 17 wherein the epithelial cell line is THP-1.
21. The method of embodiment 11 wherein the gene is A5 fatty acid desaturase (FADS1).
22. The method of embodiment 11 wherein the gene is A6 fatty acid desaturase (FADS2).
23. The method of embodiment 11 wherein the gene is fatty acid elongase.
24. The method of embodiment 11 wherein the gene is arachidonyl CoA synthetase.
25. The method of embodiment 11 wherein the gene is selected from the group consisting of cyclooxygenase 2, prostaglandin E receptor EP4, cytosolic phospholipase A2, fatty acid desaturase, prostaglandin E synthase, prostaglandin E receptor EP1, prostaglandin 12 receptor, and secretory phospholipase A2 receptor 1.
26. The method of embodiment 11 wherein the gene is selected from the group consisting of genes listed in Appendices A-E.
27. The method of embodiment 11 wherein expression of the gene is determined by detecting mRNA.
28. The method of embodiment 11 wherein expression of the gene is determined by detecting protein.
29. The method of embodiment 11 wherein expression of the gene is determined by detecting protein synthesis.
30. A method of screening macrolides to identify potential anti-inflammatory agents, comprising the steps of:
31. The method of claim 30 wherein the test macrolide increases the first level of activity of the enzyme.
32. The method of claim 30 wherein the test macrolide decreases the first level of activity of the enzyme.
33. The method of embodiment 30 wherein the step of contacting is in vitro.
34. The method of embodiment 30 wherein the step of contacting is in vivo.
35. The method of embodiment 24 wherein the step of contacting is in a cell culture.
36. The method of embodiment 33 wherein the cell culture comprises an epithelial cell line.
37. The method of embodiment 36 wherein the epithelial cell line is a macrophage cell line.
38. The method of embodiment 36 wherein the epithelial cell line is A549.
39. The method of embodiment 36 wherein the epithelial cell line is THP-1.
40. The method of embodiment 30 wherein the enzyme is A5 fatty acid desaturase (FADS1).
41. The method of embodiment 30 wherein the enzyme is A6 fatty acid desaturase (FADS2).
42. The method of embodiment 30 wherein the enzyme is fatty acid elongase.
43. The method of embodiment 30 wherein the enzyme is arachidonyl CoA synthetase.
1.9
3.8
1.8
5.1
0.4
3.1
1.9
3.8
2.4
1.9
0.5
1.9
1.8
0.6
0.7
3.3
2.9
0.5
2.6
ACE
ACOX1
ADAM17
ADAMTS1
2.3
ADAMTS1
0.5
ADCY7
ADCY7
1.9
ADRB2
AGPAT1
AGT
2.1
AIM1
AKT3
16.2
1.7
ALAD
25.9
AMPD2
APLP2
1.7
AREG
0.5
AREG
0.5
AREG
0.5
AREG
0.5
ARF6
ARF6
ARF6
4.5
ASB1
0.6
ATF3
2.2
2.6
BAI3
4.7
BCL2
BCL2
3.1
BCL3
4.1
0.6
BCRP1
2.9
3.2
3.4
3.3
3.3
22.0
11.3
3.5
2.8
11.2
C12orf8
C2
2.0
C2orf3
1.6
3.6
2.3
CASP1
5.9
2.2
CASP1
4.1
4.4
3.4
3.3
3.0
3.2
3.8
3.4
CAST
CAT
0.5
CCNA2
0.5
CCNA2
0.5
CCND3
1.6
CCNE2
0.5
CCR1
CCR1
41.5
49.6
CCRL2
5.3
CD14
2.4
CD14
1.8
CD36L1
0.5
2.6
2.8
CD97
3.4
CD97
3.4
CDK6
CDK6
CEBPA
0.2
3.8
3.8
CEP3
7.0
CKB
3.3
CKLF1
0.5
0.5
CLU
COL4A1
COL4A2
COL4A2
0.7
COL7A1
0.3
0.6
COL7A1
0.3
0.6
COL9A2
1.8
COL9A2
1.9
COMT
CPD
CRA
CRADD
0.4
CRADD
0.4
2.5
CRIP1
CSF1
CSF1
11.4
12.7
CSF3
4.3
CSF3
4.8
CSF3R
CTGF
108.1
CTSB
CTSD
CTSD
CTSG
CTSH
1.7
CXCR4
0.4
CYBB
CYC1
CYC1
CYP1A1
1.8
0.3
DAPP1
DDXL
DKFZP761N09121
1.9
DKFZp762A227
DNMT1
DSCR1
DTR
2.4
DTR
0.6
DUSP1
0.6
DUSP1
0.5
DUSP10
0.6
DUSP10
0.7
DUSP4
2.1
3.1
0.2
0.2
2.3
6.2
6.3
3.8
ECM1
1.9
ECM1
2.0
EDG1
3.1
EDN1
1.7
EDN1
1.7
3.3
4.1
EIF4EBP1
0.4
ELK1
3.3
1.6
EMILIN
EMILIN
EMS1
0.6
EMS1
EPHA2
EREG
4.5
EREG
6.6
EREG
3.8
ERG
27.1
2.7
ERG
12.8
2.5
6.4
7.6
0.3
ETV5
0.3
1.6
ETV5
0.3
F2
F8A
FAF1
FAP
0.6
FBN2
FCGR1A
2.2
FCGR2A
3.8
FCGR2A
3.8
FCGR2A
3.0
FCGR2A
2.4
3.6
FCGR2A
2.5
3.2
FGF2
3.0
3.2
FGFR1
1.7
0.7
FLJ10718
3.2
0.4
0.7
FN1
0.0
0.7
FN1
0.0
0.6
FOS
FOXF1
FOXF1
FYB
GAA
3.5
GALNAC4S-6ST
0.6
GALNAC4S-6ST
0.5
3.2
GBP1
26.3
13.0
30.2
20.6
GCH1
15.5
18.7
0.5
GGT1
GLA
GM2A
1.7
2.8
2.5
0.4
GNAS
GNAS
GNAS
GNB1
GNG11
0.4
GNG11
0.5
GNLY
GPA33
11.3
3.9
GRF2
GRF2
GRIN2C
7.9
0.2
0.7
GRO1
44.5
0.4
0.7
GRO1
33.8
0.4
44.6
GRO2
25.4
0.4
0.7
GRO3
36.2
0.4
GRO3
33.5
0.4
0.7
GRP58
GS3686
6.6
1.6
GSK3B
GSTA4
3.2
3.4
HEXA
4.1
3.9
HLA-DQB1
2.0
HLA-DRB1
2.0
HLA-DRB1
2.1
HLA-DRB1
1.8
HLA-DRB1
2.0
HLA-DRB1
2.0
HLA-DRB1
1.9
HLA-DRB3
2.0
HM74
HMGIC
HMGIY
0.5
HMGIY
0.5
HMGIY
HNRPA3
0.5
HRMT1L2
0.5
HS3ST3A1
2.3
0.6
HSPA1B
1.6
HSPA1B
1.7
HSPA1B
1.6
HSPA1B
1.6
4.1
1.0
4.1
1.1
HSPBP1
0.7
HSXIAPAF1
9.5
2.8
HSXIAPAF1
13.2
2.7
HTATIP
3.0
6.0
6.3
6.0
IDI1
4.2
IFI35
5.2
1.7
6.3
1.6
IFIT4
10.9
1.1
IFITM3
4.3
1.7
IFNGR1
2.3
IFNGR1
2.0
0.7
0.7
IGFBP3
2.8
0.2
1.5
IL10RA
9.5
2.0
IL10RA
11.8
2.1
IL13RA1
1.7
IL13RA1
1.5
11.9
0.9
13.9
1.0
IL1B
2.5
0.3
IL1B
3.3
0.2
IL1R1
2.8
0.7
5.6
5.1
3.4
IL6
13.6
IL6
13.5
IL6R
IL7R
20.6
IL7R
19.9
IL8
7.2
0.2
0.6
IMPDH1
47.1
INSIG1
0.7
5.7
IRAK2
5.3
6.9
4.2
ISG15
17.1
ISG15
32.0
1.9
ISG15
16.8
1.7
ITGA2
ITGA4
1.7
ITGA4
ITGAM
ITGAM
ITGB2
JAK2
4.6
JAK2
4.5
2.2
2.4
2.3
3.8
KIAA0173
KIAA0184
KIAA0212
2.2
0.1
2.6
3.5
2.4
KIAA1557
2.3
2.8
KRAS2
0.4
LAMB3
4.7
1.7
LAMC1
LAMP2
3.8
3.7
LGALS3
LIPA
LMO2
LOC51295
0.6
0.7
LPP
1.6
LRPAP1
LY117
LYN
5.7
4.6
5.0
3.9
MAD2L1
0.4
MAD4
2.1
2.2
MAP3K10
MAP3K4
MAP3K4
4.5
4.1
MAP3K8
MAP4K4
MAPK13
MBNL
MCM2
MCM5
MD-2
2.1
MD-2
MEF2C
MGEA5
MGLL
MIC2
MIG
15.7
MIG
13.7
MIG2
0.5
MMP10
0.5
0.6
2.8
MMP13
MMP13
1.6
MMP2
MMP2
MMP2
MMP3
3.9
0.6
MMP3
3.8
0.6
MMP7
0.4
MMP7
0.1
MRPL27
MT1L
12.3
MT3
9.4
2.8
2.6
3.0
2.3
MX1
13.2
1.8
MX2
18.3
2.2
2.4
MYLK
NBS1
3.7
NDN
NEDD4L
NEO1
NFATC3
NFE2L1
2.7
6.8
NFKB1
5.7
4.7
3.6
3.5
7.0
NFRKB
0.6
NID
5.1
NMA
4.3
NR1H3
3.3
20.4
7.0
8.1
4.8
NRG1
5.8
0.4
NRG1
5.3
0.5
0.7
2.7
OSMR
2.8
3.2
P4HA1
P4HA1
P4HA2
2.0
P4HB
PAK1
2.0
PAK1
1.8
PAK1
1.9
PAK4
PBX3
2.1
4.9
0.4
9.8
9.4
PDE7A
1.8
PDGFA
0.4
0.6
PDGFA
3.4
0.3
PFKP
PHKA1
1.7
2.2
PILR(ALPHA)
1.8
PIM1
2.4
PIM2
4.8
PIM2
4.7
PKM2
PLA2G4A
PLA2G4B
PLA2G7
2.2
PLAB
0.3
0.5
PLAB
0.3
0.5
PLAU
0.7
PLAU
0.8
0.4
PLD1
3.4
1.9
PLOD
1.9
PLOD
1.9
PLOD
1.9
PLOD
1.9
PLOD3
PLOD3
PLS3
PLXNB1
0.6
PM5
PPBP
PPFIBP1
PPFIBP1
0.6
PPIC
2.0
PPIC
2.0
PPIF
PPIH
3.5
3.2
PRKAR1A
PRKCB1
2.0
PRKCN
PRKR
4.4
1.8
PROS1
PRSS25
4.3
PTEN
1.6
PTEN
3.9
5.3
4.0
3.3
PTGER4
PTGER4
2.8
2.5
PTGS1
0.3
PTGS1
PTGS2
28.8
PTGS2
32.8
PTK2B
PTPN3
1.6
PTPRC
1.6
PTPRC
1.7
PTPRJ
PTPRO
1.7
PTX3
8.7
RAD54L
RANBP1
0.4
0.4
RASA2
RASA2
2.6
RBM3
0.7
RBMS1
RCN2
4.4
2.6
2.4
2.5
2.5
RGS14
0.4
2.0
0.3
RGS16
4.0
0.2
0.7
RGS16
3.5
0.4
0.7
8.5
ROCK2
0.4
RPS6KC1
S100A10
1.9
S100A10
1.9
S100A4
2.2
S100A4
2.0
S100A6
SARM
0.6
7.2
2.9
2.7
2.4
SCYA18
26.3
0.4
0.6
SCYA18
31.7
0.7
8.2
SCYA2
16.9
SCYA2
24.1
0.4
SCYA20
8.6
0.2
0.7
SCYA20
8.1
0.1
0.7
SCYA20
3.7
0.6
SCYA3
7.8
0.4
SCYA3
9.0
0.4
SCYA3
8.5
0.4
SCYA3
9.5
0.5
SCYA4
48.7
0.3
SCYA4
40.5
0.3
47.8
SCYB11
38.0
38.7
SCYB5
2.6
SCYB5
SDBCAG84
SELPLG
SERPINB1
3.8
SERPINB2
4.2
SERPINE1
SERPINE2
34.6
0.4
SERPINE2
26.7
0.4
SERPINE2
27.1
0.4
SERPINF1
SERPINF1
SERPINF1
1.5
2.7
11.1
SERPINH2
SERPINI1
2.6
SERPINI1
SLC25A13
SLC4A7
SLC7A5
SMPD1
SMPD1
SNK
2.4
SP110
5.3
SP110
5.4
SPP1
0.4
SPP1
0.4
SRC
10.4
SRC
10.7
SRF
3.6
ST14
ST14
1.6
STAT1
4.7
1.9
STAT1
5.2
2.1
4.3
4.3
3.4
3.3
3.1
3.5
STC1
SYK
SYK
2.5
6.9
TAX1BP1
TBL1
1.7
TBXAS1
TCF7L2
TCF8
TCFL4
5.6
TFEC
2.5
TFEC
2.5
TFPI2
4.2
0.1
TGFB3
0.4
TGFB3
0.4
0.7
TGFBR3
THBD
0.6
THBS1
0.2
THBS1
5.0
0.6
2.8
2.9
2.5
TIMP2
2.2
TIMP2
2.0
TIMP2
1.9
TIMP3
TIMP3
0.6
TM7SF2
TNF
14.9
TNF
16.6
69.9
8.2
56.8
62.1
TNFRSF21
TNFRSF21
6.0
5.7
5.4
TNFRSF6B
TNFRSF6B
0.5
TNFRSF6B
TNFRSF9
4.0
3.8
34.7
TNFSF9
3.0
TNFSF9
3.4
TP53BP1
TPST1
0.3
0.5
2.5
12.2
11.8
TRAP1
TRAP1
TREM2
0.5
2.0
TUBA3
TWIST
TXN
TXNIP
TXNIP
TXNIP
4.3
U3-55K
UCP2
1.8
UP
UP
0.6
VAV3
0.5
VAV3
0.5
XAGE-1
XBP1
3.0
YWHAQ
ZFP36
1.6
ZNF140
1.5
FN1
0.0
0.7
FN1
0.0
0.6
TFPI2
4.2
0.1
MMP7
0.1
MMP3
3.8
0.6
IGFBP3
2.8
0.2
SCYA20
8.1
0.1
0.7
RGS16
4.0
0.2
0.7
GRIN2C
7.9
0.2
0.7
CTGF
108.1
FCGR2A
3.8
THBD
0.6
CRADD
0.4
SCYA20
8.6
0.2
0.7
COL7A1
0.3
0.6
GNG11
0.4
IL8
7.2
0.2
0.6
FCGR2A
2.4
3.6
COL7A1
0.3
0.6
IL1B
3.3
0.2
CRADD
0.4
MMP10
0.5
0.6
FCGR2A
3.8
THBS1
0.2
FCGR2A
3.0
IL1B
2.5
0.3
AREG
0.5
AREG
0.5
AREG
0.5
AREG
0.5
RGS16
3.5
0.4
0.7
PLAU
0.8
TIMP2
2.2
S100A4
2.2
PLAU
0.7
IL10RA
9.5
2.0
TREM2
0.5
2.0
AGT
2.1
SCYA4
48.7
0.3
FCGR2A
2.5
3.2
TGFB3
0.4
HLA-DRB1
2.1
TIMP2
2.0
GRO2
25.4
0.4
0.7
PPIC
2.0
ECM1
2.0
HLA-DRB1
2.0
IL10RA
11.8
2.1
SCYA2
24.1
0.4
NRG1
5.8
0.4
HLA-DRB3
2.0
TIMP2
1.9
SPP1
0.4
SPP1
0.4
ECM1
1.9
PBX3
2.1
HLA-DRB1
2.0
1.9
HLA-DRB1
1.8
F2
0.7
MMP7
0.4
HSXIAPAF1
9.5
2.8
TNFRSF6B
0.5
HLA-DRB1
2.0
GNG11
0.5
S100A4
2.0
PPIC
2.0
PTGS2
32.8
HSXIAPAF1
13.2
2.7
1.8
GRO3
33.5
0.4
GRO3
36.2
0.4
GRO1
44.5
0.4
PRKCB1
2.0
FCGR1A
2.2
UP
0.6
GALNAC4S-6ST
0.5
SERPINI1
2.6
CXCR4
0.4
ADCY7
UCP2
1.8
HLA-DRB1
1.9
DTR
2.4
ADCY7
1.9
1.9
DTR
0.6
1.9
PDE7A
1.8
ADAMTS1
2.3
HMGIC
HMGIY
0.5
UP
0.5
HNRPA3
0.5
PLOD
1.9
COL9A2
1.9
PPIF
MAP3K4
PLOD
1.9
MMP13
1.6
GRO1
33.8
0.4
DKFZP761N09121
1.9
SERPINE2
34.6
0.4
EREG
4.5
SERPINE2
26.7
0.4
PLOD
1.9
TGFB3
0.4
PLOD
1.9
APLP2
1.7
CASP1
5.9
2.2
STAT1
5.2
2.1
HLA-DQB1
2.0
GM2A
1.7
HRMT1L2
0.5
SERPINE2
27.1
0.4
HMGIY
0.5
EMILIN
SCYA2
16.9
0.3
SERPINB1
3.8
MD-2
2.1
0.5
FLJ10718
3.2
0.4
0.7
COL9A2
1.8
MMP13
KIAA1557
2.3
C12orf8
HSPA1B
1.7
SCYA18
26.3
0.4
0.6
IFIT4
10.9
PTX3
8.7
RGS14
0.4
2.0
SCYA3
8.5
0.4
FGF2
3.0
INSIG1
0.7
HSPA1B
1.6
P4HA2
2.0
FGFR1
1.7
TXNIP
CTSH
1.7
GNAS
PTGER4
CD14
2.4
DUSP1
5.5
0.6
XAGE-1
ST14
SCYA3
7.8
0.4
PTGER4
PLA2G4A
CEP3
EREG
3.8
ST14
1.6
PAK1
2.0
PTGS2
28.8
C2
2.0
FOXF1
EMS1
0.6
NRG1
5.3
0.5
0.7
SCYA3
9.5
0.5
S100A10
1.9
SERPINI1
SCYA4
40.5
0.3
EREG
6.6
NFRKB
0.6
CPD
ITGA2
PIM2
NEDD4L
LOC51295
0.6
LIPA
SERPINF1
STC1
CCNE2
0.5
HMGIY
SCYB5
1.9
SERPINF1
IFNGR1
2.3
TRAP1
JAK2
4.6
CASP1
4.1
PPFIBP1
0.6
DUSP1
5.6
0.5
TXNIP
MAP3K8
4.9
GALNAC4S-6ST
0.6
1.89
2.09
1.86
1.77
1.81
2.01
1.84
1.68
1.61
1.97
1.80
1.64
1.90
1.70
1.59
2.82
2.05
1.77
1.53
1.74
1.72
1.52
1.82
1.71
1.52
2.06
1.76
1.48
1.76
1.94
1.77
1.45
1.73
1.77
1.38
1.91
1.58
1.35
1.69
1.50
1.47
1.32
2.17
1.58
1.31
1.72
1.54
1.17
1.63
1.68
1.13
2.04
1.12
1.63
1.98
1.11
1.70
1.09
1.41
1.09
2.14
1.07
1.39
1.05
1.51
1.05
1.44
1.58
1.83
1.72
0.46
1.73
2.02
2.08
2.27
0.30
1.48
1.52
0.23
3.55
0.48
0.47
0.51
0.44
1.74
0.49
1.58
0.71
2.46
1.68
1.78
0.63
0.51
0.52
1.50
1.73
1.67
1.40
0.63
0.43
2.59
1.61
1.36
1.60
1.39
0.28
0.09
0.09
0.09
1.62
1.50
1.63
1.47
1.44
1.54
1.41
1.54
1.65
0.48
1.63
1.87
1.83
1.73
0.41
0.48
0.62
1.53
1.92
1.66
1.58
0.60
1.57
0.69
1.54
0.66
1.44
1.62
1.43
0.25
1.50
0.16
0.27
0.62
0.53
0.69
0.64
0.60
0.60
0.24
0.49
0.33
0.38
0.18
0.40
0.43
0.56
0.56
0.44
1.62
1.91
1.63
1.70
0.64
1.36
2.12
1.76
2.75
0.55
1.61
1.61
1.61
1.42
1.45
1.45
1.80
0.54
1.42
0.61
0.23
0.60
0.15
0.13
0.60
0.14
0.10
2.15
0.58
0.18
0.54
1.44
0.32
0.24
0.25
0.23
1.79
1.90
0.48
0.41
0.48
0.40
0.54
0.59
1.61
1.63
1.60
0.57
0.27
4.01
0.62
0.48
0.68
1.43
0.35
0.47
0.68
0.71
0.23
0.68
1.62
0.51
2.04
0.53
1.69
0.58
0.61
0.63
0.63
1.50
0.54
2.14
0.52
2.13
0.67
0.69
0.64
0.20
0.20
0.50
0.15
0.16
0.16
0.14
0.11
0.13
0.21
0.20
0.40
1.47
1.74
0.33
0.72
1.46
0.71
0.28
0.27
0.58
2.05
1.88
0.67
1.84
1.38
0.67
0.71
0.66
2.17
0.43
2.54
2.25
0.24
1.60
0.48
1.57
0.46
1.38
0.46
0.44
0.61
0.53
0.47
0.41
0.66
0.44
1.65
1.83
0.65
0.40
1.83
1.80
2.04
1.97
0.11
0.15
0.21
0.47
0.57
0.69
0.61
1.57
0.59
1.89
0.43
0.60
1.13
0.64
1.77
1.84
0.58
1.79
1.68
0.53
1.52
0.70
0.47
1.45
0.41
1.43
0.42
1.44
1.77
1.84
0.39
0.41
2.03
1.58
0.56
0.71
0.75
1.56
0.60
0.62
1.95
1.63
0.60
0.63
1.73
2.04
1.94
0.58
0.54
0.40
0.67
1.71
0.51
0.57
1.58
0.65
1.43
2.48
0.55
1.52
1.51
2.90
2.87
0.41
1.71
0.33
1.70
1.67
1.92
2.00
0.14
0.69
0.68
0.65
0.70
0.64
0.71
0.30
0.69
0.69
0.70
0.90
0.65
0.85
0.37
2.55
0.55
0.47
0.45
0.46
0.67
0.42
0.44
0.51
1.90
0.41
0.41
0.68
1.72
1.71
1.71
0.74
0.73
0.70
0.48
1.37
0.45
1.76
0.15
1.64
1.46
1.75
1.70
1.64
0.05
2.02
0.06
2.05
1.82
1.81
2.70
2.32
1.67
1.57
1.65
0.48
0.53
1.78
2.11
1.71
1.76
2.00
1.73
0.06
1.53
0.59
1.57
2.00
0.48
2.21
0.44
1.76
1.57
1.55
0.54
0.70
1.80
0.66
2.81
1.64
0.61
1.46
0.45
0.65
0.55
0.57
1.59
1.68
0.59
1.74
1.78
0.61
1.53
2.97
1.64
0.34
0.71
1.59
1.47
1.54
1.41
1.73
0.60
0.51
0.57
1.37
1.48
2.49
2.00
1.67
1.81
0.30
0.55
0.31
1.65
0.19
1.75
1.87
1.99
1.55
1.92
1.89
1.75
1.37
1.58
1.54
1.55
1.61
1.63
0.68
0.65
0.64
0.65
1.57
0.61
1.58
0.43
2.63
1.89
0.51
0.63
1.77
0.58
0.65
2.04
1.93
1.88
1.80
2.05
0.25
0.57
1.49
1.48
0.52
0.52
0.55
0.56
0.69
1.47
1.57
1.50
0.73
1.80
0.52
0.43
0.72
0.62
2.34
2.26
2.24
2.33
1.61
2.17
1.40
0.64
0.32
0.55
0.43
0.66
2.17
2.49
2.46
2.07
1.75
1.70
0.59
0.66
0.54
6.96
2.31
1.52
2.06
0.49
0.47
1.52
1.76
1.71
1.70
0.69
0.39
0.40
0.68
1.85
1.64
0.53
1.79
1.79
0.34
0.54
0.54
0.29
0.33
0.25
0.40
0.64
0.55
0.59
0.55
1.59
0.62
2.23
2.06
0.52
0.54
0.49
0.41
1.66
0.65
0.38
Drosophila)
0.40
Drosophila)
1.89
1.62
0.66
1.60
1.59
1.53
0.34
0.33
0.64
0.56
1.74
1.67
1.76
0.49
0.08
0.08
1.41
0.41
0.42
0.28
0.70
0.72
0.39
0.33
0.57
0.56
0.63
0.50
1.46
1.81
1.65
1.98
1.59
0.65
1.74
1.80
1.73
1.47
0.64
1.66
1.82
1.52
1.79
1.76
1.91
0.68
0.61
0.63
0.05
0.06
1.68
1.38
1.58
1.39
1.38
1.33
0.65
1.34
1.66
2.89
0.72
0.70
1.44
0.47
0.59
0.60
0.68
0.72
0.46
0.57
0.63
0.66
1.51
0.62
1.41
0.51
0.53
0.44
0.65
0.43
0.47
1.85
1.79
2.12
1.91
0.26
0.64
0.69
0.46
1.67
1.52
0.46
0.62
0.54
15.15
1.61
0.64
2.38
0.57
1.84
0.59
2.10
3.72
8.82
1.52
8.93
1.43
1.72
1.71
0.40
0.34
0.36
0.38
2.87
1.95
2.94
1.93
0.23
0.27
0.27
0.57
0.59
0.63
1.70
1.90
1.92
0.63
1.87
0.68
5.38
1.98
0.58
0.59
0.62
2.05
2.09
1.48
0.62
0.36
0.37
0.45
0.45
1.44
1.61
1.88
1.71
1.42
1.83
1.73
0.08
1.49
1.46
1.45
3.10
1.38
0.52
0.67
1.64
0.67
1.39
1.82
1.78
0.36
0.64
0.68
0.61
0.53
4.04
4.89
0.68
0.66
0.67
0.70
0.41
0.67
0.53
0.67
0.42
1.44
0.32
0.47
0.46
0.65
0.50
0.41
0.36
1.69
1.65
2.09
1.82
1.62
1.90
1.52
0.73
0.64
1.68
1.60
0.56
0.50
0.38
0.37
0.38
2.40
0.56
1.84
0.67
1.64
1.50
1.61
0.50
0.51
1.97
1.66
2.37
1.95
1.77
2.36
1.82
1.62
1.71
1.78
1.72
2.00
1.79
1.71
1.92
1.74
2.41
2.39
1.70
1.84
2.02
1.67
1.61
1.76
2.10
1.55
1.79
1.61
1.94
1.63
1.50
1.82
1.66
1.68
1.64
1.87
1.56
1.63
1.72
1.97
1.51
1.81
1.46
2.09
1.69
1.50
1.70
1.68
1.92
1.50
1.00
0.75
1.71
1.85
1.77
1.82
1.49
1.98
0.58
1.67
1.73
1.66
2.26
1.64
1.54
1.41
1.61
1.75
1.74
1.67
2.17
1.86
1.50
1.52
1.40
1.73
1.64
1.52
0.67
0.50
0.50
0.65
0.58
0.55
0.65
1.72
1.49
0.56
1.49
1.55
0.66
0.68
0.72
0.69
0.68
0.47
1.56
1.35
1.50
1.45
1.82
1.56
1.52
1.70
1.53
1.53
1.66
1.87
1.46
1.46
0.62
0.64
0.63
1.69
1.79
1.85
1.45
0.67
0.67
1.80
1.59
1.36
0.66
1.43
2.04
0.64
0.62
0.65
1.67
1.72
3.36
0.73
2.02
1.91
1.75
1.42
0.38
1.46
1.38
1.53
1.52
1.59
1.70
0.68
0.64
0.65
0.69
1.44
1.85
1.39
1.64
1.60
1.42
2.04
1.46
1.84
1.56
2.03
1.67
1.91
1.86
1.93
1.71
1.78
1.79
2.39
1.77
1.75
1.89
2.10
1.71
2.18
1.50
1.52
1.72
1.73
1.59
1.46
0.61
0.67
1.58
1.66
1.68
1.73
1.60
1.85
1.59
1.51
0.58
1.43
1.89
1.75
2.42
1.79
1.56
2.28
1.46
0.65
1.72
1.63
2.13
1.76
2.07
1.46
1.78
1.89
2.13
2.63
2.45
2.04
1.51
1.39
1.73
1.55
1.72
1.80
1.66
0.73
1.44
Drosophila)
1.46
1.44
Drosophila)
1.68
1.60
1.74
2.12
2.06
1.74
0.64
0.82
1.95
0.73
0.80
0.81
1.75
1.72
1.47
1.72
1.60
1.67
1.69
1.65
1.47
1.77
2.09
1.73
1.57
1.86
1.56
1.86
2.01
1.55
0.73
1.38
1.77
1.61
1.62
1.83
1.70
2.23
1.87
1.65
1.46
0.73
1.70
1.53
1.39
1.48
1.43
1.62
2.00
1.41
1.47
1.64
1.67
1.65
2.19
1.52
0.68
0.70
1.41
1.57
1.39
1.57
1.84
1.56
1.60
1.76
1.73
1.90
1.66
1.53
1.76
1.60
1.96
1.67
0.65
1.70
1.62
0.66
0.66
0.68
1.44
0.68
0.70
1.48
0.69
0.65
0.68
0.68
0.65
0.62
0.68
0.71
1.41
1.31
1.42
0.67
1.56
0.66
1.83
1.70
1.79
0.66
0.75
0.56
0.70
0.67
0.65
1.61
1.48
1.68
0.71
1.41
1.39
0.72
1.48
1.43
0.72
0.63
0.69
0.66
2.02
1.56
0.70
1.47
1.38
1.42
1.93
1.59
1.44
0.52
0.70
0.70
1.45
0.67
1.33
0.80
0.63
1.43
1.58
1.71
0.68
1.74
1.50
0.71
1.88
0.73
0.73
0.56
0.61
0.63
0.60
0.67
0.70
0.65
0.71
1.78
1.43
1.57
0.65
0.74
1.56
1.53
1.50
1.42
0.68
0.70
0.71
0.70
0.65
1.48
1.43
0.71
0.71
1.64
1.66
0.65
0.64
*From Appendix C. For those having a plurality of SPOTIDs, a representative ID is shown.
The present application is a continuation-in-part of PCT international application No. PCT/US2005/005,401 filed Feb. 18, 2005 and designating the U.S., which claims benefit of U.S. Provisional Application Ser. No. 60/545,177 filed Feb. 18, 2004, the contents of which are incorporated hereinto by reference.
Number | Date | Country | |
---|---|---|---|
60545177 | Feb 2004 | US |
Number | Date | Country | |
---|---|---|---|
Parent | PCT/US05/05401 | Feb 2005 | US |
Child | 11505929 | Aug 2006 | US |