Patent Application
20150284812
The present patent application is a patent application regarding the results of a national government-commissioned research (Year 2010, Ministry of Education, Culture, Sports, Science and Technology, Innovation System Improvement Project, a commissioned research regarding “Formation of Chromosome Engineering Research Center associated with Drug Discovery, Development of Food Functionality Evaluation Model Animals and the like,” a patent application under Section 19 of the Industrial Technology Enhancement Act).
The present invention relates to a method for evaluating both induction of a drug-metabolizing enzyme by a test substance and cytotoxicity of the test substance, and a vector and a cell for use in said method.
The major three conditions for the usefulness of a drug are “having an effect,” “having low toxicity,” and “not accumulated in a body.” Regarding pharmaceutical product candidate compounds, absorption, distribution, metabolism, excretion, and toxicity are generally studied.
It has been known that the metabolism of a drug is carried out by various drug-metabolizing enzymes. Cytochrome P450, a representative drug-metabolizing enzyme, is an oxidase involved in 80% of drug metabolism in a living body. The CYP3A group includes major genes belonging to a cytochrome P450 family, and it is considered that the CYP3A is involved in the metabolism of approximately 50% of commercially available drugs (Non Patent Document 1).
With regard to drug-metabolizing enzymes, a phenomenon that is induction of a drug-metabolizing enzyme has been known. The induction of a drug-metabolizing enzyme is a phenomenon in which expression of a drug-metabolizing enzyme is increased by a specific drug through activation of a promoter of the drug-metabolizing enzyme and the like. Such induction of a drug-metabolizing enzyme causes a harmful drug interaction. For instance, when a first pharmaceutical agent that increases the expression of a drug-metabolizing enzyme and a second pharmaceutical agent that is different from the first pharmaceutical agent are simultaneously administered to a subject, the drug-metabolizing enzyme whose expression has been increased by the first pharmaceutical agent metabolizes the second pharmaceutical agent, and as a result, the medicinal effects of the second pharmaceutical agent are reduced. Accordingly, upon obtaining a useful drug, it is important to examine the induction of a drug-metabolizing enzyme by pharmaceutical product candidate compounds.
As a method for examining induction of a drug-metabolizing enzyme, there is a method which comprises: allowing a biological model cell comprising a vector in which a reporter gene is connected downstream of the expression control region of a drug-metabolizing enzyme gene to come into contact with a drug candidate substance; examining a change in the expression of the reporter gene in the cell between before and after the contact; and evaluating the drug candidate substance that has increased the expression of the reporter gene as a drug-metabolizing enzyme inducible substance (see Patent Documents 1 to 4). This method does not require complicated operations and can be easily carried out. However, some drug candidate substances that have been evaluated to cause induction of a drug-metabolizing enzyme by the aforementioned method do not actually cause the induction of a drug-metabolizing enzyme in living bodies. In contrast, some drug candidate substances that have been evaluated not to cause the induction of a drug-metabolizing enzyme actually cause it in living bodies. As such, conventional evaluation methods have been insufficient to ensure accuracy.
Patent Document 3 describes a vector having the expression region of CYP3A4 and a fluorescent protein. However, Patent Document 3 contains neither a description regarding the improvement of the performance of a cell with which a drug candidate substance is allowed to come into contact, nor a description regarding a fetus-specific gene.
Patent Document 4 describes the use of a CYP3A4 regulatory nucleic acid molecule and a reporter nucleic acid molecule, and contains descriptions regarding Examples in which β-galactosidase was used as such a reporter nucleic acid molecule. However, Patent Document 4 contains neither a description regarding Examples in which a fluorescent protein was used, a description regarding the improvement of the performance of a cell with which a drug candidate substance is allowed to come into contact, nor a description regarding a fetus-specific gene.
As a technique similar to the aforementioned techniques, Patent Document 5 discloses a method for evaluating the toxicity or bioavailability of a drug, using a vector having a fluorescent protein GFP as a reporter gene and comprising the promoter region of CYP3A7 that is a fetus-specific gene. However, the technique of Patent Document 5 is not intended to evaluate induction of a drug-metabolizing enzyme. Moreover, Patent Document 5 contains neither a description regarding a vector comprising a combination of the expression regions of multiple genes, nor a description regarding the improvement of the performance of a cell with which a drug candidate substance is allowed to come into contact.
Accordingly, it is an object of the present invention to evaluate the induction of a drug-metabolizing enzyme by a test substance more accurately than ever before, by simple operations, using a vector in which a reporter gene is connected downstream of the expression control region of a drug-metabolizing enzyme gene and a cell into which the aforementioned vector is introduced.
As a result of intensive studies, the present inventors have found that a major cause of inaccuracy in conventional methods is that a cell population used in the evaluation is not homogeneous and the cell population contains a large number of cells that do not cause the same induction of a drug-metabolizing enzyme as that in a living body. In addition, the inventors have also found that another cause of inaccuracy is that the expression of a reporter gene has not been sufficiently functioned in cells for evaluation, when a test substance having cytotoxicity has been used. Moreover, in conventional methods, the expression level of a reporter gene has been observed only at a specific time point after the contact with a drug, and induction of a drug-metabolizing enzyme has been evaluated. The present inventors have found that the expression level of a reporter gene by induction of a drug-metabolizing enzyme may be reduced after it has been once increased, and thus that evaluation of induction of a drug-metabolizing enzyme based on the results of the expression level after it has been reduced would be another cause of inaccuracy in conventional methods. Furthermore, the inventors have confirmed that the expression of a fetus-specific gene transiently increases when a cell is damaged by a cytotoxic substance, and thus that it becomes an indicator of the cytotoxicity of a test substance.
Based on the aforementioned findings, the present inventors have conducted further studies. As a result, the inventors have found that a cell showing high maturity suitable for an accurate drug-metabolizing enzyme induction test can be selected by introducing a vector comprising an expression control region of a drug-metabolizing enzyme gene and an expression control region of a fetus-specific gene into cells, and then by selecting cells in which the expression level of a reporter gene located downstream of the expression control region of the fetus-specific gene is low. Moreover, the inventors have also found that if such a vector is used, induction of a drug-metabolizing enzyme can be evaluated by evaluating a change in the expression level of the reporter gene located downstream of the expression control region of the drug-metabolizing enzyme gene between before and after the contact of the cell with the test substance, and further, cytotoxicity can also be evaluated by evaluating a change in the expression level of the reporter gene located downstream of the expression control region of the fetus-specific gene between before and after the contact of the cell with the test substance, so that induction of a drug-metabolizing enzyme can be evaluated more accurately than ever before based on the evaluation of cytotoxicity. When the aforementioned vector was used, both selection of cells suitable for the drug-metabolizing enzyme induction test and a test of evaluating induction of a drug-metabolizing enzyme and cytotoxicity could be carried out by simple one-step operations, and the developmental stage and quality of cells were simultaneously evaluated so that the reliability of the obtained data could be confirmed.
In particular, when a reporter gene detected by light signal is used as a reporter gene located downstream of each expression control region, the expression level of the reporter gene could be evaluated according to live imaging, simply continuously and qualitatively, without analyzing a cell extract. Accordingly, even in a case where the expression was reduced after it had been increased by induction of a drug enzyme, the drug enzyme induction test could be carried out simply and accurately, without missing the process in which the expression had been increased. Furthermore, if the vector of the present invention comprising a reporter gene detected by light signal is used, immobilization and destruction of cells and addition of a substrate, as in luciferase or β-galactosidase staining, become unnecessary, and a continuous measurement becomes possible. Thus, it has been economically advantageous.
Further, the present inventors have also found that when a hepatoma-derived cell line is used as a cell into which the vector is to be introduced, since a change in the expression level of a drug-metabolizing enzyme is extremely clearly observed depending on a difference in the maturity of cells, cells with high maturity suitable for the test can be selected more reliably and simply.
Specifically, the present invention provides a vector for producing a cell that is used for evaluation of induction of a drug-metabolizing enzyme by a test substance and cytotoxicity of the test substance, which comprises an expression control region of a fetus-specific gene, a first reporter gene, an expression control region of a drug-metabolizing enzyme gene, and a second reporter gene, wherein the first reporter gene is located downstream of the expression control region of the fetus-specific gene, and the second reporter gene is located downstream of the expression control region of the drug-metabolizing enzyme gene.
In addition, the present invention provides the above described vector, wherein the drug-metabolizing enzyme gene is cytochrome P450.
Moreover, the present invention provides the above described vector, wherein the fetus-specific gene is CYP3A7.
Furthermore, the present invention provides the above described vector, wherein the reporter gene is detected by light signal.
Further, the present invention provides the above described vector, wherein the reporter gene encodes a fluorescent protein.
Further, the present invention provides a cell used for evaluation of induction of a drug-metabolizing enzyme by a test substance and cytotoxicity of the test substance, wherein the cell comprises the above described vector.
Further, the present invention provides the above described cell, which is derived from a hepatoma-derived cell line.
Still further, the present invention provides the above described cell, which has properties of a stem cell capable of differentiating into a mature hepatocyte.
Still further, the present invention provides the above described cell, which is selected by a method comprising:
(1) a step of culturing cells containing the above described vector; and
(2) a step of selecting a cell using an expression level of the first reporter gene in the cells during the culture in the step (1) as an indicator.
Still further, the present invention provides the above described cell, wherein the culture in the step (1) is a culture for allowing cells containing the above described vector to differentiate into hepatocytes.
In addition, the present invention provides a non-human animal comprising the above described vector.
Moreover, the present invention provides a method for evaluating induction of a drug-metabolizing enzyme by a test substance and cytotoxicity of the test substance, which comprises:
(a) a step of evaluating expression of the first reporter gene in the above described cell; and
(b) a step of evaluating a change in expression of the second reporter gene in the above described cell before and after the contact of the above described cell with the test substance.
Furthermore, the present invention provides the above described method, wherein the evaluation of a change in the expression of the second reporter gene in the step (b) is based on the continuous measurement results of the expression of the second reporter gene.
Further, the present invention provides a kit for evaluating induction of a drug-metabolizing enzyme by a test substance and cytotoxicity of the test substance, wherein the kit comprises the above described vector.
Still further, the present invention provides the above described kit, further comprising a cell having properties of a stem cell capable of differentiating into a mature hepatocyte.
Using the vector of the present invention, a cell population used for simultaneously and accurately evaluating induction of a drug-metabolizing enzyme and cytotoxicity can be easily prepared. Upon preparation of such a cell population, the expression of a first reporter gene located downstream of the expression control region of a fetus-specific gene is evaluated, and if cells having a low expression level were selected, a cell population suitable for more accurate evaluation could be prepared. Moreover, since the vector of the present invention comprises both the expression control region of a drug-metabolizing enzyme gene and the expression control region of a fetus-specific gene, cells into which both of them are introduced at a constant ratio can be prepared using such a vector. With regard to the thus prepared cells, on the basis of the expression level of a reporter gene located downstream of a drug-metabolizing enzyme gene and the expression level of a reporter gene located downstream of a fetus-specific gene, the influence on cytotoxicity and the influence on the drug-metabolizing enzyme can be quantitatively evaluated. Furthermore, taking into consideration determination regarding whether or not the non-expression of the reporter gene is caused by the cytotoxicity of a test substance, the induction of a drug enzyme by the test substance can be evaluated more accurately. Further, using the vector of the present invention, the developmental stage or quality of cells can be evaluated, and as a result, cells with high maturity suitable for a drug-metabolizing enzyme induction test can be selected more reliably and easily. For example, using the vector of the present invention, a developmental stage, in which stem cells in the liver, cell lines similar thereto, induced pluripotent stem (iPS) cells, embryonic stem (ES) cells and the like differentiate into functional hepatocytes, can be monitored, and the differentiated cells can be easily selected. Whether the cells suitable for evaluation of induction of a drug-metabolizing enzyme are developed by direct differentiation from hepatocytes during the fetal period or the cells are developed from other stem cells after the birth has been unknown. This phenomenon can be clarified by pursuing whether or not the cell of the present invention expressing a first reporter gene located downstream of the expression control region of a fetal hepatocyte-specific gene can directly differentiate into a cell suitable for evaluation of induction of a drug-metabolizing enzyme over time using a second reporter gene in the vector of the present invention and also using a single cell. It has been an object to develop a cell differentiation induction method for producing cells suitable for evaluation of drug-metabolizing enzyme inducibility from stem cells such as ES cells or iPS cells. The development of such a differentiation induction method can be accelerated by clarifying the developmental stage of a hepatocyte having high metabolic function by the expression switching of a reporter gene in the vector of the present invention. Hence, according to the present invention, a vector capable of evaluating drug-metabolizing enzyme inducibility, expression switching and hepatotoxicity, and a cell prepared by introducing the above described vector into a biological model cell capable of being used for such evaluation can be provided.
The present invention provides a vector. Examples of the vector include a plasmid vector, a cosmid vector, a viral vector, and an artificial chromosome vector. Examples of the artificial chromosome vector include a yeast artificial chromosome vector (YAC), a bacterial artificial chromosome vector (BAC), a p1 artificial chromosome vector (PAC), a mouse artificial chromosome vector (MAC), and a human artificial chromosome vector (HAC).
The vector of the present invention comprises the expression control region of a fetus-specific gene.
The fetus-specific gene is a gene whose expression specifically increases during a fetal period. As such fetus-specific genes, various types of genes have been known. Examples of the fetus-specific gene include CYP3A7, Cyp3a16, α-fetoprotein, γ-glutamyl transpeptidase, GST-P, M2-PK, CK8, CK18, RL16/79, and EpCAM. CYP3A7 (cytochrome P450, family 3, subfamily A, polypeptide 7) is explained as Registration No. NM 000765 in NCBI Resources. The sequence of CYP3A7 is shown in Registration No. NM000765.3. CYP3A7 is a drug-metabolizing enzyme gene as well as a fetus-specific gene. Thus, CYP3A7 can be used as an indicator in evaluation of the induction of a drug-metabolizing enzyme, and thereby, it becomes possible to carry out more accurate evaluation. CYP3A7 is a marker for immature hepatoblasts. Using CYP3A7, liver regeneration after hepatocyte toxicity can be evaluated.
The expression control region is a region that controls the expression of a gene located downstream thereof. Examples of such an expression control region include a promoter and an enhancer. The promoter is a DNA sequence associated with initiation of the transcription of the coding region of a gene to which it binds. The enhancer controls the specificity of the bound promoter. The expression control region may comprise either a single promoter or a single enhancer, but it may also comprise both of the promoter and the enhancer.
The expression control region of a fetus-specific gene can be obtained by a well-known technique based on known information such as GenBank. Regarding CYP3A7, for example, Registration No. AF181861.1 of GenBank discloses a 1012-bp nucleotide sequence as a human cytochrome P-450IIA7 (CYP3A7) gene, a promoter region and a partial sequence. This 1012-bp nucleotide sequence is shown in SEQ ID NO: 1. Moreover, GenBank also discloses Registration No. AC069294.5 that is the nucleotide sequence of a BAC clone RP11-757A13 comprising the expression control region of CYP3A7. The nucleotide sequence disclosed as Registration No. AC069294.5 is shown in SEQ ID NO: 2. Furthermore, since the expression control region of CYP3A7 shows homology of 90% or more with the expression control region of CYP3A4, the promoter and enhancer regions of CYP3A7 are known to be in relative positions to the promoter and enhancer regions of CYP3A4 with Registration No. AF185589.1 (Bertilsson, G. et al., BBRC 280, 139-144, 2001; Sueyoshi, T and Neishi, M., Annu. Rev. Pharmacol. Toxicol. 41: 123-43, 2001). A sequence ranging from the transcription start point to the point at approximately −8000 bp of CYP3A7 can be used as an expression control region of CYP3A7. Sequences ranging from the transcription start point to −7733 to −7719 (dNR1), −7689 to −7672 (dNR2), −7287 to −7273 (dNR3), and −171 to −154 (pNP) of Registration No. AF185589.1 are known to be enhancers of CYP3A4. Four homologous regions in CYP3A7 that show homology with these sequences are also known to function as enhancers. The sequences of these 4 enhancers are shown in the following Table 1. The expression control region of CYP3A7 preferably comprises the aforementioned 4 enhancer regions. Using the sequence ranging from the transcription start point to the point at approximately −8000 bp as an expression control region of CYP3A7, the expression control region of CYP3A7 can comprise these enhancers. In addition, sequences having identity of 95%, 98%, or 99% or more with these sequences can also be used as expression control regions of CYP3A7, as long as they have a function to control the expression of a gene located downstream thereof. Herein, the identity of sequence can be calculated using analysis tools that are commercially available or are made available through electric telecommunication lines. Specifically, such sequence identity can be calculated using analysis software such as BLAST (J. Mal. Biol., 215, 403, 1990) or FASTA (Methods in Enzymology, 183, 63-69).
The vector of the present invention comprises a first reporter gene.
The reporter gene is any given nucleic acid sequence that allows a cell to present a detectable label. Examples of such a detectable label include fluorescence signal, a phosphorescence signal, a protein that is detectable in an assay, an enzyme activity, and an antigen that is detectable on a cell or in a cell. Examples of a protein encoded by such a reporter gene include fluorescent proteins such as a green fluorescent protein (GFP), a humanized Renilla green fluorescent protein (hrGEP), an enhanced green fluorescent protein (eGFP), an enhanced blue fluorescent protein (eBFP), an enhanced cyan fluorescent protein (eCFP), an enhanced yellow fluorescent protein (eYFP), and a red fluorescent protein (RFP or DsRed). More examples of a protein encoded by such a reporter gene include bioluminescent proteins such as firefly luciferase and Renilla luciferase. Further examples of a protein encoded by such a reporter gene include enzymes for converting chemiluminescent substrates, such as alkaline phosphatase, peroxidase, chloramphenicol acetyltransferase, and β-galactosidase. In the present invention, when a reporter gene detected by a light signal such as a fluorescence signal or a phosphorescence signal is used, the expression level of the reporter gene can be observed in a state in which a cell is allowed to survive, and a cell used for evaluation can be easily selected, while the cell is alive. In addition, in such a case, the reporter gene can be used in an experiment in which a test substance is continuously administered, and a change over time in the expression level of the reporter gene can be pursued in a real time. As such, a reporter gene using a light signal as a label can be preferably used as the reporter gene of the present invention.
The vector of the present invention comprises the expression control region of a drug-metabolizing enzyme gene.
The drug-metabolizing enzyme gene is a gene that encodes an enzyme metabolizing a drug in a living body. Various genes have been known as such drug-metabolizing enzyme genes. Examples of the known drug-metabolizing enzyme gene include proteins binding to cells or being present on cells and nuclear proteins, which chemically modify or isolate drugs. Specific functions of the drug-metabolizing enzyme gene, such as chemical decomposition, conversion of a drug to another compound, or transportation of a drug between other cells or in an intracellular space, are not particularly limited, as long as the drug-metabolizing enzyme gene acts to eliminate a drug from a living body or to substantially change the effects of the drug. Examples of the drug-metabolizing enzyme gene include cytochrome P450, flavin-containing monooxygenase, alcohol dehydrogenase, aldehyde dehydrogenase, monoamine oxidase, aldehyde reductase, ketone reductase, esterase, epoxy hydrolase, β-glucuronidase, sulfatase, UDP-glucuronic acid transferase, glutathione S-transferase, N-acetyltransferase, sulfotransferase, glycine conjugation enzyme, methylase, and glucose transferase. Cytochrome P450 includes a CYP3A gene group including CYP3A4, CYP3A7 and the like (which is also simply referred to as “CYP3A”). Moreover, such cytochrome P450 includes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4B1, CYP5A1, CYP8A1, CYP21, and the like. The gene having a role in transportation include OCT1, NTCP, OATP1B1, OATP1B3, OATP2B1, OAT2, MRP2, MRP3, MRP4, MRP6, MDR1, BCRP, BSEP, and the like. The nuclear receptor includes PXR, CAR, and the like.
The expression control region of a drug-metabolizing enzyme gene can be obtained by a well-known technique, based on known information such as GenBank. For example, Registration No. AF183589.1 of GenBank discloses a 11374-bp sequence as a promoter region of a human cytochrome P450 3A4 (CYP3A4) gene. A sequence ranging from the transcription start point to the point at approximately −8000 bp can be used as an expression control region of CYP3A4. This 11374-bp sequence disclosed as Registration No. AF185589.1 is shown in SEQ ID NO: 3. In particular, sequences ranging from the transcription start point to −7733 to −7719 (dNR1), −7689 to −7672 (dNR2), −7287 to −7273 (dNR3), and −171 to −154 (pNR) of Registration No. AF185589.1 are known to be enhancers of CYP3A4 (Bertilsson, G. et al., BBRC 280, 139-144, 2001; Sueyoshi, T and Neishi, M., Anna. Rev. Pharmacol. Toxicol. 41: 123-43, 2001). These sequences may be used as expression control regions of CYP3A4. In addition, sequences having identity of 95%, 98%, or 99% or more with these sequences can also be used as expression control regions of CYP3A4, as long as they have a function to control the expression of a gene located downstream thereof. Herein, the identity of sequence can be calculated using analysis tools that are commercially available or are made available through electric telecommunication lines. Specifically, such sequence identity can be calculated using analysis software such as BLAST (J. Mol. Biol., 215, 403, 1990) or FASTA (Methods in Enzymology, 183, 63-69). When the expression control region of CYP3A4 is used, high expression of a second reporter gene located downstream thereof becomes an indicator showing that the concerned cells are suitable for evaluation of induction of a drug-metabolizing enzyme. Thus, it becomes possible to prepare cells used for more accurate evaluation.
The vector of the present invention comprises a second reporter gene. The second reporter gene is desirably different from the first reporter gene.
In the vector of the present invention, the first reporter gene is located downstream of the expression control region of an early developmental stage-specific gene, and the second reporter gene is located downstream of the expression control region of a drug-metabolizing enzyme gene. If the reporter gene is positioned after the expression control region when a direction in which the reporter gene is transcribed is used as a reference, it may be determined that the reporter gene is located downstream of the expression control region. Also, when the expression control region is operably linked to the reporter gene, it may be determined that the reporter gene is located downstream of the expression control region. When experimental results are obtained in which the expression control region drives the transcription of a coding region, it may be considered that the expression control region operably binds to the coding region.
The vector of the present invention may comprise a sequence used for genetic recombination of utilizing a site-specific recombination reaction such as a Cre-loxP system. When the vector of the present invention comprises a sequence used for the Cre-loxP system, it becomes possible to introduce one copy of the entire-length vector into all cells. Thereby, a variation in the expression of a reporter gene among cells is reduced, and thus, accuracy in the evaluation is further improved.
The vector of the present invention may be obtained by ligating a nucleic acid fragment containing an expression control region, a reporter gene and the like to a vector according to a known method. In addition, the present vector may also be obtained by replacing the protein coding region (ORF) of each gene in a vector comprising the peripheral region on the genome of the genes with a reporter gene.
In the vector of the present invention, when CYP3A4 is used as a drug-metabolizing enzyme gene and CYP3A7 is used as a fetus-specific gene, the expression of the two genes is under developmental stage-specific control. Accordingly, in cells that are not suitable for evaluation of induction of a drug-metabolizing enzyme, an increase in the expression of a first reporter gene and a decrease in the expression of a second reporter gene are observed. On the other hand, in cells that are suitable for such evaluation, a decrease in the expression of a first reporter gene and an increase in the expression of a second reporter gene are observed. As such, whether or not cells are suitable for evaluation of induction of a drug-metabolizing enzyme can be determined from both sides, namely, an increase in the expression of a reporter gene and a decrease in the expression thereof. Thus, functional cells, which have high maturity of differentiation and sufficiently maintain in vivo functions, can be extremely efficiently obtained. Accordingly, in the vector of the present invention, it is preferable to use CYP3A4 as a drug-metabolizing enzyme gene and to use CYP3A7 as a fetus-specific gene.
The vector of the present invention can be used for production of cells used to evaluate the induction of a drug-metabolizing enzyme by a test substance.
The cells used to evaluate the induction of a drug-metabolizing enzyme by a test substance can be obtained by introducing the vector of the present invention into cells according to a known method such as lipofection or a microcell fusion method.
The cells into which the vector of the present invention is to be introduced are cells in tissues in a living body, in which induction of a drug-metabolizing enzyme occurs. Examples of the tissues, from which the cells into which the vector of the present invention is to be introduced are derived, include liver and small intestine. The organism, from which the cells into which the vector of the present invention is to be introduced are derived, is desirably an organism in which induction test of a drug-metabolizing enzyme is required. Examples of such an organism include mammals such as a human and a mouse. Moreover, the cells into which the vector of the present invention is to be introduced may be cultured cells, such as HepG2 cells, HepaRG cells, and Hu7 cells. The HepG2 cells are human hepatoma cells, which have been widely used as human hepatocyte model cells. The cells into which the vector of the present invention is to be introduced are preferably hepatoma-derived cell lines, such as HepaRG (registered trademark). Moreover, the cells into which the vector of the present invention is to be introduced may be pluripotent stem cells, such as embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells). Such embryonic stem cells and induced pluripotent stem cells are able to grow indefinitely, and thus, these cells are useful as a large supply source of functional cells. The cells into which the vector of the present invention is to be introduced are preferably immature cells having ability to differentiate into hepatocytes having high maturity, such as hepatoma-derived cell lines having the properties of stem cells capable of differentiating into mature hepatocytes; hepatoma-derived cell lines or stem cells in the liver in a living body; and ES cells or iPS cells.
The cells used to evaluate the induction of a drug-metabolizing enzyme by a test substance may also be cells obtained by inducing pluripotent stem cells, into which the vector of the present invention has been introduced, to differentiate into preferred tissues. For example, in the case of cells exhibiting the properties of the mature hepatocytes of an adult as their functions, the cells are induced to differentiate into hepatocytes by a method comprising obtaining SOX9-positive progenitor cells that are common in the pancreas, the liver and the small intestine from pluripotent stem cells such as iPS cells, into which the vector has been introduced, then obtaining AFP/PDX1-positive progenitor cells that are common in the pancreas and the liver, and then allowing the obtained cells to grow, and thereafter, CYP3A7/Afp/EpCAM-positive fetal hepatocytes are obtained. Then, maturation of the cells is carried out to obtain CY3A4/ALB-positive adult liver, so that the cells of interest may be obtained.
Moreover, as such cells used to evaluate the induction of a drug-metabolizing enzyme by a test substance, a chimeric animal may be produced using pluripotent stem cells into which an artificial chromosome has been introduced according to a microcell fusion method or the like, and thereafter, cells may be fractionated from tissues in the liver or the like of the produced chimeric animal and may be then used. By selecting cells having low expression of the first reporter gene from the fractionated cells, cells capable of more accurately evaluating the induction of a drug-metabolizing enzyme can be obtained. Examples of the non-human animal comprising the vector of the present invention include mammals such as bovine, miniature pigs, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and monkeys.
Cells, which comprises the vector of the present invention used to produce cells used for evaluation of the induction of a drug-metabolizing enzyme by a test substance and the cytotoxicity of the test substance, and which involves a low expression level of the gene by the expression control region of a fetus-specific gene in the vector of the present invention, have high maturity and are suitable for tests for evaluating the induction of a drug-metabolizing enzyme. Accordingly, the cell used to evaluate the induction of a drug-metabolizing enzyme by a test substance is preferably a cell selected by a method comprising: (1) a step of culturing cells containing the vector of the present invention; and (2) a step of selecting a cell, using the expression level of the first reporter gene in the cells during the culture in the step (1) as an indicator. More specifically, in step (2), a cell in which the expression level of the first reporter gene is low is selected. Among the above described cells, cells, in which the expression level of the second reporter gene by the expression control region of a drug-metabolizing enzyme gene in the vector of the present invention is high, are have higher maturity, and thus, such cells are more suitable for tests for evaluating induction of a drug-metabolizing enzyme. Accordingly, more preferably, in step (2), cells are selected, using the expression level of the second reporter gene in cells as an indicator. More specifically, in step (2), cells, in which the expression level of the second reporter gene is high, are preferably selected. Moreover, the culture in step (1) is preferably carried out to allow cells containing the vector of the present invention to differentiate into hepatocytes. The culture that is carried out to allow the cells to differentiate into hepatocytes can be carried out in a medium used for differentiation into hepatocytes. The cells containing the vector of the present invention, which are cultured in step (1), may be selected, using the expression of the first reporter gene as an indicator. Furthermore, cells may be selected in step (2), using the presence of the cells in the upper layer of the cell population as a further indicator.
The thus obtained cells can be used to evaluate the induction of a drug-metabolizing enzyme by a test substance. The test substance is a product used to verify if it causes induction of a drug-metabolizing enzyme, and an example of the test substance is a pharmaceutical product candidate compound. Examples of the test substance include, but are not limited to, pharmaceutical products, food products, chemical substances, and the metabolites thereof.
The evaluation method provided by the present invention comprises a step of evaluating the expression of the first reporter gene in the above-obtained cells used to evaluate the induction of a drug-metabolizing enzyme by a test substance (hereinafter simply referred to as “cells for evaluation”). More specifically, the evaluation method of the present invention may comprise a step of evaluating the expression of the first reporter gene in the cells and then removing cells involving high expression of the first reporter gene from the cells used for evaluation. Moreover, the evaluation method of the present invention may comprise a step of allowing a test substance to come into contact with a cell for evaluation, evaluating a change in the expression of the first reporter gene between before and after the contact, and determining that the contacted test substance has cytotoxicity when the increase in the expression is observed. The contact of the test substance with the cell for evaluation may be carried out by administering the test substance to a non-human animal comprising the vector of the present invention.
The evaluation method provided by the present invention comprises a step of evaluating a change in the expression of the second reporter gene in the cell for evaluation between before and after the contact of the cell for evaluation with the test substance. More specifically, the evaluation method of the present invention may comprise a step of determining that the contacted test substance has ability to induce a drug-metabolizing enzyme, when the expression of the second reporter gene is increased by the contact with the test substance. Preferably, a change in the expression of the second reporter gene is evaluated based on continuous measurement results of the expression of the second reporter gene. In this case, the use of the continuous measurement results is preferable in that, even in a case where the expression of the second reporter gene in the cell is decreased after it has been once increased by drug enzyme induction, the drug enzyme induction can be carried out simply and accurately, without missing the results. Such a continuous measurement is carried out, for example, for 3 days after the contact with the test substance. The continuous measurement is carried out by obtaining the results of the expression level of the reporter gene, for example, every one or two hours, and preferably every 30 minutes to 1 hour.
The evaluation method provided by the present invention may be carried out by allowing a plurality of test substances to simultaneously come into contact with cells using a plurality of wells, such as 96 wells or 384 wells, so that the present evaluation method may be carried out by high content screening (HCS) for simultaneous evaluation of reporter genes. When a reporter gene using a light signal as a label is used, the expression of the reporter gene can be simply discriminated as light signal at a time, and thus, a large number of test substances can be promptly evaluated.
The evaluation method provided by the present invention may be carried out using a kit. An example of such a kit is a kit for evaluating the induction of a drug-metabolizing enzyme by a test substance and the cytotoxicity of the test substance, wherein the kit comprises the vector of the present invention. The kit of the present invention may further comprise: cells; a medium for culturing the cell line and allowing the cells to grow; a drug for inducing differentiation of the cells into hepatocytes; a test substance; a vessel for culturing the cells; an instruction manual regarding at least one constituent of the kit; and the like. The cells comprised in the kit are preferably cells having the properties of stem cells capable of differentiating into mature hepatocytes.
Hereinafter, the present invention will be described in the following examples. However, these examples are not intended to limit the scope of the present invention.
A bacterial artificial chromosome RP11-757A13 BAC comprising the gene expression control regions and protein coding regions of CYP3A4 and CYP3A7, which was produced by inserting the gene expression control region ranging from CYP3A4 to CYP3A7 (an entire length of 123,778 bp) into the EcoRI site of a 11.6-kb BAC clone vector pBACe3.6, was used in the subsequent experiment. The nucleotide sequence of the BAC clone RP11-757A13 is disclosed as Registration No. AC069294.5 in GenBank.
(2) Substitution of Protein Coding Regions of CYP3A4 and CYP3A7 into EGFP and DsRed:
a. Production of Vector Used for Substitution into DsRed:
Using a pDsRed-Express-1 vector as a basic vector, the 5′ region (left arm, LA) of hCYP3A7 was inserted into the BglII-SalI site located immediately before the ORE of a DsRed gene, and the 3′ region (right arm, RA) of CYP3A7 was inserted into the BsaI site located immediately after a kanamycin-resistant gene, so as to construct an LA-DsRed-pA-RA CYP3A7 knock-in (hereinafter also referred to as simply “KI”) vector that is a vector used for substitution into DsRed.
b. Production of Vector Used for Substitution into EGFP
Using a pHygEGFP vector as a basic vector, the LA and PA of CYP3A4 were inserted into the BglII site comprising pA and an ampicillin-resistant gene, such that after open chain, the EGFP gene can be located between LA and RA, so as to construct an LA-EGFP-pA-RA-CYP3A4 KI vector that is a vector used for substitution into EGFP.
c. Substitution into DsRed
(a) Introduction of Bacterial Artificial Chromosome into Cells Used for Homologous Recombination
DY380 was streaked onto an LB plate, and it was then heated at 32° C. overnight. A liquid prepared by picking up colonies and subjecting them to a shaking culture (32° C., 180-200 rpm, 14 hours) was transferred into a 50-ml LB liquid, and it was then subjected to a shaking culture (32° C., 180-200 rpm, 3 to 4 hours). When the O.D. became 0.4, shaking was terminated, and the cell culture solution was then stirred in a water bath at 42° C. for 5 minutes. After that, the cell culture solution was left at rest for 5 minutes. Then, after it had been left at rest in ice for 10 minutes, DY380 was collected using a centrifuge (4° C., 3500 rpm, 10 minutes). After the collected cells were washed with MQ twice, they were then mixed with 200 ng of the aforementioned RB11-757A13 BAC DNA, followed by performing electroporation with Gene Pluser (1750 V, 25 μF, 200Ω, pulse no. 1). The resultant was shaken at 32° C. for 1.5 hours, and thereafter, it was applied onto an LB plate and was then heated at 32° C. overnight. In this way, the bacterial artificial chromosome RB11-757A13 was introduced into the DY380 cells that were cells used for homologous recombination, so as to obtain a (CYP3A4+/7+) BAC clone comprising both CYP3A4 and CYP3A7.
(b) Substitution into DsRed by Homologous Recombination
A necessary DNA fragment was cleaved from the aforementioned LA-DsRed-pA-RA CYP3A7 KI vector that was a vector used for substitution into DsRed, and 100 ng of the DNA fragment was introduced into the aforementioned CYP3A4+/7+BAC clone by electroporation. It was applied onto a drug-added LB plate. Genomic PCR was performed on the obtained kanamycin-resistant clone, using primers for confirmation of homologous recombination. The used primers are shown in Table 2. From all BAC clones, PCR products having the estimated length were obtained. Thus, a CYP3A4+/7R BAC clone, in which the ORF of CYP3A7 was substituted with DsRed, was obtained.
d. Substitution into EGFP:
The above described LA-EGFP-pA-RA-CYP3A4 KI vector was linearized with XhoI, and the obtained linear DNA was knocked in the CYP3A4 ORD region on the above-obtained CYP3A4+/7R BAC by electroporation, so that the EGFP gene was inserted therein. Using 9 pairs of primers for confirming homologous recombination, genomic PCR was carried out on the obtained ampicillin-resistant clone. As a result, it was confirmed that PCP products having the estimated length were found in all of the obtained clones. In this way, a CYP3A4G/7R BAC clone that was a BAC clone in which the ORF of CYP3A7 was substituted with DsRed and the ORE of CYP3A4 was substituted with EGFP was obtained. The primers used in the genomic PCR are shown in the above Table 2.
e. Insertion of a loxP Site:
In order to mount BAC DNA via a Cre/loxP system onto MAC (mouse artificial chromosome) or any given chromosome into which a loxP sequence had previously been inserted, a loxP sequence was knocked in the above-obtained CYP3A4G/7R BAC according to a known method (Sternberg and Hamilton, 1981; Hoess et al., 1982; Nagy, 2000).
(1) Production of loxP tg-HepG2:
A plasmid vector (VH21-12#8-3) comprising HPRT exon 1-2, loxP, and a Hyg-resistant gene as a drug selection marker was treated with the restriction enzyme (NotI) to linearize it. Lipofectamine LTX reagent was used for introduction of this vector into HepG2. First, 5 μg of VH21-12#8-3 was diluted with 2.5 ml of Opti-MEM, and 12.5 μg of PLUS reagent was added to the diluted solution. The obtained mixture was left at rest at room temperature for 5 minutes. Thereafter, 68.75 μl of LTX reagent was added to the reaction solution, and the obtained mixture was left at rest at room temperature for 25 minutes. Thereafter, the reaction solution was added onto HepG2 (10-cm dish, approximately 1×107). It was cultured in 5 ml of D-MEM that contained 10% fetal bovine serum for 1 day, and HepG2 was removed from the culture dish by treating it with trypsin, and it was then seeded on five 10-cm dishes. Hygromycin (400 μg/ml) was added to a medium, and the medium was then exchanged with a fresh one every 3 days. On Day 14 after addition of hygromycin, colonies were isolated, and loxP tg-HepG2, a transgenic HepG2 cell line having one loxP site on the long arm of chromosome 14 was obtained. Gene transfer was confirmed by PCR using a primer set that amplified a region ranging from the HPRT exon 2 terminus to a hygromycin region. Upon production, when HepG2 was cultured, D-MEM (High-glucose) that contained L-glutamine and phenol red, to which 10% fetal bovine serum was added, was used. In addition, upon subculture, cells were removed using 0.04% EDTA-added 0.1% trypsin solution.
(2) Introduction of CYP3A4G/7R BAC into loxP tg-HepG2:
In order to insert the above described CYP3A4G/7R-BAC into the above described loxP TG-HepG2 using a Cre-loxP system, lipofection was carried out using Lipofectamine LTX reagent. First, 5 μg of CYP3A4G/7R-BAC and 2 μg of pCAG-Cre were diluted with 1.25 ml of Opti-MEM, and thereafter, 6.25 μl of PLUS reagent was added to the diluted solution. The obtained mixture was left at rest at room temperature for 5 minutes. Thereafter, 25 μl of LTX reagent was added to the reaction solution, and the obtained mixture was left at rest for 25 minutes. Then, the obtained reaction solution was added onto HepG2 (6-cm dish, approximately 80% confluent). After completion of a culture for 1 day, the cells were removed from the dish by treating them with trypsin, and were then seeded on three 10-cm culture dishes. Neomycin (800 μg/ml G418) was added to a medium, and the medium was then exchanged with a fresh one every 3 days. On Day 10 after addition of neomycin, colonies were isolated, and CYP3A4G/7R BAC-introduced HepG2 cells that were a cell line prepared by introducing CYP3A4G/7R BAC into loxP tg-HepG2 were obtained. The obtainment of a stable expression cell line was confirmed by PCR using the primer sets for amplifying the EGFP and DsRed regions of a BAC vector, as shown in Table 2, and FISH analysis (
The above described CYP3A4+/7R-loxP BAC was purified, and it was then transfected together with a Cre enzyme expression vector into CHO cells retaining MAC by a lipofection method. DsRed, EGFP, and the control regions of CYP3A4 and CYP3A7 were confirmed by genomic PCR. Further, FISH analysis was carried out, and CYP3A4+/7R MAC was obtained as a clone capable of confirming MAC in the CHO cells and CYP3A4+/7R BAC mounted on the MAC (
The CYP3A4+/7R MAC in the CHO cells obtained in Example 3 was introduced into mouse ES cells (TT2F) by a microcell fusion method. Using the primers shown in Table 2, DsRed, EGFP, and the control regions of CYP3A4 and CYP3A7 were confirmed by genomic PCR. Moreover, FISH analysis was carried out, so that the BAC DNA mounted on the MAC in the TT2F cells could be confirmed (
(1) Production of Chimeric Mice from CYP3A4+/7R ES Cells:
Highly chimeric baby mice were obtained using the CYP3A4+/7R mouse ES cells obtained in Example 4. All tissues, such as liver, skin, brain, lung, small intestine, spleen, and pancreas, of individual chimeric mice on Day 1 exhibited the fluorescence of EGFP mounted on the MAC vector, and thus, it was confirmed that the CYP3A4+/7R ES cells could contribute to tissue generation. On the other hand, the fluorescence of DsRed that reflects the expression of CYP3A7 was strongly observed in the liver and the small intestine (
The chimeric mice produced in Example 5(1) were subjected to breeding to obtain a CYP3A4+/7R MAC-introduced mouse strain. The expression of DsRed was confirmed in the liver of this CYP3A4+/7R mouse on 1st day after the birth (
(1) Production of loxP tg-HepaRG:
A plasmid vector (VH21-12#8-3) comprising HPRT exon1-2, loxP, and a Hyg-resistant gene as a drug selection marker, was linearized by treating it with the restriction enzyme (NotI). Introduction of this vector into HepaRG and the obtainment of a hygromycin (400 μg/ml) resistant clone were carried out in the same manner as that in the production of loxP tg-HepG2. When HepaRG was subjected to a growing culture, only the growth-dedicated medium of Biopredic International (a 720 differentiation medium that was an MIL700 basic medium, to which ADD720 had been added) was used as a culture medium. In addition, upon subculture, cells were removed using 0.02% EDTA-added 0.005% trypsin solution. The composition of the culture medium of BIOPREDIC has been closed.
(2) Introduction of CYP3A4G/7R BAC into loxP Tg-HepaRG:
In order to insert the above described CYP3A4G/7R-BAC into the above described loxP tg-HepaRG by a Cre-loxP system, lipofection was carried out using Lipofectamine LTX reagent. Introduction of this vector into HepaRG and the obtainment of a neomycin (800 μg/ml G418) resistant clone were carried out in the same manner as in the production of loxP tg-HepG2. CYP3A4G/7R BAC-introduced HepaRG cells, which were a cell line in which CYP3A4G/7R BAC had been introduced into loxP tg-HepaRG, were obtained. The obtainment of a stable expression cell line was confirmed by PCR using the primer sets for amplifying the EGFP and DsRed regions of a BAC vector, as shown in Table 2, and FISH analysis (
The CYP3A4G/7R BAC-introduced HepG2 cells obtained in Example 2, in which the expression level of EGFP was high and the expression level of DsRed was low, were seeded in each well of a 6-well culture dish (φ 35 mm) coated with Cellmatrix (Nitta Gelatin, Inc.) at a cell density of 5×105 cells/well. From the following day for 48 hours, 50 μM or 100 μM dexamethasone (DEX) or rifampicin (RIF) was added as a GYP-inducing drug to the cells. As a result, after addition of the GYP-inducing drug, increases in the expression levels of both DsRed and EGFP were observed in the CYP3A4G/7R BAC-introduced HepG2 cells, although the expression levels of DsRed and EGFP were low when the GYP-inducing drug was not added (
Increases in the numbers of DsRed-positive cells and EGFP-positive cells, which were determined when 25 μM, 50 μM or 100 μM DEX or RIF was added to the cells for 48 hours under the same culture conditions as those in Test Example 1, were examined by FACS analysis. The loxP tg-HepG2 that was a transgenic cell line before insertion of BAC produced in Example 2 was used as a negative control, and the numbers of the aforementioned positive cells relative to the number of the negative control cells defined as 1 are shown in
The expression of CYP3A7 during the injury of hepatocytes was examined by a method of liver regeneration after partial hepatectomy performed on 70% living liver, using a humanized CYP3A model mouse into which human CYP3A4 and CYP3A7 have been incorporated, a method of damaging the liver of the same mouse as described above by administration of a CYP-inducing drug, and a method of transplanting frozen human hepatocytes into the liver injury model mouse. In the method of liver regeneration after the partial hepatectomy, mice that were fed after 70% of the liver had been excised by ligating it with a cord were produced. Thereafter, the mice were successively subjected to dissection from Days 1 to 5 to collect the liver from each mouse, and gene expression was then examined. In the method of damaging the liver by administration of a CYP-inducing drug, the liver was excised from each mouse belonging to a PCN administration group and a corn oil administration control group, and gene expression was then examined. In the method of transplanting frozen human hepatocytes into the living mouse, a part of the humanized mouse liver was obtained from PhenixBio, and gene expression was then examined. As a result, it was confirmed that the expression of CYP3A7 was transiently increased when hepatocytes were regenerated after damage had been given to the liver.
1 μM, 10 μM, 25 μM or 100 μM RIF was added to the cells for 48 hours under the same culture conditions as those in Test Example 1, and thereafter, the total area fluorescence intensity of DsRed-positive and EGFP cells was measured under a fluorescence microscope. The loxP tg-RepG2 that was a transgenic cell line before insertion of BAC produced in Example 2 was used as a negative control, and the fluorescence intensity of the aforementioned positive cells relative to the value of the negative control defined as 1 are shown in
When 100 μM DEX and RIF were added to the cells for 48 hours under the same culture conditions as those in Test Example 1, whether or not a correlation was observed between changes in the fluorescence intensity of DsRed and EGFP and changes in the gene expression levels of endogenous CYP3A4 and CYP3A7 genes was analyzed by quantitative RT-PCR. An untreated group was used as a negative control and the fluorescence intensity of DsRed and EGFP relative to the value of the negative control defined as 1 are shown in
The CYP3A4G/7R BAC-introduced HepaRG cells obtained in Example 6 were subjected to a growing culture (Growing) for 2 weeks (2 W), and the cells were then subjected to a differentiation culture (Differentiation). When the HepaRG cells were subjected to a growing culture, only the growth-dedicated medium of Biopredic International (a 710 growth medium that was an MIL700 basic medium to which ADD710 had been added) was used as a culture medium. On the other hand, when the HepaRG cells were subjected to a differentiation culture, only the differentiation-dedicated medium of Biopredic International (a 720 differentiation medium that was an MIL700 basic medium to which ADD720 had been added) was used as a culture medium. The HepaRG cells differentiate into small hepatocyte-like cells, and they appear in the upper layer of a cell population.
The CYP3A4G/7R BAC-introduced HepaRG cell clone (C3) obtained in Example 6 was seeded in a 96-well culture dish coated with Cellmatrix, and it was then subjected to a growing culture for 14 days (G14d) and then subjected to a differentiation culture for 14 days.
The fluorescence microphotographs of the cells used in Test Example 7, which were prepared by subjecting the CYP3A4G/7R BAC-introduced HepaRG cell clone (C3) to a growing culture for 14 days and then to a differentiation culture for 5 days (D5d), were analyzed in detail. The hepatic parenchymal cells in the fetal liver are hepatoblasts, and the hepatoblasts express CYP3A7 and do not express CYP3A4. After birth, CYP3A7 cannot be detected, and thus, CYP3A4 becomes a major P450 drug-metabolizing enzyme. In this process, whether the expression of CYP3A7 is shifted to the expression of CYP3A4 in a single cell, or whether cells expressing CYP3A4 can be produced from other stem cells that do not express CYP3A7, has not yet been clarified. As shown in
Changes over time in the total area fluorescence intensities of EGFP and DsRed in the CYP3A4G/7R BAC-introduced HepaRG cell clone (C3) used in Test Example 7 on Days 7 (G7d) and 14 (G14d) after the cell clone had been subjected to a growing culture, and on Days 1, 3, 5, 7, 10, and 12 (D1d, D3d, D5d, D7d, D10d, and D12d) of a differentiation culture, were analyzed under a fluorescence microscope. The results are shown in
CYP3A4 expression-inducing drugs (RIF: rifampicin, DEX: dexamethasone, CLO: clotrimazole, NIF: nifedipine, and PCN: pregnenolone 16α-carbonitrile) dissolved in 1% DMSO were each added to the CYP3A4G/7R BAC-introduced HepaRG cell clone (C3) used in Test Example 7, which was subjected to a growing culture for 14 days and then to a differentiation culture for 12 days. Forty-eight hours later (corresponding to Day 14 of differentiation culture), the total area fluorescence intensities of EGFP and DsRed were analyzed under a fluorescence microscope (
The CYP3A4+/7MAC-introduced mice produced in Example 5 strongly expressed DsRed for several weeks after the birth. However, when the mice were 8 weeks old, the expression of DsRed in the liver was significantly reduced. To such mice, 5% carbon tetrachloride dissolved in DMSO was administered, and thereafter, the expression of CYP3A7 upon cell damage was observed for 3 days. As a result, it is found that an increase in the suppressed fluorescence intensity of DsRed in the liver can be visualized under a fluorescence microscope (
Using RNAs extracted from the livers of the CYP3A4+/7R MAC-introduced mice to which 5% carbon tetrachloride had been administered in Test Example 11 and non-administered (Day 0) mice, an increase in the expression of DsRed on Days 1, 2 and 3 after the administration was analyzed by quantitative RT-PCR (
RT-PCR primers used for confirmation of gene expression in CYP3A4/7 BAC-introduced cells are shown in the following Table 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2012-232018 | Oct 2012 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2013/079058 | 10/21/2013 | WO | 00 |
