JP6788921B2 - Compounds that improve the olfactory sensitivity of nematodes - Google Patents

Description

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

C. elegans behaves as an attractant to the urine of cancer patients and as a repellent to the urine of healthy individuals. Therefore, it is possible to determine whether the target from which urine is derived is a cancer patient or a healthy person based on the tactical behavior of the nematode (Patent Document 1). More specifically, Patent Document 1 describes that a medium-scale test of 242 samples was able to detect a cancer patient with 100% sensitivity and 95% specificity.

WO2015 / 088039

The present inventors have sought a method for further enhancing taxis in the evaluation of taxis. As a result, we have shown that cineole enhances the olfactory sensitivity of C. elegans and enhances the tactical behavior of C. elegans. The present invention is an invention based on such findings.

That is, according to the present invention, the following invention is provided.
(1) A composition containing cineole for use in enhancing the olfactory sensitivity of nematodes.
(2) A composition containing cineole for use in enhancing olfactory-based tactical behavior of nematodes.
(3) The composition according to (2) above, wherein the tactical behavior is migratory movement and / or repellent behavior.
(4) A method for enhancing the olfactory sensitivity of nematodes.
A method comprising contacting a nematode with cineole, or a composition comprising cineole.
(5) A method of enhancing tactical behavior based on the sense of smell of nematodes.
A method comprising contacting a nematode with cineole, or a composition comprising cineole.
(6) The method according to (5) above, wherein the tactical behavior is migratory movement and / or repellent behavior.

FIG. 1 shows a plan view of a petri dish 1 used for evaluating the tactical behavior of nematodes. FIG. 2A shows the olfactory sensitivity of the nematode and the enhancing effect of the nematode's tactical behavior by 1,8-cineole. FIG. 2B shows the olfactory sensitivity of C. elegans and the enhancing effect of C. cineole on the tactical behavior of C. elegans. FIG. 3A shows that the decrease in nematode attraction behavior (or enhancement of repellent behavior) due to salt learning is enhanced by 1,8-cineole. FIG. 3B shows that the enhancing effect of 1,8-cineole on the reduction of nematode attraction behavior (or enhancement of repellent behavior) by salt learning disappears in the odr-3 mutant.

Specific description of the invention

As used herein, the term "subject" means a mammal, such as a primate (eg, a monkey, orangutan, gorilla, bonobo, orangutan, marmoset, and human), such as a human. As used herein, the subject is preferably a human. Further, in the present specification, the term "subject" is used to mean a healthy person, a subject suspected of having cancer, and a subject suffering from cancer.

As used herein, "cancer" means a malignant tumor. Cancers can be broadly classified into hematopoietic tumors, epithelial carcinomas (calcinoma), and non-epithelial sarcomas (sarcoma). 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 osteosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcomas, fibrosarcomas, liposarcomas, and angiosarcomas. As used herein, "cancer" includes early stage cancers such as stage 0 early stage cancers and stage 1 early stage cancers.

In the present specification, the "composition for use in cancer diagnosis" may be simply referred to as a "cancer diagnostic agent".

As used herein, the term "nematode" means Caenorhabditis elegans . C. elegans can be distributed by registering and publishing strains isolated from various environments at the Caenorhabditis Genetics Center (CGC) of the Faculty of Biological Sciences, University of Minnesota, USA. Therefore, most known strains can be obtained from CGC by those skilled in the art. Hermaphrodite can be preferably used from the viewpoint of being able to reproduce by self-fertilization.

As used herein, the term "tactical behavior" means an attracting behavior or a repelling behavior. The attracting behavior means the behavior of shortening the physical distance from a certain substance, and the repellent behavior means the behavior of increasing the physical distance from a certain substance. A substance that induces attracting behavior is called an attracting substance, and a substance that induces repellent behavior is called a repellent substance. C. elegans has the property of attracting attractants by the sense of smell and repelling them from repellent substances. The behavior of attracting an attractant is called an attracting behavior (sometimes referred to as "positive" in the present specification), and the behavior of repelling an attractant is referred to as a repellent behavior (sometimes referred to as "negative" in the present specification). .. In addition, the attracting behavior and the repelling behavior are collectively called the tactical behavior.

As used herein, the "wild strain" is a wild strain of C. elegans, and examples thereof include the N2 Bristol strain, which is a general wild strain. The wild-type strain in the present specification can be a strain that exhibits an attractive behavior in urine obtained from a subject having cancer and a repellent behavior in urine obtained from a subject not having cancer.

Analysis method of urine using the taxitactic behavior evaluation system of nematodes The analysis method of urine using the taxitactic behavior evaluation system of nematodes is to analyze a test sample (for example, urine) obtained from a subject and a nematode. This can be done by arranging them at a certain distance and observing whether the nematodes show attractive behavior or repellent behavior with respect to the test sample. Then, if the subject exhibits an attractive behavior, it can be evaluated that the subject has or may have cancer. Thus, in WO2015 / 088039, a medium-scale study of 242 samples was able to detect cancer patients with 100% sensitivity and 95% specificity.

More specifically, a method for analyzing urine using a nematode taxis behavior evaluation system is, for example,
Placing a test sample (eg, urine) obtained from a subject on a petri dish (eg, a petri dish introduced with a solid medium)
Placing a nematode at a certain distance from the test sample in the petri dish where the test sample is placed,
After placement, let the nematodes act and
If the nematode shows tactical behavior with respect to the test sample, it should be evaluated that the subject has or may have 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 tactical behavior of nematodes 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 attractive behavior and is derived from the cancer patient. If it can be evaluated and the value is negative, it can be evaluated that the test sample as a whole induces repellent behavior and is derived from a healthy person. In addition, the tactical behavior of nematodes can be evaluated using the tactical index as an index, for example, as follows.

{In the formula, A is the number of nematodes that showed an attracting behavior to the test sample, and B is the number of nematodes that showed a repellent behavior to the test sample. }

When the taximetric index is a positive value, the test sample as a whole induces attractive behavior and can be evaluated to be derived from a cancer patient, which 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 subject.

The taxitactic index means that the closer it is to 1, the greater the proportion of nematodes exhibiting attractive behavior, the closer it is to -1, the greater the proportion of nematodes exhibiting repellent behavior, and the closer it is to 0, the greater the proportion of nematodes. Means that it shows neither attracting nor repellent behavior. The larger the absolute value of the taxis index, the clearer the behavioral evaluation result.

We have found that cineole (eg, 1,8-cineole and / or 1,4-cineole) affects the olfaction of C. elegans and increases the absolute value of the taxis index.

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

According to the present invention, it is also a composition for use in enhancing the attracting behavior in the olfactory-based taxitactic behavior evaluation of C. elegans, and 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 the attracting behavior in the olfactory-based taxitactic behavior evaluation of the nematode, which comprises culturing the nematode in the presence of the cineole.

Cineol (eg, 1,8-cineole and / or 1,4-cineole) does not need to be in direct contact with the nematode. Cineol (eg, 1,8-cineole and / or 1,4-cineole) is volatile and can stimulate the nematode olfactory nerves from a distance to increase the nematode olfactory sensitivity. Therefore, cineole (eg, 1,8-cineole and / or 1,4-cineole) is applied somewhere on the inner wall of the culture vessel, where the nematodes are propagated to increase the olfactory sensitivity of the nematodes. be able to. For example, it can be attached to an inner wall such as a lid of a culture container. Also, cineole (eg, 1,8-cineole and / or 1,4-cineole) may be added to the medium.

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

In the present invention, cineole (eg, 1,8-cineole and / or 1,4-cineole) can also be used to evaluate the urinary tactical behavior of cancer patients.

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

Example 1: Evaluation of Taxis by Nematodes Smelling Cineol In this example, it is shown that cineole improves the olfactory sensitivity of nematodes.

Wild strains of C. elegans were allowed to sniff 1,8-cineole as cineole, after which the nematode taxis to benzaldehyde (BZ) or isoamyl alcohol (IAA) was evaluated.
Specifically, 1,8-cineole was diluted 100,000 times and attached to the inside of the lid of the culture vessel. After attachment, nematode eggs were placed on the nematode culture medium and grown until they became adults. C. elegans were collected and their taxis to BZ or IAA was observed according to a conventional method.

The plate used in the examples is shown in FIG. The odorant was applied to the sample placement site 5 of the plate in FIG. 1, 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. The number of nematodes present in regions 2 and 4 was counted 30 minutes after the nematodes were placed, and the taxitactic index was calculated by the following formula.
(Taxis index) = {N (2) -N (4)} / {N (2) + N (4)}
{In the equation, N (2) is the number of nematodes distributed in region 2 of FIG. 1, and N (4) is the number of nematodes distributed in region 4 of FIG. }

The taxis index means that the closer to 1 the more the nematode attracting behavior was expressed. The results were as shown in FIG. 2A.

As shown in FIG. 2A, in the nematodes cultured in the presence of 1,8-cineole, the taxitactic index increased dramatically from about 0.2 to about 0.75 with respect to BZ. For IAA, it increased dramatically from about 0.3 to about 0.75. The olfactory sensitivity to both completely different types of odorants has been improved, and 1,8-cineole causes the olfactory sensitivity of nematodes to become more sensitive, and the nematode tactical behavior (attracting behavior). It was found to improve susceptibility.

Next, a similar experiment was performed using 1,4-cineole as cineole. Specifically, 1,4-cineole was diluted 100,000 times and attached to the inside of the lid of the culture vessel. After attachment, nematode eggs were placed on the nematode culture medium and grown until they became adults. C. elegans were collected and their taxis 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, the nematodes cultured in the presence of 1,4-cineole improved the taxis index against the attractant IAA, whereas the nematodes were repellent substances. It did not change the taxitactic index for a 2-nonanone. From this, it was clarified that 1,4-cineole, like 1,8-cineole, causes olfactory sensitivity of nematodes and improves the sensitivity of taxis (attracting behavior). ..

Bornyl acetate, D-camphor, camphene, linalool, linalyl acetate, graniol, phenylethyl alcohol, citronellol, pinene, limonene, benzyl acetate, vanillin, eugenol, and myrcene are all given to the nematode running behavior in the same manner. The effects were investigated, but no significant effect was observed for these.

Example 2: Analysis using the odr-3 mutant Odr has an abnormality in the AWC nerve, which is an olfactory nerve involved in attracting behavior, in order to confirm that the responsiveness to the odorant observed in the above example is based on the sense of smell. -3 The effect of cineol on the tactical behavior of the mutant was confirmed.

C. elegans learns the salinity (NaCl) of the place where food is present and shows taxis to the place of the salt concentration. C. elegans were cultivated with or without salt concentration (concentration 50 mM) in the presence or absence of 1,8-cineole.

After that, the salt taxis with respect to the 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, cineole revealed that cineole significantly reduced the salt-taxis index. On the other hand, as shown in FIG. 3B, the taxitactic index for salt taxis was unchanged in the odr-3 mutant. Therefore, it was revealed that cineole is a modulator that changes the tactical behavior of nematodes based on the sense of smell.

Claims (6)

A composition for use in enhancing the olfactory sensitivities of nematodes, including cineole. A composition for use in enhancing olfactory-based tactical behavior of nematodes, including cineole. The composition according to claim 2, wherein the taxis behavior is migratory movement and / or repellent behavior. A method of increasing the olfactory sensitivity of nematodes
A method comprising contacting a nematode with cineole or a composition comprising cineole. A method of enhancing taxis based on the sense of smell of nematodes
A method comprising contacting a nematode with cineole or a composition comprising cineole. The method of claim 5, wherein the tactical behavior is migratory movement and / or repellent behavior.

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