JP7704407B2 - Cancer treatment composition - Google Patents

Description

The present invention relates to a composition that is effective in treating cancer.

Plasmalogen is known to promote neurogenesis, inhibit neuroinflammation caused by lipopolysaccharide (LPS), and inhibit the accumulation of amyloid beta (Aβ) protein in the brain, and is said to be effective in treating neurological diseases such as Alzheimer's disease, Parkinson's disease, depression, and schizophrenia. For example, Non-Patent Document 1 reports that memory function in mild Alzheimer's disease improved in patients who were orally administered purified plasmalogen derived from scallops.

On the other hand, the number of cancer patients in Japan is increasing year by year, and it is said that one in two Japanese people will suffer from some kind of cancer in their lifetime. In addition, there is data that one in three Japanese people will die from cancer.
Although advances in medical technology have increased the survival rate of cancer patients, there is a demand for more effective treatment methods.

Fujino T. et al, “Efficacy and Blood Plasmalogen Changes by Oral Administration of Plasmalogen in Patients with Mild Alzheimer's Disease and Mild Cognitive Impairment: A Multicenter, Randomized, Double-blind, Placebo-controlled Trial” EBioMedicine, [17] (2017) 199-205

As mentioned above, various reports have been published regarding plasmalogens, but the effects of plasmalogens on cancer have not been examined in detail.

The objective of the present invention is to provide a composition that is effective in treating cancer.

As a result of intensive research aimed at solving the above problems, the inventors discovered that plasmalogen can induce apoptosis in cancer cells and inhibit their proliferation, which led to the completion of the present invention.

That is, the present invention is as follows.
[1] A composition for inducing apoptosis in cancer cells, characterized by containing a plasmalogen.
[2] A composition for activating macrophages, comprising a plasmalogen.
[3] A composition for activating GPR21, characterized by containing a plasmalogen.
[4] A composition for destroying extracellular matrix in cancer tissue, characterized by containing a plasmalogen.
[5] A composition for inhibiting cancer cell proliferation, comprising a plasmalogen.
[6] A composition for cancer treatment comprising a plasmalogen.

[7] The composition for inducing apoptosis in cancer cells described in [1] above, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue.
[8] The composition for activating macrophages described in [2], characterized in that the plasmalogen is a plasmalogen extracted from animal tissue.
[9] The composition for activating GPR21 according to [3], characterized in that the plasmalogen is a plasmalogen extracted from animal tissue.
[10] The composition for destroying extracellular matrix in cancer tissue according to [4], characterized in that the plasmalogen is a plasmalogen extracted from animal tissue.
[11] The composition for inhibiting cancer cell proliferation described in [5] above, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue.
[12] The composition for cancer treatment described in [6] above, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue.

The composition of the present invention can induce apoptosis in cancer cells and inhibit the proliferation of cancer cells, making it effective in treating cancer.

1 shows photographs showing the size of tumors excised from mice. FIG. 1 is a graph comparing the weights of tumors excised from mice. 1 shows photographs showing induced apoptosis in mouse tumor tissues. FIG. 1 is a diagram comparing the number of apoptotic cells induced in mouse tumor tissues. 13 is a photograph showing the extracellular matrix of mouse tumor tissue stained with hematoxylin and eosin. FIG. 1 compares the relative number of disrupted extracellular matrix in mouse tumor tissues. 1 shows photographs showing the expression of the GPR21 gene in mouse tumor tissues. FIG. 1 is a diagram comparing the relative numbers of GPR21 gene-positive cells in mouse tumor tissues. FIG. 1 shows the results of detection of CD16-positive NK cells and GPR21 gene expression in mouse tumor tissues. FIG. 1 shows a comparison of the relative numbers of CD16-positive NK cells. FIG. 1 shows the results of detection of F4/80-positive activated macrophages and GPR21 gene expression. FIG. 11 is a diagram comparing the relative numbers of F4/80 positive cells.

The composition of the present invention is characterized by containing a plasmalogen.
The composition of the present invention can suppress the proliferation of cancer cells by inducing apoptosis of cancer cells and activating NK cells and macrophages. This can suppress the growth of cancer tissue (malignant tumor tissue) and reduce its size. The composition of the present invention can also activate G protein-coupled receptor 21 (GPR21), and this activation is thought to contribute to the suppression of the proliferation of cancer cells. Furthermore, the composition of the present invention can destroy the extracellular matrix found in invasive cancers and the like.

Therefore, the composition of the present invention can be used as a composition for inducing apoptosis in cancer cells, a composition for activating macrophages, a composition for activating GPR21, a composition for destroying the extracellular matrix in cancer tissues, a composition for inhibiting cancer cell proliferation, a composition for inhibiting cancer metastasis, a composition for treating cancer, etc. Specifically, for example, it can be used to alleviate symptoms and treat cancer patients.

Plasmalogen is a type of phospholipid with antioxidant properties, a type of glycerophospholipid. It is a unique subclass of glycerophospholipids characterized by having a vinyl ether bond at the sn-1 position of the glycerol backbone, and has been found in high concentrations in the cell membranes of many mammalian tissues.

The plasmalogen used in the present invention is not particularly limited as long as it is generally classified as a plasmalogen, but examples include choline-type plasmalogens, ethanolamine-type plasmalogens, inositol-type plasmalogens, and serine-type plasmalogens. Among these, choline-type plasmalogens and ethanolamine-type plasmalogens are preferred, and ethanolamine-type plasmalogens are particularly preferred.

The plasmalogen of the present invention can be extracted from animal tissue. There are no particular limitations on the animal tissue as long as it contains plasmalogen, and examples include aquatic animals such as shellfish, sea squirts, sea cucumbers, salmon, pacific saury, and bonito, and birds. Among these, shellfish, sea squirts, and birds are preferred, and shellfish are particularly preferred. Edible parts (edible parts) are preferred as the parts to be used. These animal tissues may be cut, but it is preferable to use pulverized parts, as this allows for more efficient extraction of plasmalogen.

Examples of shellfish include edible bivalve mollusks and snail shells such as scallops, mussels, and abalone, with scallops being particularly preferred. Scallops are edible bivalve mollusks belonging to the Pectenidae family, and examples of such mollusks include those belonging to the Mizuhopecten and Pecten genera. Specific examples include scallops (scientific name: Mizuhopecten yessoensis) harvested in Japan and European scallops (scientific name: Pecten maximus (Linnaeus)) harvested in Europe. Edible parts include the adductor muscle, string, etc.

Sea squirts are edible chordates belonging to the family Halocynthia, including the genera Halocynthia and Halocynthia. Specific examples include the Halocynthia ascidian (scientific name: Halocynthia roretzi) and the Halocynthia ascidian (scientific name: Halocynthia aurantium). The edible part is the flesh (fascia).

The bird is not particularly limited as long as it is an edible bird, and examples include chickens, silkie chickens, and ducks. As for the edible part, breast meat, which is rich in plasmalogens, is preferable.

Extraction of plasmalogen can be performed using water, an organic solvent, or a water-containing organic solvent, and it is preferable to use an enzyme treatment in combination. For example, ethanol extraction method and hexane extraction method can be mentioned, and ethanol extraction method is preferable.

The ethanol extraction method is not particularly limited as long as it is a method of extraction using ethanol (including hydrous ethanol), and examples of the method include those described in JP 2019-140919 A, JP 2018-130130 A, JP 2012-039472 A, JP 2010-065167 A, and JP 2010-063406 A.

The hexane extraction method is not particularly limited as long as it is a method of extraction using hexane, and examples of the method include those described in REP 2009-154309 and REP 2008-146942.

The composition of the present invention can be used as a medicine. It can also be used as a food (including so-called health foods such as foods for specified health uses, foods with nutritional functions, and foods with functional claims) that assist in cancer treatment.

The composition of the present invention may be administered orally or parenterally, such as by injection or infusion, but is preferably administered orally because it can be taken easily. When administered orally, the composition may be in the form of, for example, a tablet, capsule, powder, granule, liquid, grain, rod, plate, block, solid, round, paste, cream, caplet, gel, chewable, or stick. Of these, the capsule form is preferred.

The content of plasmalogen in the composition of the present invention may be appropriately determined within the range in which the effect is exhibited. Depending on the form, for example, the plasmalogen is preferably ^10-10 % by mass or more, more preferably 10-5% by mass or more, even more preferably ^0.1 % by mass or more, and particularly preferably 1.0% by mass or more, of the total composition of the present invention, calculated on a dry mass basis.

There is no particular limit to the amount of intake of the oral composition of the present invention, but from the viewpoint of more significantly exerting the effects of the present invention, the amount of plasmalogen intake per adult is preferably 10 ⁻⁶ μg/day or more, more preferably 1 μg/day or more, even more preferably 500 μg/day or more, and particularly preferably 1000 μg/day or more. The upper limit is, for example, 20,000 μg/day, preferably 10,000 μg/day.

The oral composition of the present invention can be stored as a daily dose in a single container or divided into, for example, 2 to 3 multiple containers so that the daily intake amount is the above-mentioned amount.

The composition of the present invention can be produced by a known method by adding other ingredients than the ingredients of the present invention, if necessary. Examples of other ingredients than the ingredients of the present invention include vitamins, minerals, proteins, peptides, amino acids, animal oils, and vegetable oils.

The present invention will now be described in detail with reference to examples.

We investigated the tumor-suppressing effect of plasmalogen administration in mice.

[Plasmalogen]
The plasmalogen (sPls) used was hexane-extracted ethanolamine-type plasmalogen (mainly ethanolamine-type plasmalogen, but also choline-type plasmalogen) prepared from scallops (scientific name: Mizuhopecten yessoensis) by the following method.

1. Add Cocurase P (manufactured by Mitsubishi Chemical Foods Corporation) and phospholipase A1 (PLA1) (manufactured by Mitsubishi Chemical Foods Corporation) to raw scallops and mix.
2. Next, add hexane/isopropanol and suction filter the supernatant.
3. Add the aqueous sodium sulfate solution and mix well.
4. Dry the upper layer using a rotary evaporator.
5. Add acetone cooled to 4°C and mix.
6. Centrifuge at 3000 rpm for 10 minutes at 4°C.
7. Discard the supernatant and collect the precipitate.
8. Dry in a desiccator overnight.

[In vivo xenograft tumor transplantation experiments]
An in vivo xenogeneic tumor transplantation experiment was carried out using severe combined immunodeficiency (SCID) mice, which lack functional T cells and B cells in the peripheral blood and exhibit severe combined immunodeficiency.

8-week-old SCID mice were orally administered sPls suspended in ultrapure water at a dose of 0.02 mg/kg/day (sPls equivalent) for 6 weeks. Ultrapure water was orally administered to the control mice for 6 weeks. Both the sPls-administered group and the control group consisted of 5 mice per group, and the mice were kept in a specific pathogen free (SPF) environment throughout the entire experimental period.

[Cancer cell transplantation]
100 μL of a cell suspension of 5×10 ⁶ cells/mL of cancer cells (human neuroblastoma SH-SY5Y cells) cultured in 10% FBS-containing DEME medium was mixed with Matrigel (basement membrane substrate, collagen type 1) and subcutaneously implanted into the back of each mouse that had been orally administered the above sPls or ultrapure water for 6 weeks. Four weeks after implantation of the SH-SY5Y cells, the tumor was excised from each mouse and its weight and size were measured.

Figure 1A shows the size of the tumors excised from each mouse in the control group and the sPls-treated group. Figure 1B shows the average weight (mg) of the excised tumors and the results of a comparison test using Student's t-test.

As shown in Figure 1A, the sPls-administered group showed a significant reduction in tumor size compared to the control group. Also, as shown in Figure 1B, the sPls-administered group showed a statistically significant reduction in tumor weight compared to the control group. Therefore, it was demonstrated that tumor cell proliferation was suppressed by administering plasmalogen.

Tissue specimens were prepared from the tumors of each mouse excised in Example 1, and the induction of apoptosis in tumor cells by plasmalogen was examined. Specifically, the following procedures were performed.

1. Tumor tissues excised from each mouse in the sPls-administered group and the control group were washed with phosphate buffered saline (PBS).
2. Fix with 4% paraformaldehyde.
3. The fixed tumor tissue is embedded in a embedding medium for frozen sectioning and frozen at -80°C.
4. Prepare tissue specimens 10 μm thick using a Cryostat Microm HM550.
5. Using an In Situ Cell Death Detection Kit by the TUNEL method (Roche Diagnostics), DNA fragmented by apoptosis in a tissue specimen is labeled with biotin-labeled nucleotides, and then stained by reacting with TUNEL and HRP-labeled streptavidin.
6. Cell nuclei are stained with DAPI.

Figure 2A shows the localization of apoptotic cells in tumor tissues of mice in the control and sPls-administered groups. Figure 2B shows the results of a comparison test using Student's t-test for the number of TUNEL-positive cells in the control and sPls-administered groups.

As shown in Figure 2A, more TUNEL-positive cells were observed in the tumor tissues of the sPls-treated group than in the control group, and as shown in Figure 2B, the number of TUNEL-positive cells was statistically significantly increased in the sPls-treated group compared to the control group.
Therefore, it was suggested that plasmalogen induces apoptosis in cancer cells and has the effect of reducing tumor size.

Cancer cells induce the promotion of type I collagen production from fibroblasts in the tumor stroma, the cross-linking of collagen fibers, and an increase in cellular fibronectin, which hardens the extracellular matrix surrounding the cancer cells. Meanwhile, the hardened extracellular matrix acts on the cancer cells as mechanical stress, and is involved in the enhancement of cancer proliferation, invasion, and metastasis.
Thus, since the interaction between cancer cells and the extracellular matrix is deeply related to the progression of tumors, the changes in the extracellular matrix caused by plasmalogen were examined for the tumor tissues of mice in the sPls administration group and the control group. Specifically, the following procedures were performed.

1. Stain the cell nuclei of the tumor tissue specimen with hematoxylin.
2. After washing with water, stain the cytoplasm with eosin.
3. Dehydrate using an ethanol series and clear using xylene.
4. Add a drop of mounting medium and mount with a cover glass to prepare a hematoxylin-eosin stained specimen.
5. Perform histological observations on the hematoxylin and eosin stained specimens using an optical microscope.

Figure 3A shows the extracellular matrix in tumor tissue stained with hematoxylin and eosin for mice in the control group and the sPls-treated group. Figure 3B shows the results of a comparative test using Student's t-test, in which the amount of disrupted extracellular matrix was quantified from tumor sites of the same area.

As shown in Figure 3A, the extracellular matrix commonly seen in invasive tumors was clearly observed in the tumor tissues of the control mice, whereas the rupture of the extracellular matrix observed during the regression phase of tumors was confirmed in the tumor tissues of the sPls-treated mice. In addition, as shown in Figure 3B, the relative number of ruptured extracellular matrices was confirmed to have increased statistically significantly.
Therefore, it was suggested that plasmalogen may be effective in preventing cancer metastasis and treating cancer.

The expression of G protein-coupled receptor 21 (GPR21), a plasmalogen receptor, was examined in the tumor tissues of mice in the sPls-administered and control groups. Specifically, the following procedures were performed.

GPR21-positive cells in mouse tumor tissues were detected by immunohistochemistry using a GPR21 antibody (Invitrogen) as the primary antibody and FITC-conjugated anti-rabbit IgG as the secondary antibody. GPR21-positive cells were observed under a fluorescence microscope (Axopskope 2, Zeiss).

Figure 4A shows the localization of GPR21-positive cells in tumor tissues of mice in the control group and the sPls-administered group. Figure 4B shows the results of a comparison test using Student's t-test of the relative numbers of GPR21-positive cells in the control group and the sPls-administered group.

As shown in Figure 4B, it was confirmed that the expression level of GPR21 in tumor tissue was statistically significantly increased in the sPls-administered group compared to the control group.

Peripheral blood lymphocytes are composed of three cell populations: T cells, B cells, and natural killer cells (NK cells), and play a central role in immune function. Expression of the CD16 antigen is associated with the cytotoxic activity of NK cells, and CD16-positive NK cells exhibit strong cytotoxic activity and play an essential role in the destruction of malignant tumor cells. We examined the increase in CD16-positive NK cells in tumor tissues caused by plasmalogen in the tumor tissues of mice in the sPls-administered and control groups. Specifically, the following procedures were performed.

CD16-positive NK cells were detected in mouse tumor tissues by immunohistochemistry using rabbit GPR21 antibody (Invitrogen) and rat CD16 antibody (BD Pharmingen). CD16-positive NK cells were observed under a fluorescent microscope (Axioskope 2, Zeiss).

Figure 5A shows the results of immunohistochemical staining of CD16-positive activated NK cells, GPR21-positive cells, and a combination of both in tumor tissues from the control group and the sPls-administered group. Figure 5B shows the results of a comparative test using Student's t-test for the relative numbers of CD16-positive cells in the control group and the sPls-administered group.

As shown in Figure 5A, an increase in CD16-positive NK cells was confirmed in the tumor tissue of the sPls-administered group. In addition, it was confirmed that the sPls receptor GPR21 was highly expressed in NK cells.
As shown in FIG. 5B, it was confirmed that the sPls administration group had a statistically significant increase in CD16-positive NK cells in the tumor tissue compared to the control group.

The increase in F4/80-positive activated macrophages in tumor tissues of mice in the sPls-administered and control groups was examined by plasmalogen. Specifically, the following procedures were performed.

Anti-F4/80 antibody (abcam, ab16911), a major marker of mature mouse macrophages, was used to detect F4/80-positive activated macrophages in mouse tumor tissues by immunohistochemistry. F4/80-positive activated macrophages were observed under a fluorescent microscope (Axioskope 2, Zeiss).

Figure 6A shows the results of immunohistochemical staining of F4/80-positive activated macrophages, GPR21-positive cells, and a combination of both in the tumor tissues of mice in the control and sPls-administered groups. Figure 6B shows the results of a comparative test using Student's t-test of the relative numbers of F4/80-positive cells in the control and sPls-administered groups.

As shown in Figure 6A, in the tumor tissue of the sPls-administered group, F4/80-positive activated macrophages were significantly increased and were found to accumulate in tumor cells. Furthermore, co-staining of F4/80-positive activated macrophages and GPR21-positive cells suggested that GPR21 was highly expressed in activated macrophages. Furthermore, as shown in Figure 6B, quantification of F4/80-positive activated macrophages showed that the number of activated macrophages was statistically significantly increased in the sPls-administered group compared to the control group.

[Formulation example]
Hard capsules were produced according to the following formulation.
Scallop extract plasmalogen 0.5mg
Cyclodextrin 3.3mg
Amino acids 1.2mg
Pine index 185.0 mg

The composition of the present invention is effective in treating cancer and is therefore industrially useful.

Claims (12)

A composition for inducing apoptosis in cancer cells, characterized by containing plasmalogen. A composition for activating macrophages, characterized by containing plasmalogen. A composition for activating GPR21, characterized by containing plasmalogen. A composition for destroying the extracellular matrix in cancer tissue, characterized by containing plasmalogen. A composition for inhibiting cancer cell proliferation, characterized by containing plasmalogen. A composition for cancer treatment that contains plasmalogen. The composition for inducing apoptosis in cancer cells according to claim 1, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue. The composition for activating macrophages according to claim 2, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue. The composition for activating GPR21 according to claim 3, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue. The composition for destroying the extracellular matrix in cancer tissue according to claim 4, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue. The composition for inhibiting cancer cell proliferation according to claim 5, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue. 7. The composition for cancer treatment according to claim 6, wherein the plasmalogen is extracted from animal tissue.