JPWO2018181880A1 - Compounds that improve the olfactory sensitivity of nematodes - Google Patents

Abstract

The present invention provides compounds that improve the olfactory sensitivity of nematodes.

Description

  The present invention provides compounds that improve the olfactory sensitivity of nematodes.

  The nematode exhibits an attracting action on the urine of a cancer patient and a repelling action on the urine of a healthy person. Therefore, it can be determined whether the subject from which urine is derived is a cancer patient or a healthy person based on the chemotactic behavior of the nematode (Patent Document 1). More specifically, Patent Literature 1 describes that a cancer patient could be detected with 100% sensitivity and 95% specificity by a medium-scale test of 242 specimens.

WO2015 / 088039

  The present inventors sought a method for further enhancing the chemotaxis behavior in the chemotaxis behavior evaluation. As a result, the present inventors have clarified that cineole improves the olfactory sensitivity of nematodes and enhances the chemotactic behavior of nematodes. The present invention is based on such knowledge.

That is, according to the present invention, the following inventions are provided.
(1) A composition for use in enhancing the olfactory sensitivity of a nematode, comprising cineole.
(2) A composition for use in enhancing chemotaxis behavior based on the olfactory sense of nematodes, including cineole.
(3) The composition according to the above (2), wherein the chemotaxis behavior is idle running behavior and / or repellent behavior.
(4) A method for enhancing the olfactory sensitivity of a nematode,
Contacting cineole or a composition containing cineole with a nematode.
(5) A method for enhancing chemotaxis behavior based on the olfactory sense of nematodes,
Contacting cineole or a composition containing cineole with a nematode.
(6) The method according to (5) above, wherein the chemotaxis behavior is idle movement and / or repelling behavior.

FIG. 1 shows a plan view of a petri dish 1 used for evaluation of chemotaxis behavior of nematodes. FIG. 2A shows the sensibility of nematode olfaction by 1,8-cineole and the effect of enhancing the chemotaxis behavior of nematode. FIG. 2B shows the effect of 1,4-cineole on the sensitivity of nematode olfaction and the chemotactic behavior of nematode. FIG. 3A shows that the decrease in nematode attracting behavior (or enhanced repellent behavior) by salt learning is enhanced by 1,8-cineole. FIG. 3B shows that the enhancement effect by 1,8-cineole on the decrease in nematode attracting behavior by salt learning (or enhanced repellent behavior) disappears in the odr-3 mutant.

Detailed Description of the Invention

  As used herein, “subject” means a mammal, eg, a primate (eg, monkey, chimpanzee, gorilla, bonobo, orangutan, marmoset, and human), eg, a human. As used herein, the subject is preferably a human. Further, in the present specification, “subject” is used in a sense that includes healthy subjects, subjects suspected of having cancer, and subjects suffering from cancer.

  As used herein, “cancer” means a malignant tumor. Cancers can be broadly classified into hematopoietic tumors, epithelial cancers (carcinoma), and non-epithelial sarcomas (sarcoma). Examples of hematopoietic tumors include leukemia, malignant lymphoma, and myeloma. Epithelial cancers include lung cancer, breast cancer, stomach cancer, colon cancer, uterine cancer, ovarian cancer, head and neck cancer, and tongue cancer. Sarcomas include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma. As used herein, “cancer” includes early stage cancers such as stage 0 early stage cancer and stage 1 early stage cancer.

  In the present specification, the “composition for use in cancer diagnosis” is sometimes simply referred to as “cancer diagnostic agent”.

As used herein, “nematode” means Caenorhabditis elegans . Nematodes can be transferred to the Caenorhabditis Genetics Center (CGC) at the University of Minnesota's Department of Biological Sciences. Thus, those skilled in the art can obtain most known strains from CGC. From the viewpoint of being able to reproduce by self-fertilization, hermaphrodite can be preferably used.

  As used herein, “running behavior” means attraction or repellent behavior. Attracting behavior means behavior that shortens the physical distance from a certain substance, and repelling behavior means behavior that widens the physical distance from a certain substance. Substances that induce attraction behavior are called attractants, and substances that induce repellent behavior are called repellent substances. Nematodes have the property of attracting attractants by olfaction and repelling repellents. The act of attracting an attracting substance is called an attracting action (sometimes referred to as “positive” in the present specification), and an action repelling from a repellent substance is referred to as a repelling action (sometimes referred to as “negative” in this specification). . In addition, attracting behavior and repelling behavior are collectively referred to as chemotaxis behavior.

  In the present specification, the “wild strain” is a wild strain of a nematode, and includes, for example, a general wild strain N2 Bristol strain. The wild strain in the present specification can be a strain that exhibits attracting behavior in urine obtained from a subject having cancer and exhibits repellent behavior in urine obtained from a subject not having cancer.

Method of analyzing urine using the nematode chemotaxis behavior evaluation system The method of analyzing urine using the nematode chemotaxis behavior evaluation system is to analyze a test sample (eg, urine) obtained from a subject and nematodes. It can be performed by locating a certain distance and observing whether the nematode exhibits an attracting action or a repelling action with respect to the test sample. If the attracting behavior is shown, the subject can be evaluated as having or possibly having cancer. In this way, WO2015 / 088039 was able to detect cancer patients with 100% sensitivity and 95% specificity by a medium-scale test of 242 specimens.

More specifically, a method for analyzing urine using a nematode chemotaxis behavior evaluation system is, for example,
Placing a test sample (eg, urine) obtained from a subject in a petri dish (eg, a petri dish into which a solid medium has been introduced);
Placing nematodes in a petri dish where the test sample is placed at a position away from the test sample;
After placement, make the nematode act,
If the nematode exhibits chemotaxis behavior on the test sample, assess that the subject is or may be affected by cancer,
Can be included.

｛式中、Ａは、被検試料に対して誘引行動を示した線虫の数であり、Ｂは、被検試料に対して忌避行動を示した線虫の数である。｝The chemotactic behavior of a nematode can be evaluated by the difference or ratio between the number of nematodes approaching the test sample and the number of nematodes far from the test sample. In the case of evaluation based on the difference in the number of nematodes, if the difference is a positive value, the test sample as a whole induces attracting behavior and is derived from a cancer patient. If it can be evaluated and is a negative value, the test sample as a whole induces repellent behavior and can be evaluated to be derived from a healthy person. Moreover, the chemotaxis behavior of a nematode can be evaluated using, for example, the chemotaxis index as an index as follows.

{In the formula, A is the number of nematodes that have attracted the test sample, and B is the number of nematodes that have repelled the test sample. }

  If the chemotaxis index is a positive value, the test sample as a whole induces attracting behavior and can be evaluated to be derived from a cancer patient and is a negative value In some cases, the test sample as a whole induces repellent behavior and can be evaluated to be derived from a healthy person.

  The chemotaxis index means that the ratio closer to 1 indicates that the nematode exhibits attracting behavior, the closer it is to -1, the greater the ratio that nematode exhibits repellent behavior, and the closer it is to 0, the nematode is closer Means no attracting or avoiding behavior. The larger the absolute value of the chemotaxis index, the clearer the action evaluation result.

  The inventors have discovered that cineole (eg, 1,8-cineole and / or 1,4-cineole) affects the nematode olfaction and increases the absolute value of the chemotaxis index.

  According to the present invention, there is provided a composition for use in enhancing the olfactory sensitivity of a nematode, comprising cineol (eg, 1,8-cineole and / or 1,4-cineole). Is done. According to the present invention, there is provided a method for enhancing the olfactory sensitivity of a nematode, comprising culturing the nematode in the presence of the cineole.

  According to the present invention, there is also provided a composition for use in enhancing attracting behavior in evaluation of chemotaxis behavior based on the olfactory sense of nematodes, such as cineole (for example, 1,8-cineole and / or 1,4-cineole). Is provided. According to the present invention, there is also provided a method for enhancing attraction behavior in evaluation of chemotaxis behavior based on the sense of smell of nematodes, comprising culturing nematodes in the presence of cineole.

  Cineol (eg, 1,8-cineole and / or 1,4-cineole) need not be in direct contact with the nematode. Cineol (eg, 1,8-cineole and / or 1,4-cineole) is volatile and can stimulate the nematode's olfactory nerve from a distance to increase the sensitivity of the nematode's olfaction. Accordingly, cineole (eg, 1,8-cineole and / or 1,4-cineole) is applied anywhere on the inner wall of the culture vessel where the nematode is propagated to increase the olfactory sensitivity of the nematode. be able to. For example, it can be attached to an inner wall such as a lid of a culture vessel. Cineol (for example, 1,8-cineole and / or 1,4-cineole) may be added to the medium.

  In the present invention, cineol (for example, 1,8-cineole and / or 1,4-cineole) diluted with a stock solution or water can be used. For example, the dilution factor can be used as 1 million times to 10,000 times. In the present invention, nematodes are present in the presence of cineole (eg, 1,8-cineole and / or 1,4-cineole) for any period of, for example, 2-4 days, 1-2 days, and 1-24 hours. Can be cultured. Cineol (eg, 1,8-cineole and / or 1,4-cineole) may be introduced into the evaluation system at the time of testing. For example, cineole (for example, 1,8-cineole and / or 1,4-cineole) is selected from the inner wall of a petri dish used for the chemotaxis behavior evaluation system, the inner wall of its lid, the surface of the solid medium, and the solid medium. Any one or more can be attached or contained.

  In the present invention, cineol (for example, 1,8-cineole and / or 1,4-cineole) can also be used for evaluating the chemotaxis behavior of urine of cancer patients.

  Hereinafter, the present invention will be described based on examples.

Example 1: Evaluation of chemotaxis behavior by nematodes smelled with cineole In this example, it is shown that cineole improves the olfactory sensitivity of nematodes.

A nematode wild strain was sniffed with 1,8-cineole as cineol, and then the chemotactic behavior of the nematode to benzaldehyde (BZ) or isoamyl alcohol (IAA) was evaluated.
Specifically, 1,8-cineole was diluted 100,000 times and adhered to the inside of the lid of the culture vessel. After attachment, the nematode eggs were placed on the nematode culture medium and grown until they became adults. Nematodes were collected and chemotaxis to BZ or IAA was observed according to a conventional method.

The plates used in the examples are shown in FIG. In FIG. 1, the odorant was applied to the sample placement site 5 of the plate, the nematode was placed on the nematode placement site 3 of the plate, and then the behavior of the nematode was observed at 23 ° C. Thirty minutes after the nematode was placed, the number of nematodes present in region 2 and region 4 was counted, and the chemotaxis index was determined by the following equation.
(Taxis) = {N (2) −N (4)} / {N (2) + N (4)}
{Where N (2) is the number of nematodes distributed in region 2 in FIG. 1, and N (4) is the number of nematodes distributed in region 4 in FIG. }

  As the chemotaxis index is closer to 1, it means that the attracting behavior of the nematode is strongly expressed. The result was as shown in FIG. 2A.

  As shown in FIG. 2A, nematodes cultured in the presence of 1,8-cineole have a dramatic increase in the chemotaxis index from about 0.2 to about 0.75 for BZ, For IAA, it increased dramatically from about 0.3 to about 0.75. Olfactory sensitivity is improved for both different types of odorants, and 1,8-cineole is responsible for increasing the sensitivity of nematode olfactory and the chemotaxis behavior of nematode (attractive behavior). It has been shown to improve sensitivity.

Next, the same experiment was conducted using 1,4-cineole as cineole. Specifically, 1,4-cineole was diluted 100,000 times and adhered to the inside of the lid of the culture vessel. After attachment, the nematode eggs were placed on the nematode culture medium and grown until they became adults. Nematodes were collected and chemotaxis to IAA and 2-nonanone was observed according to a conventional method. The experiment was performed twice.
As a result, as shown in FIG. 2B, nematodes cultured in the presence of 1,4-cineole improved the chemotaxis index against IAA as an attractant, whereas it was a repellent substance. There was no change in the chemotaxis index for a certain 2-nonanone. From this, it was clarified that 1,4-cineole, like 1,8-cineole, raises the sensitivity of nematode olfactory and improves the sensitivity of chemotaxis behavior (attraction behavior). .

  In addition, bornyl acetate, D-camphor, camphene, linalool, linalyl acetate, graniol, phenylethyl alcohol, citronellol, pinene, limonene, benzyl acetate, vanillin, eugenol, and myrcene are similarly given to the behavior of nematodes. Although the effects were examined, no significant effects were observed for these.

Example 2: Analysis using odr-3 mutant In order to confirm that the responsiveness to the odorant observed in the above example is based on the olfactory sense, there is an abnormality in the AWC nerve, which is the olfactory nerve involved in the attraction behavior The effect of cineole on the chemotaxis behavior of the -3 mutant was confirmed.

  The nematode learns the salt concentration (NaCl) of the place where the food exists, and shows chemotaxis to the salt concentration part. Nematodes were allowed to learn the salt concentration (concentration 50 mM) in the presence or absence of 1,8-cineole or propagated without learning.

  Thereafter, salt chemotaxis against salt concentration (100 mM) was evaluated based on a conventional method. The results were as shown in FIGS. 3A and 3B.

  As shown in FIG. 3A, salt chemotaxis was found to be greatly reduced by cineole. On the other hand, as shown in FIG. 3B, the chemotaxis index related to salt chemotaxis did not change in the odr-3 mutant. Therefore, it became clear that cineole is a modulator that changes the chemotaxis behavior of nematodes based on the sense of smell.

Claims (6)

  A composition for use in enhancing the olfactory sensitivity of a nematode, comprising cineole.   A composition for use in enhancing chemotaxis behavior based on the sense of smell of nematodes, comprising cineole.   The composition according to claim 2, wherein the chemotaxis behavior is idle movement and / or repelling behavior. A method of increasing the olfactory sensitivity of a nematode,
Contacting a nematode with cineole or a composition comprising cineole. A method for enhancing chemotaxis behavior based on the sense of smell of nematodes,
Contacting a nematode with cineole or a composition comprising cineole.   The method according to claim 5, wherein the chemotaxis behavior is idle movement and / or repelling behavior.

Images