WO2022168971A1 - 哺乳動物における着床率の調節方法、哺乳動物における着床率の向上剤、及び医薬 - Google Patents

哺乳動物における着床率の調節方法、哺乳動物における着床率の向上剤、及び医薬 Download PDF

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WO2022168971A1
WO2022168971A1 PCT/JP2022/004638 JP2022004638W WO2022168971A1 WO 2022168971 A1 WO2022168971 A1 WO 2022168971A1 JP 2022004638 W JP2022004638 W JP 2022004638W WO 2022168971 A1 WO2022168971 A1 WO 2022168971A1
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Prior art keywords
implantation rate
amino acid
acid sequence
implantation
mammals
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English (en)
French (fr)
Japanese (ja)
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尚史 三輪
良平 齋藤
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Saitama Medical University
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Saitama Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • 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.
  • a fertilized egg (fertilized egg or embryo) in the fallopian tube progresses through the fallopian tube and reaches the uterus while differentiating.
  • a series of processes until the embryo (blastula stage) adheres to the endometrial epithelium, invades the endometrium and is encapsulated is called implantation.
  • ART assisted reproductive technology
  • the pregnancy success rate with ART is 15-30%, and there is still room for improvement. Many of the unsuccessful cases are thought to be due to implantation failure, and there is a strong need to elucidate the molecular basis of the implantation process and to identify substances that regulate the implantation rate.
  • Dicalcin A substance called Dicalcin, which is a factor that regulates fertilization efficiency, has been identified from eggs of Xenopus laevis (see Non-Patent Document 1).
  • Dicalcin is known to bind to glycoproteins that constitute the egg protective membrane (the extracellular matrix surrounding the egg) and to control the orientation of extracellular matrix filaments throughout the egg protective membrane.
  • 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, which is a low-molecular-weight calcium-binding protein, and is known to exist in humans, mice, pigs, and the like. Dicalcin or a partial peptide thereof has also been reported to effectively inhibit metastasis of human cancer cells (see Patent Document 3).
  • the object of the present invention is to solve the above-mentioned conventional problems and to achieve the following objectives. That is, the present invention provides a new method capable of regulating the implantation rate in mammals, a new implantation rate improver capable of improving the implantation rate in mammals, and the improvement of the implantation rate.
  • An object of the present invention is to provide a medicament containing an agent.
  • the present inventors found that a peptide consisting of the amino acid sequence of SEQ ID NO: 1, which is derived from the amino acid sequence of dicalsin, significantly reduces the implantation rate. It was found that the sugar chain portion of glycolipid, one of the molecules, significantly increases the implantation rate.
  • Means for solving the above problems are as follows. Namely ⁇ 1> (I) a compound represented by the following structural formula (1), (II) a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1, and (III) an amino acid sequence represented by SEQ ID NO: 1 , at least one selected from the group consisting of 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.
  • a method for modulating implantation rate in mammals ⁇ 2> An agent for improving the implantation rate in a mammal, comprising the compound represented by the structural formula (1).
  • ⁇ 3> A medicament for improving the implantation rate in a mammal, the medicament comprising the implantation rate improving agent according to ⁇ 2>.
  • ⁇ 4> A method for improving implantation rate, comprising administering the drug according to ⁇ 3> to an individual.
  • a new method capable of solving the above-mentioned conventional problems, regulating the implantation rate in mammals, and a new implantation rate capable of improving the implantation rate in mammals. and the agent for improving the implantation rate.
  • FIG. 1 is a diagram showing the results of Test Example 1.
  • FIG. 2A is a diagram showing the control results of Test Example 2.
  • FIG. 2B is a diagram showing the results when a peptide (DC peptide) consisting of the amino acid sequence represented by SEQ ID NO: 1 of Test Example 2 was used.
  • 2C is a graph of the implantation rate of Test Example 2.
  • FIG. 3 shows the results of observation of DiI-labeled spheroids of Test Example 3.
  • FIG. 4A is a diagram showing the results of Test Example 4.
  • FIG. 4B is a graph of the implantation rate of Test Example 4.
  • FIG. 5 is a graph of the implantation rate of Test Example 5.
  • FIG. 5 is a graph showing the implantation rate of Test Example 5.
  • the method for regulating implantation rate in mammals of the present invention comprises (I) a compound represented by the following structural formula (1), (II) a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1, and (III) Among the amino acid sequences represented by SEQ ID NO: 1, selected from the group consisting of peptides 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 At least one of the above is used, and other configurations are used as necessary.
  • adjustment of the implantation rate includes both increasing the implantation rate and decreasing the implantation rate.
  • the mammal is not particularly limited and can be appropriately selected depending on the purpose.
  • it may be a human or a non-human mammal.
  • Mammals other than humans are not particularly limited and can be appropriately selected according to the purpose. Examples thereof include bovine, porcine, sheep, goat, horse, dog, and cat.
  • ⁇ (II) Peptide consisting of the amino acid sequence represented by SEQ ID NO: 1> A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 below is a peptide derived from a partial amino acid sequence of dicalsin. The peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 has the effect of lowering the implantation rate (inhibiting implantation), as shown in the section of Examples described later.
  • QLDFSEFLNLI SEQ ID NO: 1
  • 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 intended purpose. geranylgeranylation, etc.), phosphorylation, acetylation, addition of sugar chains (N-glycosylation, O-glycosylation, etc.).
  • 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 according to the purpose. For example, it can be obtained by chemical synthesis.
  • 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 lowering the implantation rate can be obtained, and can be appropriately selected according to the purpose.
  • Daily doses include 0.1 mg to 1 mg per kg of body weight.
  • ⁇ (III) 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 in the amino acid sequence represented by SEQ ID NO: 1>
  • the amino acid sequences represented by SEQ ID NO: 1 the 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, Similar to the peptide represented by 1, it has the effect of lowering the implantation rate (inhibiting implantation).
  • the number of the "several amino acids" is not particularly limited as long as it has the effect of lowering the implantation rate, and can be appropriately selected according to the purpose. be done.
  • 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 may be modified.
  • the modification is not particularly limited and can be appropriately selected depending on the intended purpose. geranylgeranylation, etc.), phosphorylation, acetylation, addition of sugar chains (N-glycosylation, O-glycosylation, etc.).
  • a 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 in the amino acid sequence represented by SEQ ID NO: 1 can be appropriately selected according to the purpose, and can be obtained, for example, by chemical synthesis.
  • amino acid sequences represented by SEQ ID NO: 1 the amount of the 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 , As long as the effect of lowering the implantation rate is obtained, there is no particular limitation, and it can be appropriately selected according to the purpose. .
  • 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 a glycolipid, which is one of the target molecules of dicalsin.
  • the compound represented by the structural formula (1) has the action of increasing the implantation rate (enhancing implantation), as shown in the section of Examples described later.
  • the structure of the sugar in 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). .
  • the compounds represented by the structural formula (1) also include those in the form of salts.
  • the type of the salt is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include sodium salts.
  • the compound represented by the structural formula (1) can also be used in a state of being bound to other constituents such as lipids such as ceramide.
  • the method for obtaining the compound represented by the structural formula (1) is not particularly limited and can be appropriately selected according to the purpose.
  • commercially available products can be used.
  • the amount of the compound represented by the structural formula (1) to be used is not particularly limited as long as the effect of increasing the implantation rate is obtained, and can be appropriately selected according to the purpose. Amounts include 0.2 mg to 5 mg per kg of body weight.
  • At least one administration method to mammals selected from the group consisting of peptides 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 is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include uterine administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration and oral administration.
  • the method of adjusting the implantation rate is preferably a method of improving the implantation rate using the compound represented by the structural formula (1).
  • the other configuration is not particularly limited as long as it does not impair the effects of the present invention, and can be appropriately selected according to the purpose.
  • the usage amount and administration method of the other constituents are not particularly limited, and can be appropriately selected according to the purpose.
  • the agent for improving the implantation rate in mammals of the present invention is for improving the implantation rate in mammals, and contains at least the compound represented by the above-described structural formula (1), and if necessary Contains other ingredients.
  • ⁇ Compound Represented by Structural Formula (1)> The details of the compound represented by the structural formula (1) are as described in the item of the method for adjusting the implantation rate of the present invention. Since the compound represented by the structural formula (1) can effectively improve the implantation rate in mammals, it is suitable as an active ingredient of an agent for improving the implantation rate in mammals.
  • the content of the compound represented by the structural formula (1) in the agent for improving implantation rate in mammals is not particularly limited, and can be appropriately selected according to the purpose. Further, the agent for improving the implantation rate in mammals may be the compound itself represented by the structural formula (1).
  • ⁇ Other ingredients> Other ingredients in the agent for improving the implantation rate in mammals are not particularly limited and can be appropriately selected depending on the intended purpose. are mentioned.
  • the content of the other components in the agent for improving the implantation rate in mammals is also not particularly limited, and can be appropriately selected according to the purpose.
  • the mammal to which the agent for improving the implantation rate in mammals is applied is not particularly limited and can be appropriately selected according to the purpose. It can be an animal. Examples of mammals other than humans include those described in the item of the method for regulating the implantation rate of the present invention.
  • the agent for improving the implantation rate in mammals improves the implantation rate by introducing it into the body by, for example, uterine administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or oral administration. can be done.
  • the method of introduction is not particularly limited, and can be appropriately selected from conventionally known methods according to the purpose.
  • the amount of the agent for improving the implantation rate in mammals is not particularly limited, and can be appropriately selected according to the age and body weight of the individual to be administered, the degree of desired effect, and the like.
  • the amount of the compound represented by the structural formula (1)) includes 0.2 mg to 5 mg per 1 kg of body weight per day.
  • the agent for improving implantation rate in mammals of the present invention may be used alone, or may be used in combination with an agent for improving implantation rate in other mammals.
  • the medicament of the present invention is a medicament for improving the implantation rate in mammals, and contains at least the agent for improving the implantation rate in mammals of the present invention and, if necessary, other ingredients.
  • the mammal is not particularly limited, and includes those described in the item of the agent for improving the implantation rate in the mammal of the present invention.
  • the agent for improving the implantation rate in mammals contains the compound represented by the structural formula (1), it can effectively improve the implantation rate in mammals. That is, the agent for improving the implantation rate in mammals can be suitably used as a medicine for improving the implantation rate in mammals.
  • the content of the agent for improving the implantation rate in the mammal in the pharmaceutical is not particularly limited, and can be appropriately selected according to the purpose. Further, the medicine may consist only of the agent for improving the implantation rate in the mammal.
  • ingredients in the pharmaceutical are not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include pharmaceutically acceptable carriers. Also, the content of the other ingredients in the medicine is not particularly limited, and can be appropriately selected according to the purpose.
  • the dosage form of the medicament is not particularly limited, and can be appropriately selected according to the desired administration method.
  • Oral liquids oral liquids, syrups, elixirs, etc.
  • injections solutions, suspensions, solids for dissolution for use, etc.
  • ointments patches, gels, creams, external powders, sprays, inhalers powders and the like.
  • excipients and, if necessary, additives such as binders, disintegrants, lubricants, coloring agents, flavoring agents, etc. are added to the active ingredient, and can be manufactured by
  • the excipient include lactose, white sugar, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.
  • the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinylpyrrolidone, and the like.
  • Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose.
  • Examples of the lubricant include refined talc, stearate, borax, polyethylene glycol and the like.
  • Examples of the coloring agent include titanium oxide and iron oxide.
  • Examples of the flavoring/flavoring agent include sucrose, orange peel, citric acid, and tartaric acid.
  • the oral liquid preparation can be produced, for example, by adding additives such as flavoring agents, buffering agents, stabilizers, etc. to the active ingredient and by a conventional method.
  • additives such as flavoring agents, buffering agents, stabilizers, etc.
  • the flavoring/flavoring agent include sucrose, orange peel, citric acid, and tartaric acid.
  • buffering agent include sodium citrate.
  • the stabilizer include tragacanth, gum arabic, and gelatin.
  • a pH adjuster, a buffer, a stabilizer, a tonicity agent, a local anesthetic, etc. are added to the active ingredient, and subcutaneously, intramuscularly, or intravenously administered by a conventional method. etc. can be manufactured.
  • the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like.
  • the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like.
  • the tonicity agent include sodium chloride and glucose.
  • the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.
  • the ointment can be produced, for example, by blending the active ingredient with known bases, stabilizers, wetting agents, preservatives and the like, and mixing the mixture in a conventional manner.
  • the base include liquid paraffin, white petrolatum, bleached beeswax, octyldodecyl alcohol, and paraffin.
  • the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, and propyl parahydroxybenzoate.
  • the patch can be produced, for example, by applying a cream, gel, paste or the like as the ointment to a known support by a conventional method.
  • the support include woven fabrics and nonwoven fabrics made of cotton, staple fiber, and chemical fibers, films of soft vinyl chloride, polyethylene, polyurethane, and the like, foam sheets, and the like.
  • the method of administering the drug is not particularly limited.
  • either local administration or systemic administration can be selected depending on the dosage form of the drug, the condition of the individual to be administered, etc.
  • local administration is preferred. preferable.
  • the active ingredient of the drug (the compound represented by the structural formula (1)) can be administered by direct injection into the uterus. Conventionally known methods such as injection can be appropriately used for the injection.
  • the dose of the drug is not particularly limited, and can be appropriately selected according to the age, body weight, degree of desired effect, etc. of the individual to be administered.
  • the amount of the compound to be administered) includes 0.2 mg to 5 mg per kg of body weight per day.
  • the number of administrations of the medicine is not particularly limited, and can be appropriately selected according to the purpose.
  • the timing of administration of the drug is not particularly limited and can be appropriately selected according to the purpose.
  • the medicament of the present invention may be used in combination with a medicament having an effect of improving the implantation rate in other mammals.
  • the present invention also relates to a method for improving the implantation rate (method for preventing or treating implantation failure, etc.), which comprises administering the pharmaceutical to an individual.
  • the mammal is not particularly limited, and includes those described in the item of the agent for improving the implantation rate in the mammal of the present invention.
  • a drug having an effect of improving the implantation rate in other mammals may be further administered.
  • the present invention provides a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are substituted or deleted, or one An agent for reducing implantation rate in mammals (implantation inhibitor in mammals) containing at least one selected from the group consisting of peptides consisting of amino acid sequences in which one or several amino acids are inserted or added. related.
  • the agent for reducing the implantation rate in mammals includes a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 It contains at least one selected from the group consisting of peptides consisting of amino acid sequences with substitutions, deletions, insertions or additions of one or several amino acids, and optionally other components.
  • the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the agent for reducing the implantation rate in mammals, and one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are substituted or deleted.
  • the details of the peptide consisting of an amino acid sequence with deletion, insertion or addition of one or several amino acids are as described in the item of the method for regulating the implantation rate of the present invention.
  • One or several amino acids in the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 1 in the agent for reducing implantation rate in mammals are substituted.
  • the total content of the peptide consisting of an amino acid sequence with deletion, insertion or addition of one or several amino acids is not particularly limited and can be appropriately selected depending on the purpose.
  • the agent for reducing the implantation rate in mammals is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1. may consist of at least one peptide selected from the group consisting of peptides consisting of amino acid sequences in which is substituted or deleted, or one or several amino acids are inserted or added.
  • ingredients in the agent for reducing the implantation rate in mammals are not particularly limited and can be appropriately selected depending on the intended purpose. are mentioned.
  • the content of the other components in the agent for reducing the implantation rate in mammals is not particularly limited, and can be appropriately selected according to the purpose.
  • the mammal to which the agent for lowering the implantation rate in mammals is applied is not particularly limited, and examples thereof include those described in the item of the agent for improving the implantation rate in mammals of the present invention.
  • the method for introducing the agent for lowering the implantation rate in mammals into the body is not particularly limited and can be appropriately selected according to the purpose. Methods similar to those described in the item of agents, etc. can be used.
  • the amount of the implantation rate-lowering agent used in mammals is not particularly limited, and can be appropriately selected according to the age and body weight of the individual to be administered, the degree of desired effect, and the like.
  • the total amount of the peptide consisting of an amino acid sequence in which is inserted or added is, for example, 0.1 mg to 1 mg per 1 kg of body weight per day.
  • the agent for lowering the implantation rate in mammals may be used alone, or may be used in combination with an agent for lowering the implantation rate in other mammals.
  • the medicament for reducing the implantation rate in the mammal contains at least an agent for reducing the implantation rate in the mammal, and further contains other ingredients as necessary.
  • the mammal is not particularly limited, and includes those described in the item of the agent for improving the implantation rate in the mammal of the present invention.
  • the details of the agent for lowering the implantation rate in mammals are as described in the item of the agent for lowering the implantation rate in mammals.
  • the content of the agent for lowering the implantation rate in mammals in the pharmaceutical for lowering the implantation rate in mammals is not particularly limited, and can be appropriately selected according to the purpose. Further, the medicine may consist only of the agent for reducing the implantation rate in the mammal.
  • ingredients in the pharmaceutical for reducing the implantation rate in mammals are not particularly limited and can be appropriately selected according to the purpose. and those described in the item of pharmaceuticals. Also, the content of the other ingredients in the medicine for reducing the implantation rate in the mammal is not particularly limited, and can be appropriately selected according to the purpose.
  • the dosage form of the medicament for reducing the implantation rate in mammals is not particularly limited and can be appropriately selected according to the purpose. Examples include those described in the item of pharmaceuticals.
  • the administration method of the drug for reducing the implantation rate in the mammal is not particularly limited and can be appropriately selected depending on the purpose. Examples include the methods described in the section on drugs.
  • the dosage of the drug for reducing the implantation rate in mammals is not particularly limited, and can be appropriately selected according to the age and body weight of the individual to be administered, the degree of desired effect, etc.
  • Active ingredient the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and the amino acid sequence represented by SEQ ID NO: 1, one or several amino acids are substituted or deleted, or one or several
  • the total amount of a peptide consisting of an amino acid sequence in which 1 amino acid is inserted or added is, for example, 0.1 mg to 1 mg per 1 kg of body weight per day.
  • the number of administrations of the medicine for reducing the implantation rate in the mammal is not particularly limited, and can be appropriately selected according to the purpose.
  • the timing of administration of the drug for reducing the implantation rate in mammals is not particularly limited and can be appropriately selected according to the purpose. Examples include the stage before fertilization and the stage after fertilization. , can also be administered at both stages.
  • the medicament for lowering the implantation rate in mammals may be used in combination with a medicament that has the effect of lowering the implantation rate in other mammals.
  • the present invention also relates to a method for reducing implantation rate (contraception method), which comprises administering to an individual a pharmaceutical for reducing the implantation rate in said mammal.
  • the mammal is not particularly limited, and includes those described in the item of the agent for improving the implantation rate in the mammal of the present invention.
  • a drug having an effect of lowering the implantation rate in other mammals may be further administered.
  • FIG. 1 The results are shown in FIG.
  • the upper left shows the result of staining the nucleus
  • the upper right shows the result of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 fluorescently labeled with rhodamine
  • the lower left shows the result of merging the upper left and upper right results.
  • the lower right shows a bright-field image.
  • the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 binds to choriocarcinoma cells (BeWo cells).
  • Test Example 2 In vitro implantation assay
  • a cell cluster (spheroid) of a choriocarcinoma cell line (BeWo; obtained from JCRB Cell Bank) was used, and as an endometrium model, a confluent endometrial cancer cell line (Ishikawa; obtained from JCRB Cell Bank) was used. and used a co-culture system of these cells.
  • BeWo cells ( ⁇ 1 ⁇ 10 5 ) were cultured on PrimeSurface (Sumitomo Bakelite) to form spheroids (200-300 ⁇ m).
  • PrimeSurface Silicon-Surface
  • spheroids 200-300 ⁇ m
  • a certain amount of spheroids severe tens/well
  • a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (final concentration 20 ⁇ M, room temperature, 20 minutes).
  • ISK Ishikawa
  • the cell population was fixed (4% paraformaldehyde/phosphate buffer (PBS), room temperature, 10 minutes), washed with PBS to remove unattached spheroids, and stained with crystal violet.
  • PBS paraformaldehyde/phosphate buffer
  • the implantation index (implantation rate) was set to (number of stained cells)/(number of seeded cells) ⁇ 100, and the control (when 10 ⁇ M BSA was used instead of peptide) was set to 100, and each experimental value was standardized. . Experiments were performed with at least n ⁇ 3, and significant differences were analyzed by Unpaired Student's t-test.
  • FIG. 2A shows the control results
  • FIG. 2B shows the results when the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 was used
  • FIG. 2C shows the implantation rate graph.
  • the arrows in FIGS. 2A and 2B indicate spheroids.
  • FIGS. 2A to 2C it was confirmed that the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 inhibits implantation.
  • Test Example 3 DiI labeling of spheroids
  • Figure 3 shows the results of observing DiI-labeled spheroids.
  • the left is a bright field image and the right is a fluorescence image.
  • Test Example 4 In vitro implantation assay using DiI-labeled spheroids
  • 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 ⁇ M, room temperature, 20 minutes). After removing excess peptide, spheroids were plated on Ishikawa (ISK). After about 16 hours, the cell population was fixed (4% paraformaldehyde/phosphate buffer (PBS), room temperature, 10 minutes), washed with PBS to remove non-adherent spheroids, and then examined by fluorescence microscopy to reveal cells. counted the number.
  • PBS paraformaldehyde/phosphate buffer
  • the implantation index (implantation rate) was set to (number of stained cells)/(number of seeded cells) ⁇ 100, and the control (when 10 ⁇ M BSA was used instead of peptide) was set to 100, and each experimental value was standardized. . Experiments were performed with at least n ⁇ 3, and significant differences were analyzed by Unpaired Student's t-test.
  • FIG. 4A the left side shows the control results
  • the right side shows the results when the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 was used
  • FIG. 4B shows a graph of the implantation rate.
  • the triangle mark in FIG. 4 shows a spheroid. As shown in FIGS. 4A and 4B, it was confirmed that the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 significantly inhibited implantation.
  • Test Example 5 In vitro implantation assay using DiI-labeled spheroids
  • the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 used in Test Example 4 was converted into a compound represented by the following structural formula (manufactured by Elicityl) (sodium salt of the compound represented by structural formula (1), hereinafter referred to as In vitro implantation assay was performed in the same manner as in Test Example 4, except that the amount used was 50 ⁇ M at the final concentration.
  • the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 derived from dicalsin and the oligosaccharide represented by the structural formula (1), which is one of the target molecules of dicalsin can be used as implantation regulators. Possibilities were shown. In addition, the effect of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the compound represented by structural formula (1) was obtained at 20 to 50 ⁇ M, and it was also confirmed that the effect was obtained at a low dose. rice field.
  • the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the compound represented by Structural Formula (1) are low molecular weight (about 1 kDa) and are endogenous substances, so that side effects do not occur. expected to be unlikely.
  • the compound represented by the structural formula (1) can be suitably used as a compound for increasing the implantation rate of mammals such as humans or for increasing the fertility of superior domestic animals.
  • a peptide consisting of the amino acid sequence represented by 1, and one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are substituted or deleted, or one or several amino acids are inserted or
  • a peptide consisting of the added amino acid sequence can be suitably used as a contraceptive compound aiming at inhibition of implantation after fertilization is established.
  • Embodiments of the present invention include, for example, the following. ⁇ 1> (I) a compound represented by the following structural formula (1), (II) a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1, and (III) an amino acid sequence represented by SEQ ID NO: 1 , at least one selected from the group consisting of 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.
  • a method for modulating implantation rate in mammals ⁇ 2> The method according to ⁇ 1>, wherein the compound represented by the structural formula (1) is used to improve the implantation rate.
  • ⁇ 3> An agent for improving the implantation rate in a mammal, comprising the compound represented by the structural formula (1).
  • ⁇ 4> A medicament for improving the implantation rate in a mammal, the medicament comprising the implantation rate improving agent according to ⁇ 3>.
  • ⁇ 5> A method for improving implantation rate, comprising administering the drug according to ⁇ 4> to an individual.

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6445313A (en) * 1987-06-30 1989-02-17 Baiokaabu Ab Contraceptive and diagnostic method
WO2019102710A1 (ja) * 2017-11-21 2019-05-31 尚史 三輪 ヒト癌細胞転移阻害薬およびヒト癌細胞判定薬

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WO2011123041A1 (en) * 2010-04-01 2011-10-06 Karolinska Institutet Innovation Ab Fertilization prediction and promotion

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6445313A (en) * 1987-06-30 1989-02-17 Baiokaabu Ab Contraceptive and diagnostic method
WO2019102710A1 (ja) * 2017-11-21 2019-05-31 尚史 三輪 ヒト癌細胞転移阻害薬およびヒト癌細胞判定薬

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU XIN-MEI, DING GUO-LIAN, JIANG YING, PAN HONG-JIE, ZHANG DAN, WANG TING-TING, ZHANG RUN-JU, SHU JING, SHENG JIAN-ZHONG, HUANG H: "Down-Regulation of S100A11, a Calcium-Binding Protein, in Human Endometrium May Cause Reproductive Failure", JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, vol. 97, no. 10, 1 October 2012 (2012-10-01), The Endocrine Society, US, pages 3672 - 3683, XP055956881, ISSN: 0021-972X, DOI: 10.1210/jc.2012-2075 *

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