Method for controlling implantation rate in mammal, agent for increasing implantation rate in mammal, and medicine

Abstract

A method for controlling implantation rate in a mammal, said method comprising using at least one selected from the group consisting of: (I) a compound represented by structural formula (1); (II) a peptide comprising the amino acid sequence represented by SEQ ID NO: 1; and (III) a peptide comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 1 by substitution or deletion of one to several amino acids or insertion or addition of one to several amino acids.

Description

Method for regulating implantation rate in mammals, agent for improving implantation rate in mammals, and medicine

[0001] The present invention relates to a method for regulating implantation rate in mammals, an agent for improving implantation rate in mammals, and a medicine for improving implantation rate in mammals.

[0002] The fertilized egg (zygote or embryo) in the fallopian tube progresses through the fallopian tube while differentiating and reaches the uterus. The process in which the embryo (blastula stage) adheres to the endometrial epithelium, invades the endometrium, and is encapsulated is called implantation. Although it has been suggested that complex and ingenious interactions between the embryo and the endometrium are important for implantation to occur, many aspects remain unknown.

[0003] Due to the recent social penetration of assisted reproductive technology (ART), the number of ART babies in Japan reached 56,617 per year, meaning that approximately 1 in 16.7 of all babies born that year were born as a result of advanced infertility treatment (FY2017).

[0004] However, the pregnancy success rate with ART is only 15-30%, leaving room for improvement. Many unsuccessful cases are thought to be due to implantation disorders, and there is a strong need to elucidate the molecular basis of the implantation process and identify substances that regulate the implantation rate.

[0005] A method using oligosaccharides has been proposed as a method for controlling the implantation of mammalian embryos, including humans, into the uterus (see Patent Document 1). However, this proposal only specifically demonstrates that the use of oligosaccharides inhibits implantation, but does not demonstrate any improvement in the implantation rate.

[0006] Furthermore, a method using galectin as a component for increasing the implantation rate has been proposed (see Patent Document 2). However, in this proposal, the effective concentration of galectin is as high as 160 μM, and a component that can improve the implantation rate at a lower dose is desired.

[0007] In addition, a substance called dicalcin, a factor that regulates fertilization efficiency, has been identified in Xenopus eggs (see Non-Patent Document 1). Dicalcin is known to bind to glycoproteins that make up the egg protective membrane (the extracellular matrix surrounding the egg) and control the orientation of extracellular matrix filaments throughout the egg protective membrane. Furthermore, molecular phylogenetic analysis of dicalcin in mammals revealed that S100A11 is a homologous protein (see Non-Patent Document 2). Dicalcin is a member of the S100 protein family, a low-molecular-weight calcium-binding protein, and is known to exist in humans, mice, pigs, etc. Dicalcin or its partial peptides has also been reported to effectively inhibit the metastasis of human cancer cells (see Patent Document 3).

[0008] Japanese Patent Application Laid-Open No. 64-045313 Japanese Patent Application Laid-Open No. 2019-23208 International Publication No. 2019 / 102710

[0009] Miwa et al. , J. Biol. Chem. , 285, 15627-15636 (2010) Hanaue et al. , Mol. Reprod. Dev. , 78, 91-103 (2011)

[0010] The present invention aims to solve the above-mentioned conventional problems and achieve the following objectives: Namely, the present invention aims to provide a new method for regulating the implantation rate in mammals, a new agent for improving the implantation rate in mammals, and a medicine containing the agent for improving the implantation rate.

[0011] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they discovered that a peptide consisting of the amino acid sequence of SEQ ID NO: 1, which is derived from the amino acid sequence of dicalcin, significantly reduces the implantation rate, and that the sugar chain portion of glycolipid, one of the target molecules of dicalcin, significantly increases the implantation rate.

[0012] The means for solving the above problems are as follows: <1> A method for regulating implantation rate in a mammal, characterized by using at least one selected from the group consisting of (I) a compound represented by the following structural formula (1), (II) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and (III) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added. <2> An agent for improving implantation rate in mammals, comprising the compound represented by structural formula (1). <3> A pharmaceutical for improving implantation rate in mammals, comprising the agent for improving implantation rate according to <2>. <4> A method for improving implantation rate, comprising administering the pharmaceutical according to <3> to an individual.

[0013] According to the present invention, it is possible to solve the above-mentioned problems of the prior art, and to provide a new method for regulating the implantation rate in mammals, a new agent for improving the implantation rate in mammals, and a pharmaceutical containing the agent for improving the implantation rate.

[0014] FIG. 1 is a diagram showing the results of Test Example 1. FIG. 2A is a diagram showing the results of the control in Test Example 2. FIG. 2B is a diagram showing the results of Test Example 2 when a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (DC peptide) was used. FIG. 2C is a graph showing the implantation rate in Test Example 2. FIG. 3 is a diagram showing the results of observing DiI-labeled spheroids in Test Example 3. FIG. 4A is a diagram showing the results of Test Example 4. FIG. 4B is a graph showing the implantation rate in Test Example 4. FIG. 5 is a graph showing the implantation rate in Test Example 5.

[0015] (Method for regulating implantation rate in mammals) The method for regulating implantation rate in mammals of the present invention uses at least one compound selected from the group consisting of (I) a compound represented by the following structural formula (1), (II) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and (III) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added, and further uses other compositions as necessary. In the present invention, regulating the implantation rate includes both increasing the implantation rate and decreasing the implantation rate.

[0016] <Mammals> The mammals are not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, humans or non-human mammals. The non-human mammals are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include cows, pigs, sheep, goats, horses, dogs, and cats.

[0017] <(II) Peptide consisting of the amino acid sequence represented by SEQ ID NO: 1> The peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is a peptide derived from a part of the amino acid sequence of dicalcin. As shown in the Examples section below, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 has the effect of reducing the implantation rate (inhibiting implantation). QLDFSEFLNLI (SEQ ID NO: 1)

[0018] In the present invention, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 may be modified. The modification is not particularly limited and can be appropriately selected depending on the purpose, and examples include amidation, addition of a lipid chain (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation, acetylation, addition of a sugar chain (N-glycosylation, O-glycosylation, etc.), etc.

[0019] The method for obtaining the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is not particularly limited and can be appropriately selected depending on the purpose. For example, it can be obtained by chemical synthesis.

[0020] The amount of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 to be used is not particularly limited as long as the effect of reducing the implantation rate is obtained, and can be appropriately selected depending on the purpose. For example, the daily dosage may be 0.1 mg to 1 mg per kg of body weight.

[0021] (III) Peptides consisting of an amino acid sequence in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added, from the amino acid sequence represented by SEQ ID NO: 1 A peptide consisting of an amino acid sequence in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added, from the amino acid sequence represented by SEQ ID NO: 1, has the effect of reducing the implantation rate (inhibiting implantation), similar to the peptide represented by SEQ ID NO: 1. In the present specification, the number of the "several amino acids" is not particularly limited as long as it has the effect of reducing the implantation rate, and can be appropriately selected depending on the purpose, and may be, for example, 2 to 5.

[0022] In the present invention, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added, may be modified. The modification is not particularly limited and can be appropriately selected depending on the purpose, and examples include amidation, addition of a lipid chain (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation, acetylation, addition of a sugar chain (N-glycosylation, O-glycosylation, etc.), etc.

[0023] The method for obtaining a peptide consisting of an amino acid sequence in which one or several amino acids are substituted or deleted, or one or several amino acids are inserted or added, from the amino acid sequence represented by SEQ ID NO: 1, is not particularly limited and can be appropriately selected depending on the purpose, and can be obtained by, for example, chemical synthesis.

[0024] The amount of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added, is not particularly limited as long as the effect of reducing the implantation rate is obtained, and can be appropriately selected depending on the purpose. For example, the daily dosage may be 0.1 mg to 1 mg per kg of body weight.

[0025] <Compound Represented by Structural Formula (1)> The compound represented by the following structural formula (1) is a compound (hereinafter sometimes referred to as "oligosaccharide") based on a sugar chain present in glycolipid, which is one of the target molecules of dicalcin. As shown in the Examples section below, the compound represented by the structural formula (1) has the effect of increasing the implantation rate (enhancing implantation). The sugar structure of the compound represented by the structural formula (1) is, from left to right, N-acetylneuraminic acid-galactose-N-acetylgalactosamine-galactose-glucose (Neu5Ac-Gal-GalNAc-Gal-Glu).

[0026] In the present invention, the compound represented by the structural formula (1) also includes salt forms. The type of salt is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include sodium salts. In addition, the compound represented by the structural formula (1) can also be used in a state bound to other components such as lipids, such as ceramide.

[0027] The method for obtaining the compound represented by the structural formula (1) is not particularly limited and can be appropriately selected depending on the purpose. For example, a commercially available product can be used.

[0028] The amount of the compound represented by the structural formula (1) used is not particularly limited as long as the effect of increasing the implantation rate is obtained, and can be appropriately selected depending on the purpose. For example, the daily dosage may be 0.2 mg to 5 mg per kg of body weight.

[0029] The method of administration to at least one mammal selected from the group consisting of (I) the compound represented by structural formula (1), (II) the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and (III) the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added, is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include intrauterine administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, and oral administration.

[0030] The method for controlling the implantation rate is preferably a method for improving the implantation rate by using a compound represented by the structural formula (1).

[0031] <Other Components> The other components are not particularly limited as long as they do not impair the effects of the present invention and can be selected appropriately depending on the purpose, and examples thereof include (I) the compound represented by structural formula (1), (II) the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and (III) a substance having an effect of regulating implantation rate other than a peptide consisting of an amino acid sequence in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added, in the amino acid sequence represented by SEQ ID NO: 1. The amount and administration method of the other components are not particularly limited as long as they do not impair the effects of the present invention and can be selected appropriately depending on the purpose.

[0032] (Agent for improving implantation rate in mammals) The agent for improving implantation rate in mammals of the present invention is intended to improve implantation rate in mammals, and contains at least the compound represented by the structural formula (1) described above, and further contains other components as necessary.

[0033] <Compound Represented by Structural Formula (1)> Details of the compound represented by structural formula (1) are as described in the section on the method for regulating implantation rate of the present invention. The compound represented by structural formula (1) can effectively improve the implantation rate in mammals, and is therefore suitable as an active ingredient of an agent for improving the implantation rate in mammals. The content of the compound represented by structural formula (1) in the agent for improving the implantation rate in mammals is not particularly limited and can be selected appropriately depending on the purpose. Furthermore, the agent for improving the implantation rate in mammals may be the compound represented by structural formula (1) itself.

[0034] <Other Components> The other components in the agent for improving implantation rate in mammals are not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include the same as the other components in the medicament of the present invention described below. The content of the other components in the agent for improving implantation rate in mammals is also not particularly limited and can be selected appropriately depending on the purpose.

[0035] <Mammals> The mammals to which the agent for improving implantation rate in mammals is applied are not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, humans or mammals other than humans. Examples of the mammals other than humans include those described in the section on the method for adjusting implantation rate of the present invention.

[0036] The agent for improving implantation rate in mammals can improve implantation rate by introducing it into the body, for example, by uterine administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, oral administration, etc. The method of introduction is not particularly limited and can be appropriately selected from conventionally known methods depending on the purpose, and examples thereof include injection. The amount of the agent for improving implantation rate in mammals to be used is not particularly limited and can be appropriately selected depending on the age and body weight of the subject individual, the degree of desired effect, etc., and examples thereof include the amount of the active ingredient (compound represented by the structural formula (1)) in a daily dose of 0.2 mg to 5 mg per kg of body weight.

[0037] The agent for improving implantation rate in mammals of the present invention may be used alone or in combination with other agents for improving implantation rate in mammals.

[0038] (Medicine) The medicine of the present invention is a medicine for improving implantation rate in a mammal, and contains at least the agent for improving implantation rate in a mammal of the present invention described above, and further contains other ingredients as necessary. The mammal is not particularly limited, and examples thereof include those described in the section on the agent for improving implantation rate in a mammal of the present invention described above.

[0039] <Agent for improving implantation rate in mammals> Details of the agent for improving implantation rate in mammals are as described above in the section on the agent for improving implantation rate in mammals of the present invention.

[0040] The agent for improving implantation rate in mammals contains the compound represented by the structural formula (1), and therefore can effectively improve implantation rate in mammals. That is, the agent for improving implantation rate in mammals can be suitably used as a medicine for improving implantation rate in mammals.

[0041] The content of the agent for improving implantation rate in mammals in the pharmaceutical is not particularly limited and can be appropriately selected depending on the purpose. In addition, the pharmaceutical may consist solely of the agent for improving implantation rate in mammals.

[0042] <Other Components> The other components in the pharmaceutical are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include pharmaceutically acceptable carriers, etc. The content of the other components in the pharmaceutical is also not particularly limited and can be appropriately selected depending on the purpose.

[0043] <Dosage Form> The dosage form of the pharmaceutical is not particularly limited and can be appropriately selected depending on, for example, the desired administration method. Examples include oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations to be dissolved when used, etc.), ointments, patches, gels, creams, external powders, sprays, inhalation powders, etc.

[0044] The oral solid preparation can be prepared by a conventional method by adding, for example, an excipient to the active ingredient, and optionally additives such as a binder, disintegrant, lubricant, colorant, and flavoring agent. Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Examples of the disintegrant include dry starch, sodium alginate, powdered agar, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose. Examples of the lubricant include purified talc, stearates, borax, and polyethylene glycol. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring and odor correcting agents include sucrose, orange peel, citric acid, and tartaric acid.

[0045] The oral liquid preparation can be produced by a conventional method by adding additives such as flavorings, buffers, and stabilizers to the active ingredient. Examples of flavorings include sucrose, orange peel, citric acid, and tartaric acid. Examples of buffers include sodium citrate. Examples of stabilizers include tragacanth, gum arabic, and gelatin.

[0046] The injection preparation can be prepared by adding, for example, a pH adjuster, a buffer, a stabilizer, an isotonicity agent, a local anesthetic, etc. to the active ingredient, and then using a conventional method to prepare an injection preparation for subcutaneous, intramuscular, intravenous, etc. Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate, etc. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, etc. Examples of the isotonicity agent include sodium chloride, glucose, etc. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride, etc.

[0047] The ointment can be prepared by, for example, blending the active ingredient with known bases, stabilizers, humectants, preservatives, etc., and mixing them in a conventional manner. Examples of the bases include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, and propyl parahydroxybenzoate.

[0048] The patch can be produced, for example, by applying the ointment as a cream, gel, paste, etc. to a known support by a conventional method. Examples of the support include woven fabrics made of cotton, staple fiber, or chemical fiber, nonwoven fabrics, films made of soft vinyl chloride, polyethylene, polyurethane, etc., and foam sheets.

[0049] <Administration> The method of administering the pharmaceutical is not particularly limited, and either local administration or systemic administration can be selected depending on, for example, the dosage form of the pharmaceutical, the condition of the individual to be administered, etc., but local administration is preferred. For example, in local administration, the active ingredient of the pharmaceutical (the compound represented by the structural formula (1)) can be administered by direct injection into the uterus. For the injection, conventionally known methods such as injection can be used as appropriate.

[0050] The dosage of the medicament is not particularly limited and can be appropriately selected depending on the age and body weight of the individual to be administered, the degree of the desired effect, etc. For example, the amount of the active ingredient (the compound represented by the structural formula (1)) may be 0.2 mg to 5 mg per kg of body weight as a daily dosage. Furthermore, the number of times the medicament is administered is also not particularly limited and can be appropriately selected depending on the purpose.

[0051] The timing of administration of the pharmaceutical agent is not particularly limited and can be appropriately selected depending on the purpose. For example, administration can be performed at the pre-fertilization stage, the post-fertilization stage, or both stages.

[0052] The pharmaceutical agent of the present invention may be used in combination with other pharmaceutical agents that have the effect of improving implantation rates in mammals.

[0053] Since the medicament contains an agent for improving implantation rate in the mammal, administration to an individual can effectively improve the implantation rate and prevent or treat implantation disorders, etc. Therefore, the present invention also relates to a method for improving implantation rate (a method for preventing or treating implantation disorders, etc.) comprising administering the medicament to an individual. The mammal is not particularly limited, and examples include those described in the section on the agent for improving implantation rate in mammals of the present invention. Furthermore, the method for improving implantation rate may further include administration of a medicament that has the effect of improving implantation rate in other mammals.

[0054] (Agent for Reducing Implantation Rate in Mammals) As described above, the (II) peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the (III) peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added, have the effect of reducing the implantation rate (inhibiting implantation). Therefore, the present invention also relates to an agent for reducing the implantation rate in mammals (an inhibitor of implantation in mammals) comprising at least one peptide selected from the group consisting of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and peptides consisting of the amino acid sequence represented by SEQ ID NO: 1, in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added.

[0055] The agent for reducing implantation rate in mammals contains at least one peptide selected from the group consisting of peptides consisting of the amino acid sequence represented by SEQ ID NO: 1 and peptides consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence represented by SEQ ID NO: 1 have been substituted or deleted, or one or more amino acids have been inserted or added, and may further contain other components as necessary.

[0056] Details of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 and the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added, in the agent for reducing implantation rate in mammals, are as described above in the section on the method for regulating implantation rate of the present invention. The total content of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 and the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added, in the agent for reducing implantation rate in mammals is not particularly limited and can be selected appropriately depending on the purpose. Furthermore, the agent for reducing implantation rate in mammals may consist of at least one peptide selected from the group consisting of the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 and the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added.

[0057] The other components in the agent for reducing implantation rate in mammals are not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include the same as the other components in the medicament of the present invention described above. The content of the other components in the agent for reducing implantation rate in mammals is also not particularly limited and can be selected appropriately depending on the purpose.

[0058] There are no particular limitations on the mammals to which the agent for reducing implantation rate in mammals can be applied, and examples include those described in the section on the agent for improving implantation rate in mammals of the present invention.

[0059] The method for introducing the agent for reducing implantation rate in a mammal into the body is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include the same methods as those described in the section on the agent for improving implantation rate in a mammal of the present invention above. The amount of the agent for reducing implantation rate in a mammal to be used is also not particularly limited and can be selected appropriately depending on the age and body weight of the individual to be administered, the degree of the desired effect, and the like, and examples thereof include a daily dose of 0.1 mg to 1 mg per kg of body weight as the total amount of the active ingredients (the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added).

[0060] The agent for reducing implantation rate in mammals may be used alone or in combination with other agents for reducing implantation rate in mammals.

[0061] (Drug for reducing implantation rate in mammals) As described above, the (II) peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the (III) peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added, have the effect of reducing the implantation rate (inhibiting implantation). Therefore, the present invention also relates to a drug for reducing the implantation rate in mammals.

[0062] The pharmaceutical for reducing the implantation rate in a mammal contains at least an agent for reducing the implantation rate in a mammal, and further contains other ingredients as necessary. The mammal is not particularly limited, and examples thereof include those described in the section on the agent for improving the implantation rate in a mammal of the present invention.

[0063] Details of the agent for reducing implantation rate in mammals are as described above in the section on agents for reducing implantation rate in mammals.

[0064] The content of the agent for reducing implantation rate in a mammal in the pharmaceutical for reducing implantation rate in a mammal is not particularly limited and can be appropriately selected depending on the purpose. Furthermore, the pharmaceutical may consist solely of the agent for reducing implantation rate in a mammal.

[0065] The other components in the pharmaceutical for reducing the implantation rate in mammals are not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include those described in the section on pharmaceuticals for improving the implantation rate in mammals. Furthermore, the content of the other components in the pharmaceutical for reducing the implantation rate in mammals is also not particularly limited and can be selected appropriately depending on the purpose.

[0066] The dosage form of the pharmaceutical for reducing the implantation rate in mammals is not particularly limited and can be selected appropriately depending on the purpose, and examples include those described in the section on pharmaceuticals for improving the implantation rate in mammals.

[0067] The method of administering the pharmaceutical agent for reducing the implantation rate in the mammal is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include the methods described in the section on pharmaceutical agents for improving the implantation rate in the mammal.

[0068] The dosage of the medicament for reducing the implantation rate in a mammal is not particularly limited and can be appropriately selected depending on the age and body weight of the subject individual, the degree of the desired effect, etc., and examples thereof include a daily dosage of 0.1 mg to 1 mg per kg of body weight as the total amount of the active ingredients (the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added). Furthermore, the number of times the medicament for reducing the implantation rate in a mammal is administered is also not particularly limited and can be appropriately selected depending on the purpose.

[0069] The timing of administration of the pharmaceutical agent for reducing the implantation rate in the mammal is not particularly limited and can be selected appropriately depending on the purpose. For example, the timing can be the pre-fertilization stage, the post-fertilization stage, or both stages.

[0070] The above-mentioned medicine for reducing the implantation rate in mammals may be used in combination with a medicine having an effect of reducing the implantation rate in other mammals.

[0071] The pharmaceutical for reducing the implantation rate in a mammal contains an agent for reducing the implantation rate in the mammal, and thus, when administered to an individual, it can effectively reduce the implantation rate and inhibit implantation. Therefore, the present invention also relates to a method for reducing the implantation rate (a method of contraception) characterized by administering the pharmaceutical for reducing the implantation rate in the mammal to an individual. The mammal is not particularly limited, and examples include those described in the section on the agent for improving the implantation rate in a mammal of the present invention. Furthermore, the method for reducing the implantation rate may further include administering a pharmaceutical having an effect of reducing the implantation rate in other mammals.

[0072] Test examples of the present invention will be explained below, but the present invention is not limited to these test examples.

[0073] (Test Example 1: Peptide Binding Experiment) Choriocarcinoma cell line (BeWo; obtained from JCRB Cell Bank) cultured on a glass plate was fixed (4% paraformaldehyde / phosphate buffer (PBS), room temperature, 10 minutes), washed with PBS, treated with sheep serum, and then reacted with a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (QLDFSEFLNLI) fluorescently labeled with rhodamine (hereinafter sometimes referred to as "DC peptide"; final concentration 20 μM) (4°C overnight). After washing, the cells were analyzed using a confocal microscope (Zeiss, Jena, Germany). Nuclei were stained with DAPI. Bovine serum albumin was used as a control.

[0074] The results are shown in Figure 1. In Figure 1, the upper left panel shows the results of staining nuclei, the upper right panel shows the results of a peptide fluorescently labeled with rhodamine and consisting of the amino acid sequence represented by SEQ ID NO: 1, the lower left panel shows the merged results of the upper left and upper right panels, and the lower right panel shows a bright-field image. As shown in Figure 1, it was confirmed that the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 binds to choriocarcinoma cells (BeWo cells).

[0075] Test Example 2: In vitro implantation assay A cell mass (spheroid) of a choriocarcinoma cell line (BeWo; obtained from JCRB Cell Bank) was used as an embryo model, and a confluent endometrial cancer cell line (Ishikawa; obtained from JCRB Cell Bank) was used as an endometrial model, and a co-culture system of these cells was used.

[0076] BeWo cells (~1 x 10 ５ ) were cultured on PrimeSurface (Sumitomo Bakelite) to form spheroids (200-300 μm). First, a certain amount of spheroids (several dozen per well) was pretreated with a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (final concentration 20 μM, room temperature, 20 minutes). After removing excess peptide, the spheroids were seeded on Ishikawa (ISK) plates. After approximately 16 hours, the cell population was fixed (4% paraformaldehyde / phosphate buffer (PBS), room temperature, 10 minutes), washed with PBS, and unadhered spheroids were removed. After staining with crystal violet, the cells were counted. The implantation index (implantation rate) was calculated as (number of stained cells) / (number of seeded cells) × 100, and each experimental value was normalized to 100 for the control (10 μM BSA used instead of peptide). The experiment was carried out with at least n≧3, and significant differences were analyzed by Unpaired Student's t-test.

[0077] The results are shown in Figures 2A to 2C. Figure 2A shows the results for the control, Figure 2B shows the results when a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 was used, and Figure 2C is a graph of the implantation rate. The arrows in Figures 2A and 2B indicate spheroids. As shown in Figures 2A to 2C, it was confirmed that implantation was inhibited by the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0078] (Test Example 3: DiI labeling of spheroids) Spheroids prepared in the same manner as in Test Example 2 were reacted with a lipophilic carbocyanine dye (indocarbocyanine perchlorate (DiI)), which is a fluorescent dye (room temperature, 20 minutes), the excess dye was removed, and the spheroids were suspended in a liquid medium and used in Test Examples 4 and 5 described below.

[0079] The results of observing DiI-labeled spheroids are shown in Figure 3. In Figure 3, the left image is a bright-field image, and the right image is a fluorescent image.

[0080] Test Example 4: In Vitro Implantation Assay Using DiI-Labeled Spheroids The DiI-labeled spheroids prepared in Test Example 3 were pretreated with a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (final concentration 20 μM, room temperature, 20 minutes). After removing excess peptide, the spheroids were seeded on Ishikawa (ISK). After approximately 16 hours, the cell population was fixed (4% paraformaldehyde / phosphate buffer (PBS), room temperature, 10 minutes), washed with PBS, and non-adhered spheroids were removed. The cell number was then counted by fluorescence microscopy. The implantation index (implantation rate) was calculated as (number of stained cells) / (number of seeded cells) × 100, and each experimental value was normalized to 100 for the control (10 μM BSA used instead of peptide). Experiments were performed with at least n≧3, and significant differences were analyzed using an unpaired student's t-test.

[0081] The results are shown in Figures 4A and 4B. In Figure 4A, the left side shows the results for the control, the right side shows the results when a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 was used, and Figure 4B is a graph of the implantation rate. The triangles in Figure 4 indicate spheroids. As shown in Figures 4A and 4B, it was confirmed that implantation was significantly inhibited by the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0082] (Test Example 5: In vitro implantation assay using DiI-labeled spheroids) An in vitro implantation assay was performed in the same manner as in Test Example 4, except that the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 used in Test Example 4 was replaced with a compound represented by the following structural formula (manufactured by Elicityl) (sodium salt of the compound represented by structural formula (1); hereinafter, this may be referred to as "oligosaccharide"), and the amount used was set to a final concentration of 50 μM.

[0083] The results are shown in Figure 5. As shown in Figure 5, it was confirmed that the compound (oligosaccharide) represented by structural formula (1) significantly enhanced implantation.

[0084] These results suggest that the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 derived from dicalcin and the oligosaccharide represented by structural formula (1), which is one of the target molecules of dicalcin, may be useful as implantation regulators. Furthermore, the effects of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the compound represented by structural formula (1) were observed at 20-50 μM, confirming that they are effective even at low doses. Furthermore, because the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the compound represented by structural formula (1) are small molecules (approximately 1 kDa) and endogenous substances, the possibility of side effects is expected to be low.

[0085] The compound represented by the structural formula (1) can be suitably used as a compound for increasing the implantation rate in mammals such as humans or for increasing the fertility of premium livestock, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added can be suitably used as a contraceptive compound aimed at inhibiting implantation after fertilization has been established.

[0086] Examples of aspects of the present invention include the following: <1> A method for regulating implantation rate in a mammal, characterized by using at least one selected from the group consisting of (I) a compound represented by the following structural formula (1), (II) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and (III) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been substituted or deleted, or one or several amino acids have been inserted or added. <2> The method according to <1>, which is a method for improving the implantation rate using the compound represented by the structural formula (1). <3> An agent for improving the implantation rate in mammals, comprising the compound represented by the structural formula (1). <4> A pharmaceutical for improving the implantation rate in mammals, comprising the agent for improving the implantation rate according to <3>. <5> A method for improving the implantation rate, comprising administering the pharmaceutical according to <4> to an individual.

Claims

1. A method for regulating implantation rate in a mammal (excluding humans), characterized by using at least one selected from the group consisting of (I) a compound represented by the following structural formula (1), (II) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and (III) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added. 【Chemistry 1】

2. The method according to claim 1, which is a method for improving the implantation rate by using a compound represented by the structural formula (1).

3. An agent for improving implantation rate in mammals, comprising a compound represented by the following structural formula (1): 【Chemistry 2】

4. A pharmaceutical for improving implantation rate in a mammal, comprising the agent for improving implantation rate according to claim 3.

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