Medicine for improving state of pregnancy, and use thereof

Abstract

To provide a medicine for improving the state of pregnancy.SOLUTION: The present invention provides a pharmaceutical composition for improving pregnancy conditions in a human subject with pregnancy complications, comprising tacrolimus or a pharmaceutically acceptable salt thereof as an active ingredient, where the human subject has a history of repeated implantation failure (RIF), infertility, or pregnancy-induced hypertension.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a drug for improving pregnancy conditions, including infertility and / or miscarriage, comprising a specific immunosuppressant component, and use of the drug. [Background technology]

[0002] There are many people who want to have children, but ultimately give up because they are unable to have a normal pregnancy or birth. If we classify the stages of not being able to have a normal pregnancy or birth, Infertility can be divided into two categories: a condition in which the implantation of a fertilized egg into the endometrium does not proceed properly and pregnancy does not occur (infertility in the broad sense), and a condition in which the implantation of a fertilized egg into the endometrium proceeds properly, but the child is not conceived due to miscarriage, stillbirth, etc. (infertility in the broad sense).

[0003] When infertility or recurrent pregnancy loss becomes established, it can be treated as so-called infertility or recurrent pregnancy loss. Currently, the treatment of infertility or recurrent pregnancy loss is selected based on the cause. For example, in the case of infertility or recurrent pregnancy loss caused by blood coagulation abnormalities, diagnostic methods and treatment indicators exist, and treatment methods are almost established. However, there is a certain percentage of infertility or recurrent pregnancy loss whose cause cannot be clearly identified, which is one of the reasons why treatment is often difficult. For example, among severe infertility, there are cases of implantation disorder in which pregnancy does not result in pregnancy no matter how many times IVF treatment is performed, and among severe recurrent pregnancy loss, there are quite a few cases that are resistant to the currently performed recurrent pregnancy loss treatment. There is no special treatment for severe infertility with unknown causes, but for severe recurrent pregnancy loss, for example, antiplatelet therapy (low-dose aspirin administration), anticoagulant therapy (heparin administration), steroid therapy (PSL; prednisolone), high-dose gamma globulin therapy (IVIG), etc. may be performed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Uemura Y, Suzuki M, Liu TY, Narita Y, Hirata S, Ohyama H, Ishihara O, Matsushita S. Int Immunol. 2008;20:405-12. [Non-Patent Document 2] Saito S, Nakashima A, Shima T, Ito M. Am J Reprod Immunol. 2010;63:601-10. [Non-Patent Document 3] Kwak-Kim JY, Chung-Bang HS, Ng SC, Ntrivalas EI, Mangubat CP, Beaman KD, Beer AE, Gilman-Sachs A. Hum Reprod. 2003;18:767-73. [Non-Patent Document 4] Ng SC, Gilman-Sachs A, Thaker P, Beaman KD, Beer AE, Kwak-Kim J. Am J Reprod Immunol. 2002 Aug;48 (2):77-86. [Non-Patent Document 5] Yamaguchi K, Hisano M, Isojima S, Irie S, Arata N, Watanabe N, Kubo T, Kato T, Murashima A. J Med Virol. 2009;81:1923-8. Summary of the Invention [Problem to be solved by the invention]

[0005] A fertilized egg or fetus is a foreign body (a kind of foreign antigen) to the mother. Therefore, it is believed that during the period from implantation of the fertilized egg, maintenance of pregnancy to birth, changes occur in the immune system of the mother in order to continue to accept the fertilized egg or fetus without eliminating it from the mother. For example, in the intrauterine decidua, it is believed that one of the important factors for continuing pregnancy is the promotion of suppression of the immune system, mainly cellular immunity, so that the fertilized egg or fetus is not eliminated as a foreign body (Non-Patent Documents 1 to 5). However, the immune system is very complicated, and many details of the changes in the immune system in the mother from implantation of the fertilized egg to birth remain to be elucidated.

[0006] Due to these circumstances, there is no existing immunosuppressant therapy for infertility treatment.

[0007] In the treatment of recurrent pregnancy loss, the above-mentioned steroid (PSL) therapy and high-dose gamma globulin therapy (IVIG) are thought to have a suppressive effect on the immune system. However, both treatments have been reported to have various undesirable effects on the mother and fetus. Furthermore, IVIG therapy has problems such as the risk of unknown infectious diseases due to the use of plasma fraction preparations (purified from pooled plasma of more than 1,000 people) and a short duration of action.

[0008] Meanwhile, preliminary research by the present inventors has gradually revealed that some infertility patients have enhanced cellular immunity before pregnancy, and are presumed to have a rejection reaction to the fertilized egg at the time of implantation, and that some recurrent pregnancy loss patients have no abnormalities in their immune status before implantation, but are presumed to have a rejection reaction to the fetus due to a marked increase in cellular immunity after implantation (after pregnancy is established). In either case, the main cause is thought to be insufficient suppression mechanisms (immune tolerance) of the immune system that attacks the fertilized egg or fetus.

[0009] Based on these findings, the inventors of the present application hypothesized that the reception of fertilized eggs or embryos carrying alloantigens on the endometrium may be an immune response very similar to that of organ transplantation, and after verifying from various angles, they discovered a drug that is effective in sufficiently improving infertility and miscarriage, while suppressing undesirable effects on the mother and fetus, which led to the invention of the present application.Furthermore, they discovered that this drug can also be useful in improving pregnancy conditions other than infertility and miscarriage, which led to the invention of the present application. [Means for solving the problem]

[0010] In order to solve the above problems, the present invention includes the following aspects.

[0011] [ka]

[0012] A drug for improving a pregnancy condition, comprising a compound represented by the above general formula (I) or a pharma- ceutical acceptable salt thereof as an active ingredient (wherein R 1 and R 2 , R 3 and R 4 , R 5 and R 6 each adjacent pair of R independently represents (a) two adjacent hydrogen atoms, or 2 may be an alkyl group, or (b) may form an additional bond between each of the bonded carbon atoms; R 7 represents a hydrogen atom, a hydroxy group, a protected hydroxy group, or, together with an alkyl, an oxo group; R 8 and R 9 R independently represents a hydrogen atom or a hydroxyl group; 10represents a hydrogen atom, an alkyl group, an alkyl group substituted with one or more hydroxy groups, an alkenyl group, an alkenyl group substituted with one or more hydroxy groups, or an alkyl group substituted with an oxo group; X represents an oxo group, (hydrogen atom, hydroxy group), (hydrogen atom, hydrogen atom), or a group represented by the formula -CH2O-; Y represents an oxo group, (hydrogen atom, hydroxy group), (hydrogen atom, hydrogen atom), or a group represented by the formula N-NR 11 R 12 Or N-OR 13 A group represented by R 11 and R 12 each independently represents a hydrogen atom, an alkyl group, an aryl group, or a tosyl group; R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 22 and R 23 each independently represents a hydrogen atom or an alkyl group; R 24 is an optionally substituted ring which may contain one or more heteroatoms; n is 1 or 2, and in addition to the above meanings, Y, R 10 and R 23 may represent, together with the carbon atom to which they are attached, a saturated or unsaturated 5- or 6-membered ring containing a nitrogen atom, a sulfur atom and / or an oxygen atom, and the heterocyclic group may be substituted by one or more groups selected from an alkyl group, a hydroxy group, an alkyloxy group, a benzyl group, a group represented by the formula -CH2Se(C6H5), and an alkyl group substituted by one or more hydroxy groups. Effect of the Invention

[0013] The present invention can improve pregnancy conditions, including infertility and / or pregnancy loss. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below.

[0015] 1. Drugs (pharmaceutical compositions) (Active ingredient) The drug according to the present invention contains, as an active ingredient, a compound represented by the following general formula (I) (hereinafter, these may be collectively referred to as compound (I)) or a pharma- ceutically acceptable salt thereof.

[0016] Compound (I) is a macrolide compound. Macrolide compounds are macrocyclic lactones, and are a general term for compounds with 12 or more ring members.

[0017] [ka]

[0018] (In the formula, R 1 and R 2 , R 3 and R 4 , R 5 and R 6 each adjacent pair of (a) represents two adjacent hydrogen atoms or R 2 may be an alkyl group, or (b) an additional bond may be formed between each of the bonded carbon atoms; R 7 represents a hydrogen atom, a hydroxy group, a protected hydroxy group, or may combine with alkyl to represent an oxo group; R 8 and R 9 each independently represents a hydrogen atom or a hydroxyl group; R 10 represents a hydrogen atom, an alkyl group, an alkyl group substituted with one or more hydroxy groups, an alkenyl group, an alkenyl group substituted with one or more hydroxy groups, or an alkyl group substituted with an oxo group; X is an oxo group, (hydrogen atom, hydroxy group), (hydrogen atom, hydrogen atom), or a group represented by the formula -CH2O-; Y is an oxo group, (hydrogen atom, hydroxy group), (hydrogen atom, hydrogen atom), or a group of the formula N-NR 11R 12 Or N-OR 13 A group represented by: R 11 and R 12 each independently represents a hydrogen atom, an alkyl group, an aryl group, or a tosyl group; R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 22 and R 23 each independently represents a hydrogen atom or an alkyl group; R 24 is an optionally substituted ring which may contain one or more heteroatoms; n represents 1 or 2.

[0019] In addition to the above meanings, Y, R 10 and R 23 may represent, together with the carbon atom to which they are attached, a saturated or unsaturated 5- or 6-membered ring containing a nitrogen atom, a sulfur atom and / or an oxygen atom, and the heterocyclic group may be substituted by one or more groups selected from an alkyl group, a hydroxy group, an alkyloxy group, a benzyl group, a group represented by the formula -CH2Se(C6H5), and an alkyl group substituted by one or more hydroxy groups.

[0020] Preferred R 24 Examples of the alkyl group include cyclo(C5-7)alkyl groups which may have an appropriate substituent, such as the following groups: (a) 3,4-dioxo-cyclohexyl group; (b) 3-R 20 -4-R 21 -cyclohexyl group, Among them, R 20 is hydroxy, alkyloxy, oxo, or OCH2OCH2CH2OCH3, and R 21is hydroxy, -OCN, alkyloxy, heteroaryloxy which may have an appropriate substituent, -OCHOCHCHOCH, protected hydroxy, chloro, bromo, iodo, aminooxalyloxy, azide group, p-tolyloxythiocarbonyloxy, or R 25 R 26 CHCOO-(wherein, R 25 represents an optionally protected hydroxy group or a protected amino group, and R 26 is a hydrogen atom or methyl, or R 20 and R 21 together form the oxygen atom of an epoxide ring); or (c) cyclopentyl groups, which are substituted by methoxymethyl, optionally protected hydroxymethyl, acyloxymethyl (in which the acyl moiety is an optionally quaternized dimethylamino group or an optionally esterified carboxy group), one or more optionally protected amino and / or hydroxy groups, or aminooxalyloxymethyl. A preferred example is the 2-formyl-cyclopentyl group.

[0021] Each definition used in the general formula (I), specific examples thereof, and preferred embodiments thereof will be explained in detail below.

[0022] "Lower" shall mean a group having 1 to 6 carbon atoms, unless otherwise specified.

[0023] Preferred examples of the alkyl moiety of the "alkyl group" and "alkyloxy group" include straight-chain or branched-chain aliphatic hydrocarbon residues, such as lower alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, neopentyl, and hexyl.

[0024] Preferred examples of the "alkenyl group" include straight-chain or branched-chain aliphatic hydrocarbon residues containing one double bond, such as lower alkenyl groups such as vinyl, propenyl (allyl, etc.), butenyl, methylpropenyl, pentenyl, and hexenyl. Examples of such groups include the aryl group.

[0025] Preferred examples of the "aryl group" include phenyl, tolyl, xylyl, cumenyl, mesityl, naphthyl, and the like.

[0026] Preferred protecting groups for the "protected hydroxy group" and "protected amino" include 1-(lower alkylthio)(lower)alkyl groups such as lower alkylthiomethyl groups such as methylthiomethyl, ethylthiomethyl, propylthiomethyl, isopropylthiomethyl, butylthiomethyl, isobutylthiomethyl, and hexylthiomethyl, more preferably C1-C4 alkylthiomethyl groups, and most preferably methylthiomethyl groups; tri-substituted silyl groups such as tri(lower)alkylsilyl groups, e.g., trimethylsilyl, triethylsilyl, tributylsilyl, tertiary butyl-dimethylsilyl, tritertiary butylsilyl, etc.; lower alkyl-diarylsilyl groups, e.g., methyl-diphenylsilyl, ethyl-diphenylsilyl, propyl-diphenylsilyl, tertiary butyl-diphenylsilyl, etc.; more preferably, tri(C1-C4)alkylsilyl groups and C1-C4 alkyldiphenylsilyl groups; most preferably, tertiary butyl-dimethylsilyl groups and tertiary butyl-diphenylsilyl groups; acyl groups such as aliphatic acyl groups, aromatic acyl groups and aromatic substituted aliphatic acyl groups derived from carboxylic acids, sulfonic acids and carbamic acids; etc.

[0027] Examples of the aliphatic acyl group include lower alkanoyl groups which may have one or more suitable substituents such as carboxy, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, carboxyacetyl, carboxypropionyl, carboxybutyryl, and carboxyhexanoyl; For example, cyclo(lower)alkyloxy(lower)alkanoyl groups which may have one or more suitable substituents such as lower alkyl, such as cyclopropyloxyacetyl, cyclobutyloxypropionyl, cycloheptyloxybutyryl, menthyloxyacetyl, menthyloxypropionyl, menthyloxybutyryl, menthyloxypentanoyl, menthyloxyhexanoyl, etc.; Camphorsulfonyl group; Examples of the alkylcarbamoyl group include carboxy(lower)alkylcarbamoyl groups such as carboxymethylcarbamoyl, carboxyethylcarbamoyl, carboxypropylcarbamoyl, carboxybutylcarbamoyl, carboxypentylcarbamoyl, carboxyhexylcarbamoyl, and the like; and lower alkylcarbamoyl groups having one or more suitable substituents such as carboxy or protected carboxy, such as tri(lower)alkylsilyl(lower)alkyloxycarbonyl(lower)alkylcarbamoyl groups such as trimethylsilylmethoxycarbonylethylcarbamoyl, trimethylsilylethoxycarbonylpropylcarbamoyl, triethylsilylethoxycarbonylpropylcarbamoyl, tert-butyldimethylsilylethoxycarbonylpropylcarbamoyl, trimethylsilylpropoxycarbonylbutylcarbamoyl, and the like.

[0028] Examples of the aromatic acyl group include aroyl groups which may have one or more suitable substituents such as nitro, such as benzoyl, toluoyl, xyloyl, naphthoyl, nitrobenzoyl, dinitrobenzoyl, and nitronaphthoyl; Examples of the sulfonyl group include arenesulfonyl groups which may have one or more suitable substituents such as halogen, such as benzenesulfonyl, toluenesulfonyl, xylenesulfonyl, naphthalenesulfonyl, fluorobenzenesulfonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl, and iodobenzenesulfonyl.

[0029] Examples of the aliphatic acyl group substituted with an aromatic group include aryl(lower)alkanoyl groups which may have one or more suitable substituents such as lower alkyloxy or trihalo(lower)alkyl, such as phenylacetyl, phenylpropionyl, phenylbutyryl, 2-trifluoromethyl-2-methoxy-2-phenylacetyl, 2-ethyl-2-trifluoromethyl-2-phenylacetyl, and 2-trifluoromethyl-2-propoxy-2-phenylacetyl.

[0030] Among the above acyl groups, more preferred acyl groups are C1-C4 alkanoyl groups which may have a carboxyl group and acyl groups having two (C1-C4) alkyl groups in the cycloalkyl moiety. Examples of the alkyl group include a cyclo(C5-C6)alkyloxy(C1-C4)alkanoyl group having one or more nitro groups, a camphorsulfonyl group, a carboxy(C1-C4)alkylcarbamoyl group, a tri(C1-C4)alkylsilyl(C1-C4)alkyloxycarbonyl(C1-C4)alkylcarbamoyl group, a benzoyl group which may have one or two nitro groups, a benzenesulfonyl group having a halogen, and a phenyl(C1-C4)alkanoyl group having C1-C4 alkyloxy and a trihalo(C1-C4)alkyl, and among these, the most preferred ones include acetyl, carboxypropionyl, menthyloxyacetyl, camphorsulfonyl, benzoyl, nitrobenzoyl, dinitrobenzoyl, iodobenzenesulfonyl and 2-trifluoromethyl-2-methoxy-2-phenylacetyl.

[0031] Preferred examples of the "saturated or unsaturated 5- or 6-membered heterocyclic group containing a nitrogen atom, a sulfur atom and / or an oxygen atom" include a pyrrolyl group, a tetrahydrofuryl group, and the like.

[0032] The "heteroaryl which may have suitable substituents" in the "heteroaryloxy which may have suitable substituents" includes those exemplified as the group R1 of the compound represented by the formula in EP-A-532,088, and for example, 1-hydroxyethylindol-5-yl is preferred. The disclosure of that document is incorporated herein by reference.

[0033] The compound (I) and its pharma- ceutically acceptable salts used in the present invention have excellent immunosuppressive action, antibacterial activity, and other pharmacological activities, and therefore are useful for the treatment and prevention of organ or tissue transplant rejection, graft-versus-host reaction, autoimmune diseases, infectious diseases, and the like, as disclosed in, for example, EP-A-184162, EP-A-323042, EP-A-423714, EP-A-42 No. 7680, EP-A-465426, EP-A-480623, EP-A-532088, EP-A-532089, EP-A-569337, EP-A-626385, WO89 / 05303, WO93 / 05058, WO96 / 31514, WO91 / 13889, WO91 / 19495, WO93 / 5059, etc., and methods for producing these compounds are also disclosed. The disclosures of these publications are incorporated herein by reference.

[0034] In particular, the compounds designated FR900506 (=FK506, tacrolimus), FR900520 (ascomycin), FR900523 and FR900525 are derived from Streptomyces genus, e.g. Streptomyces tsukubaensis No. 9993 (deposit: Biotechnology and Human-Related Technology Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan (former name: Microbial Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry), date of deposit: October 5, 1984, accession number: Biotechnology and Human-Related Technology Institute No. 927) or Streptomyces hygroscopicus subsp. yakushimaensis (Streptomyces yakushimaensis) No. 7238 (deposit institution: National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-3, Machihigashi, Tsukuba-shi, Ibaraki-ken, Japan, date of deposit: January 12, 1985, accession number: Bioscience and Human-Technology Accession No. 928) (EP-A-0184162), and in particular, FK506 (generic name: tacrolimus), represented by the structural formula below, is a representative compound.

[0035] [ka]

[0036] Chemical Name: 17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone.

[0037] Among the compounds (I), more preferred are those represented by R 3 and R 4 , R 5 and R 6 each adjacent pair of carbon atoms form an additional bond between the carbon atoms to which they are attached, R 8 and R23 are independently hydrogen atoms, R 9 is a hydroxy group, R 10 is a methyl, ethyl, propyl or allyl group; X is (hydrogen atom, hydrogen atom) or oxo group, Y is an oxo group; R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 22 are methyl groups, R 24 is 3-R 20 -4-R 21 -cyclohexyl group, Among them, R 20 is hydroxy, alkyloxy, oxo, or -OCH2OCH2CH2OCH3, and R 21 is hydroxy, -OCN, alkyloxy, heteroaryloxy which may have an appropriate substituent, 1-tetrazolyl or 2-tetrazolyl, -OCHOCHOCHOCHOCH, protected hydroxy, chloro, bromo, iodo, aminooxalyloxy, azide group, p-tolyloxythiocarbonyloxy, or R 25 R 26 CHCOO-(wherein, R 25 represents an optionally protected hydroxy group or a protected amino group, and R 26 is a hydrogen atom or methyl), or R 20 and R 21 taken together form the oxygen atom of an epoxide ring, and n is 1 or 2.

[0038] Preferred compounds (I) include tacrolimus, ascomycin or derivatives thereof, such as 33-epi-chloro-33-desoxyascomycin, as described in Example 66a of EP 427,680.Other preferred compounds (I) include the compound described in Example 6d of EP 569337 and the compound described in Example 8 of EP 626385.

[0039] Compounds described in EP-0184162, EP323042, EP424714, EP427680, EP465426, EP474126, EP480623, EP484936, EP532088, EP532089, EP569337, EP626385, WO89 / 05303, WO93 / 05058, WO96 / 31514, WO91 / 13889, WO91 / 19495, WO93 / 5059, WO96 / 31514 and the like are also mentioned as preferred examples of the macrolide compound (I), and the disclosures thereof are incorporated herein by reference.

[0040] In another embodiment of the present invention, the pharmaceutical agent according to the present invention comprises, as an active ingredient, the compound shown below or a pharma- ceutically acceptable salt thereof.

[0041] In the pharmaceutical composition of the present invention, the compound acting as an active ingredient may be used in place of the above-mentioned compound (I) or a salt thereof or in combination with the above-mentioned compound (I) or a salt thereof, and may include cyclosporins such as cyclosporins A, B, D, etc., which are described in Merck Index (12th Edition) No. 2821, the disclosure of which is incorporated herein by reference.

[0042] In the pharmaceutical preparation of the present invention, preferred macrolide compounds that can be used as an active ingredient in place of the above-mentioned compound (I) or a salt thereof, or in combination with the above-mentioned compound (I) or a salt thereof, include rapamycin and its derivatives described in Merck Index (12th Edition) No. 8288. Preferred examples include rapamycin and its derivatives described in formula A on page 1 of WO95 / 16691, in which the hydroxyl at the 40-position is -OR. 1(where R 1 include O-substituted derivatives substituted with hydroxyalkyl, hydroalkyloxyalkyl, acylaminoalkyl and aminoalkyl, such as 40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin and 40-O-(2-acetaminoethyl)-rapamycin.

[0043] These O-substituted derivatives can be prepared by reacting rapamycin (or dihydro- or deoxorapamycin) with a leaving group (e.g., RX, where R is an organic radical desired for the O-substituent, such as an alkyl, aryl, or benzyl moiety, and X is CC l3 They can be prepared by reaction with an organic radical bonded to a leaving group such as C(NH)O or CF3SO3).

[0044] The condition is that X is CC l3 When X is C(NH)O, this may be under acidic or neutral conditions, for example in the presence of trifluoromethanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid or their corresponding pyridinium or substituted pyridinium salts, or when X is CF3SO3, in the presence of a base such as pyridine, substituted pyridines, diisopropylethylamine or pentamethylpiperidine.The most preferred rapamycin derivative is 40-O-(2-hydroxy)ethyl rapamycin, as described in WO94 / 09010, the disclosure of which is incorporated herein by reference.

[0045] Compound (I) and rapamycin and its derivatives have a similar basic structure, ie, a tricyclomacrolide structure, and at least one similar biological property (eg, immunosuppressive activity).

[0046] The pharma- ceutically acceptable salts of Compound (I), cyclosporine, rapamycin and its derivatives are non-toxic, pharma-ceutically acceptable conventional salts, including salts with inorganic or organic bases such as alkali metal salts, e.g., sodium salts, potassium salts, etc.; alkaline earth metal salts, e.g., calcium salts, magnesium salts, etc.; ammonium salts; and amine salts, e.g., triethylamine salts, N-benzyl-N-methylamine salts, etc.

[0047] In the compound (I) of the present invention, one or more pairs of stereoisomers such as conformers or optical isomers and geometric isomers resulting from asymmetric carbon atoms and double bonds may exist, and such conformers or isomers are also included in the scope of the compound of the present invention.

[0048] Compound (I) may also form a solvate, which is also included in the scope of the present invention. Preferred solvates include hydrates and ethanolates.

[0049] (Other optional ingredients) In addition to the above-mentioned active ingredient, the drug of the present invention may contain one or more therapeutically active substances that have a therapeutic effect against other diseases, disorders, and conditions, so long as there is no risk of inhibiting the activity of the active ingredient and they are not harmful to the subject to which they are administered (hereinafter also referred to as the patient).

[0050] The therapeutic agent may also contain a pharma- ceutically acceptable carrier that is not harmful to the subject of administration. The carrier that can be used may be solid, semi-solid, or liquid, and may include, but is not limited to, any of water, electrolyte solutions, and sugar solutions. Furthermore, the therapeutic agent may contain an auxiliary agent. Examples of the auxiliary agent include lubricants, stabilizers, preservatives, emulsifiers, thickeners (viscosifiers), colorants, flavors (flavoring agents), excipients, preservatives, buffers, flavorings, suspending agents, emulsifiers, solubilizing agents, and lubricants.

[0051] (Dosage form) The dosage form may be a tablet, a capsule, a pill, a granule, a powder, a syrup, a suppository, a lozenge, a pellet, an emulsion, a suspension, or other known forms.Among these, for example, as an oral administration formulation, it is preferable to use any of a tablet, a capsule, a pill, a granule, a powder, a liquid, a syrup, and a jelly, more preferably any of a tablet, a capsule, and a granule, and even more preferably a tablet.In addition, as described later, it may be formulated as a parenteral administration formulation such as an injection, a suppository, and a transdermal absorption type formulation.

[0052] (Drug manufacturing method) The drug according to the present invention can be produced by utilizing a known production method. For example, the active ingredient and any other ingredients are separately produced, and then the ingredients are mixed to obtain the desired content.

[0053] (Target of drug administration) =Target species= The subjects of administration of the drug of the present invention include mammals, birds, reptiles, amphibians, etc., among which mammals are preferred. Mammals include humans and non-human animals such as livestock such as cows, horses, pigs, and sheep, and pets such as dogs, cats, rats, and rabbits, and preferably humans. Birds include poultry such as chickens, ducks, and geese.

[0054] =Poor pregnancy condition= The subject to which the drug of the present invention is administered is the above subject with poor pregnancy. Here, poor pregnancy is a concept that includes not only those classified as disease or illness, but also poor pregnancy compared to normal pregnancy. That is, poor pregnancy includes not only poor conception and fetus conditions in the mother, but also poor health conditions of the mother itself during pregnancy. During pregnancy refers to any period from the establishment of pregnancy (synonymous with pregnancy establishment) to delivery, including the entire period of early pregnancy, mid pregnancy, and late pregnancy. Here, early pregnancy refers to the period from the establishment of pregnancy to the 13th week, mid pregnancy refers to the period from the 14th week to the 27th week, and late pregnancy refers to the period from the 28th week to the delivery date. Furthermore, poor pregnancy includes poor health conditions of the mother after delivery and health conditions of the mother during and after delivery that appear as disorders in the newborn after delivery.

[0055] The type of pregnancy defect is not particularly limited, but specifically includes, for example, at least one selected from 1) infertility, 2) miscarriage, and 3) pregnancy defect other than infertility or miscarriage (e.g., pregnancy complications, etc.). Among these, as described below, pregnancy defects caused by immune abnormalities in the subject of administration are more suitable as defects to be improved by administration of the drug of the present invention.

[0056] = Infertility, infertility = In this specification, the term "infertility" is used in a broad sense, referring to a state in which the mother's body is in a difficult state to conceive compared to a normal state, and is a concept that includes infertility. Infertility here refers to a case in which pregnancy is not achieved within one year without contraception after wishing to have a child.

[0057] In one embodiment, the subject with infertility to which the agent of the present invention is administered may be undergoing existing infertility treatment, such as artificial insemination, in vitro fertilization, and administration of an ovulation inducer.

[0058] Among these, specific methods of in vitro fertilization include in vitro fertilization embryo transfer (IVF-ET), in which collected eggs and sperm are fertilized in a petri dish and the fertilized eggs are returned to the uterus, and intracytoplasmic sperm injection (ICSI), in which sperm are directly injected into eggs under a microscope to perform fertilization. IVF-ET further includes fresh embryo transfer (fresh ET), in which fresh embryos are used for transfer, and frozen-thawed embryo transfer (FET), in which embryos frozen and stored after collection are frozen and thawed for transfer. The subject may be a person who has not been able to become pregnant even after multiple embryo transfers or intracytoplasmic sperm injections (no clear increase in hCG is observed), and it can also be suitably applied to a person who has not been able to become pregnant even after three or more, four or more, or five or more treatments.

[0059] Causes of infertility, including subfertility, include egg factors, fallopian tube factors, uterine factors, cervical factors, and immune factors. Specific examples of egg factors include ovulation disorders caused by antiprolactinemia, polycystic ovarian syndrome, various stresses, dieting, and premature ovarian insufficiency. Specific examples of fallopian tube factors include blockage, stenosis, or adhesion of the fallopian tube caused by chlamydia salpingitis and perisalpingitis, and specific examples of uterine factors include uterine fibroids, endometrial polyps, congenital malformations of the uterus, and intrauterine adhesion (Asherman's syndrome). Specific examples of cervical factors include cervicitis, abnormal or insufficient mucus secretion from the cervix, and the like. Specific examples of immune factors include production of antisperm antibodies. Here, the term "unexplained infertility" refers to infertility that does not fall under any of the above factors and has no identified cause. It is believed that infertility of unknown cause includes infertility caused by immune abnormalities (including immune cell abnormalities and autoimmune diseases such as antiphospholipid syndrome), and subjects with such infertility of unknown cause are also suitable subjects for administration of the drug of the present invention (see also Examples). In particular, subjects with infertility whose main cause is thought to be the inhibition of implantation of a fertilized egg or embryo into the endometrium due to immune abnormalities, with or without autoimmune diseases, are suitable subjects for administration of the drug of the present invention.

[0060] In this specification, "failure to conceive" refers to a state in which the fetus is unable to develop in the mother's uterus after pregnancy, or a state in which the fetus shows delayed or poor development, and is a concept that includes recurrent pregnancy loss. Here, recurrent pregnancy loss refers to a case in which pregnancy (including natural pregnancy, artificial insemination, and in vitro fertilization) is established, but a miscarriage or stillbirth occurs twice or more times, resulting in no child being born. Note that the concept of "failure to conceive" in this specification also includes the experience of one miscarriage, premature birth, or stillbirth, or two repeated miscarriages, premature births, or stillbirths.

[0061] Causes of infertility, including recurrent pregnancy loss, include genetic factors, anatomical factors, endocrine factors, coagulation factors, and autoimmune factors. Specific examples of genetic factors include marital chromosomal abnormalities and fetal chromosomal abnormalities. Specific examples of anatomical factors include myoma and uterine morphological abnormalities. Specific examples of secretory factors include luteal phase defect, hyperprolactinemia, thyroid dysfunction, and blood sugar abnormalities. Specific examples of coagulation factors include blood coagulation abnormalities. Specific examples of autoimmune factors include production of antiphospholipid antibodies (antiphospholipid syndrome). Here, infertility of unknown cause refers to infertility that does not fall under any of the above factors and has no identified cause. Unexplained infertility is thought to include infertility caused by immune abnormalities (including immune cell abnormalities and autoimmune diseases other than antiphospholipid syndrome), and subjects with such infertility of unknown cause and subjects with infertility caused by autoimmune factors (antiphospholipid syndrome) are also suitable subjects for administration of the drug of the present invention (see also Examples). In particular, subjects with infertility thought to be primarily caused by 1) immune abnormalities leading to an enhanced immune mechanism that eliminates fertilized eggs or embryos as foreign bodies from the mother's body, and / or 2) immune abnormalities leading to defective placental construction are suitable subjects for administration of the agents of the present invention.

[0062] As mentioned above, infertility and infertility associated with autoimmune diseases other than antiphospholipid syndrome are also targets for administration of the drug of the present invention. Examples of such autoimmune diseases include systemic lupus erythematosus, ANCA-associated vasculitis (cryoglobulinemic vasculitis, IgA vasculitis, hypocomplementemic urticarial vasculitis, polyarteritis nodosa, Kawasaki disease, anti-GBM disease, Takayasu's arteritis, giant cell arteritis, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis), and malignant rheumatoid arthritis. Autoimmune diseases including antiphospholipid syndrome can cause defective construction of the placenta through the formation of thrombi, etc. Defective construction of the placenta can be accompanied by vasculitis of the placenta, inflammation of the placenta, etc., in addition to the formation of thrombi. By administering the drug of the present invention, it is expected that the undesirable activity of autoantibodies can be suppressed and defective construction of the placenta can be improved.

[0063] = Poor pregnancy status other than infertility or miscarriage = In this specification, "poor pregnancy condition other than infertility or miscarriage" includes, for example, pregnancy-induced hypertension, thrombosis, infarction, cardiac failure (peripartum cardiomyopathy, etc.), and pulmonary edema, and other diseases or disorders (complications of pregnancy, etc.) that occur with pregnancy, but also includes conditions that are poorer than normal pregnancy conditions, in addition to those classified as diseases or disorders. Among these, subjects who are likely to develop pregnancy-induced hypertension (for example, those who have a history of developing pregnancy-induced hypertension) are more suitable for administering the drug of the present invention. Although the mechanism is not entirely clear, this treatment improves the immunological relationship between mother and child, making placenta construction smooth and reducing maternal stress, which is expected to prevent pregnancy complications.

[0064] Pregnancy-induced hypertension refers to a syndrome that occurs during pregnancy, mainly in the late stages of pregnancy and up to 12 weeks after birth, and is characterized by high blood pressure or high blood pressure and symptoms associated with high blood pressure, such as edema and proteinuria. Pregnancy-induced hypertension is further classified into gestational hypertension, preeclampsia, aggravated preeclampsia, and eclampsia. Pregnancy-induced hypertension is recurrent, and if a woman has a history of pregnancy-induced hypertension in a previous pregnancy, she is at a very high risk of developing pregnancy-induced hypertension in the next pregnancy.

[0065] Causes of pregnancy-induced hypertension include vascular endothelial disorder due to placental ischemia and hypoxia. Therefore, pregnancy-induced hypertension can lead to poor placental construction. As a result, fetal growth restriction, placental abruption, fetal disfunction, and fetal death can occur.

[0066] In the case of patients with pregnancy-induced hypertension, administration of the drug of the present invention is expected to suppress the rejection of fetal components, trophoblasts (a type of immune disorder suppression), and promote the reconstruction of endometrial spiral arteries into larger spiral arteries, thereby promoting the construction of a good placenta.

[0067] =Age of the person to whom the drug is to be administered= The age of the subject of administration may be any age that allows pregnancy. In the case of a human subject, for example, the subject is preferably 16 to 50 years old, more preferably 16 to 45 years old, and even more preferably 16 to 40 years old. If the subject is 16 years old or older, the consent of the subject can be obtained. In addition, in the case of an infertility patient, if the subject is 40 years old or younger, the possibility of pregnancy is higher, so that the effect of the drug according to the present invention can be obtained more efficiently. The age of the subject of administration is not limited to this, but for example, it is 30 to 45 years old, 33 to 43 years old, 33 to 41 years old, or 33 to 39 years old.

[0068] =A more preferable example of a subject to which the drug is administered= The subjects to which the above-mentioned drugs are administered preferably have no diseases, disorders or conditions other than the above-mentioned poor pregnancy condition, and more preferably have no diseases, disorders or abnormalities related to the uterus, such as submucosal myoma, endometrial polyps, uterine cavity adhesions, congenital abnormalities in the uterus, and hydrofallopian tubes. In addition, it is preferable that the subjects do not have diseases, disorders or conditions that are at high risk of being seriously affected by the drugs of the present invention. Examples of such diseases include chronic infections.

[0069] = Drug administration target and Th1 / Th2 cell ratio = In one embodiment, the Th1 / Th2 cell ratio in a subject to which the above-mentioned drug is administered may be determined and used to determine whether or not the drug should be administered to the subject and the dosage of the drug (see also the Examples for details of this process). For example, the Th1 / Th2 cell ratio in a subject to which the drug is administered is elevated compared to a healthy subject. In a more specific example, a subject in which the Th1 / Th2 cell ratio is elevated by 10% to 800% compared to a healthy subject may be a preferred subject to which the drug of the present invention is administered, a subject in which the Th1 / Th2 cell ratio is elevated by 10% to 400% may be a more preferred subject to which the drug of the present invention is administered, and a subject in which the Th1 / Th2 cell ratio is elevated by 10% to 200% may be a further preferred subject to which the drug of the present invention is administered. As for specific numerical values ​​of the Th1 / Th2 cell ratio, a subject in which the Th1 / Th2 cell ratio is 10 to 50 may be a preferred subject to which the drug of the present invention is administered, a subject in which the Th1 / Th2 cell ratio is 10 to 40 may be a more preferred subject to which the drug of the present invention is administered, and a subject in which the Th1 / Th2 cell ratio is 10 to 30 may be a further preferred subject to which the drug of the present invention is administered. In the examples described later, the average Th1 / Th2 cell ratio in healthy subjects was about 7-8, while the highest Th1 / Th2 cell ratio in the treatment group was about 30-40.

[0070] Here, a healthy individual is an individual who is not accompanied by a pregnancy defect that is the object of improvement. For example, when the object is to improve infertility, it refers to an individual who is at least not accompanied by infertility, and when the object is to improve infertility, it refers to an individual who is at least not accompanied by infertility. A healthy individual preferably refers to an individual who is not accompanied by a pregnancy defect that is the object of improvement, and does not develop other known diseases or illnesses (although there may be pregnancy defects other than the object of improvement). More preferably, a healthy individual refers to an individual who is not accompanied by any of the above-mentioned pregnancy defects, and even more preferably, it refers to an individual who is not accompanied by any of the above-mentioned pregnancy defects, and does not develop other known diseases or illnesses.

[0071] The determination of the Th1 / Th2 cell ratio is not particularly limited, but is preferably performed between immediately before (the day, the day before, or 2 days before) and 6 months before the scheduled date of administration of the drug, more preferably immediately before to 3 months before the scheduled date of administration of the drug, even more preferably immediately before to 2 months before the scheduled date of administration of the drug, and particularly preferably immediately before to 1 month before the scheduled date of administration of the drug. In a more specific example, when the subject of administration of the drug undergoes treatment such as IVF-ET or ICSI, it is preferable to determine the Th1 / Th2 cell ratio in the menstrual cycle immediately before, 2, 3, 4, 5, or 6 times before the treatment, more preferably to determine the Th1 / Th2 cell ratio in the menstrual cycle immediately before or 2 times before, and even more preferably to determine the Th1 / Th2 cell ratio in the menstrual cycle immediately before. In addition, considering the influence of the menstrual cycle on the immune state, it is preferable to determine the Th1 / Th2 cell ratio in a healthy person and a subject of administration of the drug at the same timing in the menstrual cycle. Furthermore, it is preferable to determine the Th1 / Th2 cell ratio during the hypothermic period of the menstrual cycle (from menstruation to ovulation).

[0072] More specifically, but not limited to, for example, the Th1 / Th2 cell ratio in a subject with infertility can be determined at least 1 or 2 days to 180 days before implantation of a fertilized egg or transplanted embryo, more preferably 1 or 2 days to 60 days before, and even more preferably 2 days to 30 days before, before the planned date of administration of the drug. More specifically, but not limited to, for example, the Th1 / Th2 cell ratio in a subject who may have a poor pregnancy condition other than infertility (such as miscarriage) can be determined preferably 0 to 60 days, more preferably 0 to 30 days, even more preferably 0 to 20 days or 0 to 15 days, and particularly preferably 0 to 10 days after confirmation of pregnancy, before the planned date of administration of the drug.

[0073] The Th1 / Th2 cell ratio may be a value in any tissue in the body of a subject to which an agent is administered, but is preferably a value in peripheral blood.

[0074] =A specific example of a person to whom the drug is to be administered= <Infertility> An example of a particularly preferred patient is a patient who meets the following criteria 1) to 2), more preferably 3), and even more preferably 4) to 6). 1) Patients with severe infertility with no clear cause who have undergone four or more embryo transfers of fertilized eggs (regardless of whether they are fresh, frozen, early embryos, or blastocysts) that have been confirmed to be of good morphology but have not achieved a scientific pregnancy (increase in hCG levels). 2) Patients under 40 years of age. 3) Subjects undergo immunological testing (screening) during the hypothermic period before pregnancy when they are in good health (from menstruation to ovulation) and are found to have a high Th1 / Th2 cell ratio. 4) Patients without active infection. 5) Patients without persistent HBV, HCV, or HIV infection. 6) Patients with no history of hypersensitivity to any component of tacrolimus.

[0075] <Infertility> An example of a particularly preferred patient is a patient who meets the following criteria 1) or 2) (including patients with unexplained recurrent pregnancy loss), and it is more preferable that the patient also meets criteria 3) to 5). 1) Patients with severe recurrent pregnancy loss who do not show obvious abnormal findings related to recurrent pregnancy loss in serological tests. 2) Patients with treatment-resistant habitual miscarriage (including antiphospholipid syndrome). 3) Patients without active infection. 4) Patients who do not have persistent HBV, HCV, or HIV infection. 5) Patients with no history of hypersensitivity to any component of tacrolimus.

[0076] <General> Although not particularly limited, patients who are continuously receiving tacrolimus or a derivative thereof for medical purposes other than improving the condition of pregnancy (e.g., patients who have undergone organ transplantation and are continuously receiving tacrolimus) may be excluded from the subjects of administration of the drug of the present invention.

[0077] (Route of administration / method of administration) The administration method (administration route) of the drug of the present invention can be appropriately determined depending on the age, condition, and treatment period of the subject. Specifically, either oral administration or parenteral administration may be used, but oral administration is preferred (oral administration is shown in the example). Examples of parenteral administration include injection administration, administration as a suppository, and administration as a transdermal absorption type preparation. Examples of types of injection administration include intramuscular, intraperitoneal, subcutaneous, intravenous, and local injection. In addition, the drug of the present invention can be administered via various routes such as transdermal, nasal, vaginal, and rectal.

[0078] (Dosage) The dosage of the drug varies depending on the type and severity of the disease, disorder or condition of the patient receiving the drug, various test results, and the type of active ingredient of the drug. Furthermore, the dosage of the drug also varies depending on the age of the patient to be treated, the number of times the treatment is performed by the treatment method of the present invention, and various test results. As an example, the drug of the present invention is administered at a dosage that is lower than the dosage when used as an immunosuppressant in the treatment of living body transplantation and immune system diseases in terms of the content of the active ingredient contained in the drug. For example, when the subject of administration of the drug is a human, the amount of the active ingredient is preferably within the range of 0.5 to 5 mg or 1 to 5 mg, more preferably within the range of 0.5 to 4.5 mg or 1 to 4.5 mg, even more preferably within the range of 0.5 to 4 mg or 1 to 4 mg, even more preferably within the range of 0.5 to 3.5 mg or 1 to 3.5 mg, even more preferably within the range of 0.5 to 3 mg or 1 to 3 mg, and most preferably within the range of 1 to 2 mg per day. In the following, unless otherwise specified, descriptions regarding drug dosages apply to human subjects, and the dosages are expressed as the amount of active ingredient.

[0079] Although not particularly limited, in the case of oral administration, the number of administrations per day is preferably 1 to 4 times, more preferably 1 to 3 times, and even more preferably 1 to 2 times.

[0080] If necessary, the above-mentioned Th1 / Th2 cell ratio (see also Examples) may be determined in advance, and the dosage may be determined based on the numerical value of the ratio. In addition, in the case of continuous administration for a certain period or more during pregnancy and after childbirth, including treatment of infertility, the Th1 / Th2 cell ratio may be measured at regular intervals during the treatment period, and the dosage may be determined based on the result of each new measurement.

[0081] As an example of determining the dosage based on the numerical value of the Th1 / Th2 cell ratio, when the Th1 / Th2 cell ratio is 10 or more, the amount of the active ingredient is preferably within the range of 0.5 to 5 mg or 1 to 5 mg, more preferably within the range of 0.5 to 4.5 mg or 1 to 4.5 mg, even more preferably within the range of 0.5 to 4 mg or 1 to 4 mg, even more preferably within the range of 0.5 to 3.5 mg or 1 to 3.5 mg, even more preferably within the range of 0.5 to 3 mg or 1 to 3 mg, and most preferably within the range of 1 to 2 mg per day. As another example, when the Th1 / Th2 cell ratio is within the range of 10 or more and 13 or less, the amount of the active ingredient is preferably within the range of 0.5 to 3 mg or 1 to 3 mg, more preferably within the range of 0.5 to 2 mg or 1 to 2 mg, and even more preferably within the range of 0.5 to 1 mg per day. As another example, when the Th1 / Th2 cell ratio is within the range of more than 13 and not more than 16 (e.g., not more than 15.8), the amount of the active ingredient is preferably within the range of 0.5 to 4 mg or 1 to 4 mg, more preferably within the range of 0.5 to 3.5 mg or 1 to 3.5 mg, and even more preferably within the range of 1 to 3 mg or 1.5 to 2.5 mg per day. As another example, when the Th1 / Th2 cell ratio is 15 or more (e.g., when it exceeds 15.8), the amount of the active ingredient is preferably within the range of 0.5 to 5 mg or 1 to 5 mg, more preferably within the range of 0.5 to 3.5 mg or 1 to 3.5 mg, and even more preferably within the range of 2 to 3.5 mg or 2.5 to 3.5 mg per day.

[0082] The dosage of the drug can also be increased according to the number of times the improvement method according to the present invention is performed (number of pregnancies). For example, in the treatment of infertility (one embodiment of improvement), the daily dosage is increased with each treatment from the dosage in the previous treatment, preferably by 0.5 to 3 mg (amount of active ingredient; the same applies below), more preferably by 0.5 to 2 mg, and even more preferably by 0.5 to 1 mg. In other words, the daily dosage of the drug administered from the start to the end of the first treatment period is increased by 0.5 to 3 mg in the second treatment period following the first treatment period. This is repeated for each single treatment period until pregnancy is established.

[0083] In the treatment of infertility (one embodiment of improvement), the number of treatments allowed is preferably 2 to 5 times, more preferably 2 to 4 times. In the repeated treatment, it is preferable that the daily dosage at the end of the final treatment period does not exceed the dosage preferred in the treatment method of the present invention (up to 5 mg of active ingredient per day).

[0084] In addition, in the treatment of infertility (one embodiment of improvement), the Th1 / Th2 cell ratio may be measured at regular intervals during pregnancy, and each time the Th1 / Th2 cell ratio increases, the daily dosage may be increased by preferably 0.5 to 3 mg (amount of active ingredient; the same applies below), more preferably 0.5 to 2 mg, and even more preferably 0.5 to 1 mg.

[0085] The above dosage amounts are applicable to women in general, but when the subject is a woman whose body weight is significantly different from the standard weight (for example, a woman weighing less than 45 kg or more than 75 kg), the above dosage amounts are divided by 60 (kg) to determine the dosage per kg of body weight, and the actual dosage amount may be adjusted according to the subject's actual body weight.

[0086] In addition, when a drug containing rapamycin or a derivative thereof as an active ingredient is used, an appropriate dosage can be set according to the type of the active ingredient, and as an example, in addition to applying the above-mentioned dosage, a dosage of 2 / 3 of the above-mentioned dosage can be adopted. When a drug containing cyclosporine or a derivative thereof as an active ingredient is used, an appropriate dosage can be set according to the type of the active ingredient, and as an example, a dosage of 1000 mg or less, preferably 60 mg or less per day of the active ingredient can be adopted.

[0087] (Time and duration of drug administration) The timing and duration of administration of the agent of the present invention varies depending on the disease, disorder, or condition to be improved. When the human subject is infertility, the agent is preferably administered for a period from at least 1 or 2 days to 60 days, more preferably 2 to 30 days, and even more preferably 2 to 15 days, before implantation of the fertilized egg or transplanted embryo into the endometrium, to preferably 0 to 100 days, more preferably 0 to 15 days (0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days) after implantation. When the subject is a patient undergoing artificial insemination or in vitro fertilization treatment, the treatment is performed over a period of preferably 1 or 2 days to 60 days, more preferably 2 to 30 days, and even more preferably 2 to 15 days, prior to sperm injection or embryo transfer, and preferably 0 to 100 days, more preferably 0 to 15 days after (0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after) sperm injection or embryo transfer.

[0088] Furthermore, when the human subject is experiencing infertility, it is preferable for the compound to be administered continuously, preferably from the day the establishment of pregnancy is confirmed until 200 days later, more preferably from the day the establishment of pregnancy is confirmed until 300 days later, and even more preferably from the day the establishment of pregnancy is confirmed until delivery.

[0089] Furthermore, when the human subject is at risk of experiencing pregnancy complications (such as pregnancy-induced hypertension), it is preferable for the drug to be administered continuously, preferably from the day the establishment of pregnancy is confirmed until 200 days later, more preferably from the day the establishment of pregnancy is confirmed until 300 days later, even more preferably from the day the establishment of pregnancy is confirmed until delivery, and most preferably from the day the establishment of pregnancy is confirmed until 60 days after delivery.

[0090] The dosage can also be changed appropriately at each stage of early, middle and late pregnancy. For example, the dosage of the drug per day can be gradually reduced by 0.5 to 1 mg each time the pregnancy progresses from early, middle and late pregnancy. Alternatively, the drug can be administered only in the early pregnancy and discontinued from the middle pregnancy onwards, or the drug can be administered until the middle pregnancy but discontinued thereafter, or the drug can be administered only in the early and late pregnancy and discontinued from the middle pregnancy onwards.

[0091] Furthermore, the duration of successive administration may be adjusted depending on whether the pregnancy status test results during each period of pregnancy are good or not. After 100 days from the establishment of pregnancy, the mother enters the stable pregnancy period. Therefore, by reducing the drug dosage as much as possible while still achieving sufficient therapeutic effect, the side effects of the drug on the mother and fetus can be minimized, and the physical and physical burden on the subject can be minimized.

[0092] 2. How to improve your pregnancy A method for improving a pregnancy condition using the drug of the present invention (one embodiment of which is a method for treating a poor pregnancy condition) is also within the scope of the present invention. Hereinafter, the details of the method for improving a pregnancy condition of the present invention will be described.

[0093] In one embodiment, the method for improving a pregnancy condition of the present invention includes a step of administering the drug of the present invention to a subject (administration step). Improving a pregnancy condition means improving a poor pregnancy condition to a good or normal condition, and is a concept that also includes preventing a pregnancy condition from becoming poor. Specific examples of a poor pregnancy condition include pregnancy complications such as infertility, miscarriage, and pregnancy-induced hypertension syndrome. Diseases, disorders, and conditions included in the concept of a poor pregnancy condition are as described above in the section (Subject to which the drug is administered) of [1. Drug (pharmaceutical composition)]. In addition, the drug to be administered contains a compound represented by the above general formula (I) or a pharmaceutical acceptable salt thereof as an active ingredient, or contains cyclosporine, rapamycin, or a derivative thereof (including pharmaceutical acceptable salts thereof, etc.) as an active ingredient. The drug according to the present invention also includes those described above in the section (1. Drug (pharmaceutical composition)]. Furthermore, the subject to which the treatment method of the present invention is applied is as described above in the section (Subject to which the drug is administered) of [1. Drug (pharmaceutical composition)].

[0094] In a further embodiment, the treatment method of the present invention may further include, in addition to the administration step, 1) a step of measuring the Th1 / Th2 cell ratio, and / or 2) a step of performing artificial insemination (injection of sperm into the uterus) or transplanting a fertilized egg or embryo obtained by in vitro fertilization into the endometrium. Each step will be described in more detail below.

[0095] (Administration Step) The administration step is a step of administering the agent according to the present invention to a subject.

[0096] When the method of the present invention is a method (including a treatment method) for improving infertility in a human subject, the drug administration period in the administration step is preferably from 1 or 2 days to 60 days, more preferably 2 to 30 days, and even more preferably 2 to 15 days before implantation of a fertilized egg or a transplanted embryo into the endometrium, to preferably 0 to 100 days, and more preferably 0 to 15 days after implantation. When the human subject is a patient who has undergone artificial insemination or in vitro fertilization treatment, the drug administration is preferably from 1 or 2 days to 60 days, more preferably 2 to 30 days, and even more preferably 2 to 15 days before sperm injection or embryo transfer, to preferably 0 to 100 days, and more preferably 0 to 15 days after sperm injection or embryo transfer.

[0097] When the method of the present invention is a method (including a treatment method) for improving infertility in a human subject, the drug administration period in the administration step is preferably from the day the establishment of pregnancy is confirmed to 200 days thereafter, more preferably from the day the establishment of pregnancy is confirmed to 300 days thereafter, and even more preferably from the day the establishment of pregnancy is confirmed to delivery.

[0098] When the method of the present invention is a method for improving (including preventing) pregnancy complications such as pregnancy-induced hypertension in a human subject, the drug administration period in the administration step is preferably from the day the establishment of pregnancy is confirmed to 200 days thereafter, more preferably from the day the establishment of pregnancy is confirmed to 300 days thereafter, even more preferably from the day the establishment of pregnancy is confirmed to delivery, and most preferably from the day the establishment of pregnancy is confirmed to 60 days after delivery.

[0099] Administration during the above period may be continuous (i.e., daily administration during the period) or intermittent (i.e., administration may be absent during the period), and the details are as described in (Timing and Period of Administration of Drugs) under [1. Drugs (Pharmaceutical Compositions)]. Specific examples of intermittent administration include administration at regular intervals of a specified number of days (1 day, 2 days, 3 days), administration with one or more predetermined suspension periods consisting of multiple days, and the like.

[0100] The dosage is as described in the (Dosage) section of [1. Drugs (pharmaceutical compositions)].

[0101] (Step of measuring Th1 / Th2 cell ratio) In a further embodiment, the method of the present invention may further include a step of measuring the Th1 / Th2 cell ratio. The determined Th1 / Th2 cell ratio may be used to determine whether or not a drug should be administered to the subject and the dosage of the drug, as described in the section [1. Drug (pharmaceutical composition)]. The step of measuring the Th1 / Th2 cell ratio is carried out before the administration step. Here, "before the administration step" refers to the period between administrations in one administration period, but also refers to the period before the first administration in one administration period (treatment period).

[0102] When the method of the present invention is a method for improving infertility, the step of measuring the Th1 / Th2 cell ratio can be carried out, for example, preferably between 1 or 2 days and 180 days, more preferably between 1 or 2 days and 60 days, and even more preferably between 2 days and 30 days before implantation of the fertilized egg or transplanted embryo, and can be carried out before the planned date of administration of the drug.

[0103] When the treatment method of the present invention is a treatment method for improving poor pregnancy conditions other than infertility, including pregnancy complications such as infertility and pregnancy-induced hypertension, the step of measuring the Th1 / Th2 cell ratio can be carried out preferably from 0 to 60 days, more preferably from 0 to 30 days, even more preferably from 0 to 20 days or from 0 to 15 days, and particularly preferably from 0 to 10 days after confirmation of pregnancy, and can be carried out before the planned date of administration of the drug.

[0104] (The process of performing artificial insemination or implanting a fertilized egg or embryo obtained by in vitro fertilization into the uterine lining) In applying the method for improving a pregnancy condition using the drug of the present invention, the subject (including a human subject) may be undergoing artificial insemination (injection of sperm into the uterus) or a treatment of transplanting a fertilized egg or embryo obtained by in vitro fertilization into the endometrium. A particularly preferred embodiment of the present invention is to improve a pregnancy condition related to an implanted fertilized egg or transplanted embryo by undergoing such treatment. Specific methods of in vitro fertilization include IVF-ET and ICSI. IVF-ET further includes fresh embryo transfer (fresh ET) in which a fresh embryo is used for transplantation, and frozen-thawed embryo transfer (FET) in which an embryo frozen and stored after collection is frozen and thawed for transplantation, and among these, FET is preferred. By administering the drug of the present invention, the efficiency of pregnancy establishment and birth of subjects who have undergone such treatment can be improved, and further, the onset of pregnancy complications can be suppressed.

[0105] However, pregnancy may be established naturally in a subject with infertility or infertility, and improving the pregnancy condition in a natural pregnancy is also within the scope of the present invention.

[0106] 4. Other Aspects The present invention also encompasses within its scope the use of at least one compound selected from the group consisting of compound (I), rapamycin or a derivative thereof, cyclosporine or a derivative thereof, and pharma- ceutical acceptable salts thereof in the manufacture of a medicament for improving a pregnancy condition.

[0107] [5. Summary] That is, the scope of the present invention includes the following embodiment examples. 1) A drug for improving a pregnancy condition, comprising a compound represented by the above general formula (I) (compound (I)) or a pharma- ceutical acceptable salt thereof as an active ingredient. 2) The agent according to 1), wherein the compound is tacrolimus or a pharma- ceutically acceptable salt thereof. 3) A drug according to 1) or 2), which improves poor pregnancy conditions caused by immune abnormalities. 4) The drug according to any one of 1) to 3), which improves at least one adverse pregnancy condition selected from the group consisting of infertility, miscarriage, and pregnancy-induced hypertension. 5) The drug according to 4), wherein the infertility and miscarriage are accompanied by an autoimmune disease. 6) The drug according to 5), wherein the autoimmune disease is antiphospholipid syndrome. 7) The drug according to any one of 1) to 6), wherein the amount of the active ingredient is 5 mg or less per day administered to the subject of treatment. 8) The drug according to 7), wherein the amount of the active ingredient is within the range of 1 mg or more and 3 mg or less per day. 9) The agent according to any one of 1) to 8), which is administered to a subject having an elevated Th1 / Th2 cell ratio compared to a healthy subject. 10) The drug described in any one of 1) to 9), which is administered to a subject undergoing transplantation of an embryo obtained by in vitro fertilization. 11) A drug described in any of 1) to 9), which is administered during the period from at least 1 or 2 days prior to implantation of a fertilized egg or transplanted embryo into the endometrium to day 0 after implantation when targeting infertility as an abnormality in pregnancy, and which is administered during the period from the establishment of pregnancy to at least day 200 when targeting an abnormality in pregnancy other than infertility. 12) A method for improving a condition of pregnancy, comprising the step of administering a drug for improving a condition of pregnancy, which contains the above-mentioned compound (I) or a pharma- ceutically acceptable salt thereof as an active ingredient. 13) The method according to 12), wherein the drug is administered in any of the forms or modes described in 2) to 11) above. EXAMPLES

[0108] Example 1 <Problems of this embodiment> The inventors evaluated the effect of immunosuppressive treatment with tacrolimus in patients with recurrent implantation failure (RIF) and a high ratio of T helper (Th)1 / Th2 cytokine-producing cells (hereinafter also referred to as "Th1 / Th2 cell ratio").

[0109] <Research methods used in this example> This was a prospective cohort study of treatment in RIF patients (n=42) with a high peripheral blood Th1 (CD4+ / IFN-γ+) / Th2 (CD4+ / IL-4+) cell ratio at Sugiyama Clinic between November 2011 and October 2013. Twenty-five patients were treated with tacrolimus (treatment group) and 17 patients were not treated (control group).

[0110] The treatment group received tacrolimus for a total of 16 days, starting 2 days before embryo transfer (ET) and ending the day of pregnancy test. The daily dose of tacrolimus (1-3 mg) was determined based on the Th1 / Th2 cell ratio.

[0111] <Result> The medical pregnancy rate in the treatment group was 64.0%, significantly higher than that in the control group (0%) (P<0.0001). In the treatment group, the miscarriage rate was 6.3%, and the live birth rate was 60.0% (P<0.0001). No serious side effects from tacrolimus were observed in the treatment group. None of the patients experienced any obstetric complications during pregnancy.

[0112] <Conclusion> Immunosuppressant treatment with tacrolimus improved reproductive outcomes in women with recurrent implantation failure and a high peripheral blood Th1 / Th2 cell ratio.

[0113] (Introduction) In recent years, the incidence of in vitro fertilization (IVF) and embryo transfer (ET) has increased worldwide. Accordingly, the number of women experiencing multiple IVF failures, including recurrent implantation failure, is increasing. During IVF / ET, an embryo is transferred into the uterine cavity between 2 and 5 days after fertilization. Pregnancy is established when the embryo, a semi-allograft, successfully implants in the maternal decidua, accompanied by the establishment of maternal immune tolerance (Ref. 2). The establishment of an appropriate immune response at the time of implantation is key to successful implantation. Thus, immune etiology appears to play an important role in RIF after IVF / ET.

[0114] T helper (Th)1 / Th2 cells play an important role in immune responses, such as immune rejection and immune tolerance (Ref. 3). It is generally agreed that pregnancy is associated with Th2 dominance, whereas Th1 immune responses are associated with embryo rejection (Ref. 4, 5). The mechanisms underlying embryo rejection are thought to be similar to the rejection reaction in allogeneic transplantation (Ref. 6). Embryos transferred during IVF / ET may fail to implant due to immune responses similar to the rejection reaction in allogeneic transplantation.

[0115] In the past few decades, graft survival rates have improved dramatically (Ref. 7). This success is largely attributable to the development of novel immunosuppressants. Tacrolimus (Prograf, Astellas Pharma, Tokyo) is one of the major immunosuppressants used after allogeneic organ transplantation to reduce the risk of rejection of the transplanted organ by reducing the allogeneic responsiveness of the immune system of the transplant recipient (Ref. 8). Tacrolimus has been reported to suppress immune rejection of allografts and promote their survival by suppressing alloantigen-induced lymphocyte proliferation, generation of cytotoxic T cells, expression of IL-2 receptors, and production of T cell-derived soluble mediators such as IL-2 and IFN-γ (Ref. 9). Tacrolimus has also been reported to effectively control graft-versus-host disease and other immune disorders such as rheumatoid arthritis, albeit conditions involving different T lymphocyte subsets (Refs. 10, 11). Women with RIF have an enhanced Th1 immune response with an elevated Th1 / Th2 cell ratio in peripheral blood (Reference 4). Therefore, immunosuppressants such as tacrolimus may improve implantation rates and pregnancy outcomes in women with a history of RIF after ART cycles, especially those with an enhanced Th1 immune response. In this study, we investigated the therapeutic effect of tacrolimus on women with RIF and an elevated Th1 / Th2 cell ratio in peripheral blood.

[0116] Materials and Methods (Research Subjects) A total of 81 patients with a history of ≥5 RIF after IVF / ET cycles at the Department of Reproductive Medicine of Sugiyama Clinic (Tokyo, Japan) were consecutively enrolled in this study between November 2011 and February 2014. The study was approved by the Institutional Review Board of Sugiyama Clinic. Signed informed consent was obtained from all patients prior to participating in this study.

[0117] Patients were selected based on their ability to undergo IVF / ET and a history of at least five failed IVF / ET cycles in which embryos of good morphological and developmental quality were transferred into a properly prepared endometrium (endometrial thickness ≥8 mm). Patients were evaluated by transvaginal ultrasound, tubal patency, and hysteroscopy before the index ART cycle. None of the participants had submucosal fibroids, endometrial polyps, uterine adhesions, congenital uterine anomalies, or tubal hydrops. Furthermore, none of the participants had a history of autoimmune disease. Women who were pregnant or had chronic medical or inflammatory conditions were excluded. Women who had a miscarriage in a previous IVF cycle, had undergone an IVF cycle, or had been vaccinated within the past 3 months were excluded. Women with acquired or congenital thrombophilia were excluded.

[0118] To assess baseline values ​​of Th1 / Th2 cell ratio, blood was collected between cycle day (CD) 5 and 10 before entering the index ART cycle. 42 patients had elevated Th1 / Th2 cell ratio (≥10.3) and 39 patients had normal Th1 / Th2 cell ratio (<10.3). The range of normal Th1 / Th2 cell ratio was established using 28 women with a history of normal births, either naturally conceived or by artificial insemination using husband's sperm. Blood was collected between CD5 (CD 5) and CD10 of the menstrual cycle. The level of Th1 / Th2 cell ratio was determined by the mean plus one standard deviation method, and a Th1 / Th2 cell ratio equal to or exceeding 10.3 was classified as elevated Th1 / Th2 cell ratio.

[0119] (Analysis of Th1 and Th2 cells) A total of 10 ml of venous blood was collected to assess baseline Th1 / Th2 cell ratios, which were determined by detecting intracellular interferon (IFN)-γ and IL-4 production.

[0120] Specific staining of lymphocytes was performed by incubating whole blood with anti-CD4-PC5 or anti-CD8-PC5-conjugated monoclonal antibodies (mAbs) (Beckman Coulter, Fullerton, Ca, USA). Red blood cells (RBCs) were removed by hemolysis (using FACS Lysing solution; Becton Dickinson, BD 134 Biosciences, Franklin Lake. NJ, USA) and lymphocytes were analyzed using flow cytometry (FACSCalibur; Becton Dickinson). Activated whole blood samples were surface stained with anti-CD4-PC5-conjugated mAbs, followed by hemolysis of RBCs and specific intracellular staining with FastImmune™ IFN-γ-FITC / IL-4-PE (Becton Dickinson) according to the manufacturer's instructions. Th1 cells are CD4+ cells with intracellular IFN-γ but without intracellular IL-4. + Th2 cells are defined as CD4+ lymphocytes with intracellular IL-4 but without intracellular IFN-γ. + The ratio of intracellular IFN-γ positive Th cells to intracellular IL-4 positive Th cells was expressed as the Th1 / Th2 cell ratio.

[0121] (Treatment with Tacrolimus (Prograf®)) Of the 42 patients with an elevated Th1 / Th2 cytokine ratio (≥10.3) during the index IVF / ET cycle, 25 were treated with tacrolimus (Prograf®, Astellas Pharma, Tokyo, Japan) (treatment group), and the remaining (n=17) were not treated (control group). Patients in the treatment group received tacrolimus starting 2 days before ET and continuing for a total of 16 days until pregnancy test. The daily dose of tacrolimus ranged from 1 to 3 mg / day depending on the degree of elevation of the Th1 / Th2 cell ratio. That is, patients (n=12) with a mildly elevated Th1 / Th2 cell ratio (≥10.3 and <13.0) were treated with 1 mg tacrolimus / day. Patients (n=8) with a mildly elevated Th1 / Th2 cell ratio (>13.0 and <15.8) were treated with 2 mg tacrolimus / day. Patients (n=5) with highly elevated Th1 / Th2 cell ratios (>15.8) were treated with tacrolimus at 3 mg per day.

[0122] (IVF-ET treatment) Ovarian stimulation and oocyte retrieval (OPU) were performed routinely as previously reported by the inventors (Reference 12). Our mild stimulation protocol was as follows: the patient received 50 mg of clomiphene citrate (Serophen®, Merck Serono, Tokyo) per day for 5 days between days 3 and 7 of the menstrual cycle. The patient also received 225 international units (IU) of rec-FSH (Gonal-F®, Merck Serono, Tokyo,) on days 4, 6, and 8 of the menstrual cycle. On day 10 of the menstrual cycle, the dominant follicle was stimulated with 100 mg of clomiphene citrate (Serophen®, Merck Serono, Tokyo, Tokyo). When the follicles reached a diameter of ≥17 mm, 10,000 IU of human chorionic gonadotropin (hCG; Gonadotropin, Mochida, Tokyo) was injected or 300 μg of buserelin acetate (Buserecur, Fuji Pharma, Tokyo) was administered intranasally, and OPU was performed 35 h later. Additional rec-FSH (150 IU per day) was administered as needed based on follicular growth. Depending on semen parameters, intercytoplasmic sperm injection (ICSI) or conventional co-culture techniques were used. Embryos were placed into the patient's uterus via the cervix using a flexible catheter (Kitazato ET catheter, Kitazato Supply, Shizuoka, Japan). In all patients, one or two embryos were transferred. Fresh ET was performed on day 3. The remaining embryos were cryopreserved by vitrification (Ref. 13). On the day of ET and 5 days after ET, 125 mg of hydroxyprogesterone caproate (Progestone Depot-S, Fuji Pharma, Tokyo) was injected for luteal support. 14 days after ET, chlormadinone acetate (Lutoral®, Shionogi, Osaka) was orally administered. For freeze-thaw ET (FET) cycles, either natural ovulation cycles or hormone replacement cycles were used for endometrial conditioning. When natural ovulation cycles were used, the day of ET was the 3rd day after ovulation. The same progesterone was supplemented as in the fresh ET cycle. When hormone replacement cycles (HRC) were used, endometrial conditioning for ET was performed with conjugated estrogen (Premarin 0.625 mg, Wyeth, Tokyo, Japan) and transdermal estradiol (Estrana TAPE 0.72 mg, Hisamitsu Pharmaceutical, Tokyo, Japan). These treatments were performed from day 3 or day 1 of the menstrual cycle without bleeding until the day of the urine pregnancy test. Progesterone (100 mg / oil; Progestone Depot-S, Fuji Pharmaceutical, Tokyo, Japan) was administered starting from day 12 of the menstrual cycle.Three days after initiating progesterone treatment, the embryos were thawed and surviving embryos were transferred (Reference 14).

[0123] All patients underwent transfer of one or two morphologically good quality embryos (MGEs) 3 days after oocyte retrieval. For frozen embryo cycles, 3-day-old frozen-thawed embryos were transferred 3 days after ovulation in natural ovulation cycles or 3 days after progesterone administration in HRC. MGEs were defined as having 7 or more blastomeres and less than 10% incidence of fragmentation 3 days after oocyte retrieval (Ref. 13). Pregnancy testing was performed 14 days after ET. Clinical pregnancy was defined as development of the embryonic sac by transvaginal ultrasound 21 days after ET. Pregnancy was considered ongoing when a normal fetus was observed by transvaginal ultrasound at the 12th week of gestation. The primary and secondary outcomes of this study were clinical pregnancy and live birth, respectively.

[0124] (statistical analysis) Statistical analyses were performed using StatView, version 5 (SAS Institute Inc., Cary, NC). Continuous variables were analyzed by Wilcoxon signed-rank test. Categorical variables were analyzed by chi-square analysis or as suggested by Fisher's exact test. Differences were considered statistically different when the probability was less than 0.05.

[0125] <Result> (Research group) Age, parity, and infertility history did not differ between the treatment and control groups (Table 1). The mean age of the treatment group was 36.2 ± 2.5 (mean ± SD), which was similar to that of the control group (36.1 ± 4.1). ART treatment indicators in both groups were comparable. Approximately one-quarter of patients in the treatment group had a history of a positive pregnancy test but no neonatal birth. A similar trend was observed in the control group. The number of previous ET attempts, number of embryos transferred, and number of MGEs transferred in the treatment group, expressed as means and standard deviations (SD), were 5.8 ± 2.9, 8.6 ± 5.8, and 5.0 ± 2.1, respectively, which were also not significantly different from the control group (5.9 ± 2.7, 7.4 ± 3.4, and 4.9 ± 2.0, respectively).

[0126] (Th1 / Th2 cell ratio) The results of the analysis of Th1 and Th2 cells are shown in Table 1. The percentage of IFN-γ-producing CD4+ T lymphocytes (Th1) was 27.7±9.4 and 26.7±7.3 (mean±SD) in the treatment group and the control group, respectively, with no significant difference. The percentage of IL-4-producing CD4+ T lymphocytes (Th2) was 1.8±0.6 and 1.7±0.6 (mean±SD) in the treatment group and the control group, respectively, with no significant difference. The Th1 / Th2 cell ratio was 16.1±7.0 in the treatment group, which was similar to that in the control group (16.7±5.2).

[0127] (Treatment results) The results of ART treatment in both groups are summarized in Table 2. The number of embryos transferred per cycle was 1.4 ± 0.5 in the treatment group and 1.4 ± 0.5 in the control group (P = NS). The percentage of MGEs was 68.9% in the treatment group, comparable to that in the control group (70.8%). No pregnancy was achieved in the control group, whereas 16 of 25 patients (64%) in the treatment group had a positive pregnancy test and progressed to clinical pregnancy with tacrolimus treatment. There were no biochemical losses, and the implantation rate was 45.7% (16 / 35). There were 12, 8, and 5 patients receiving 1 mg, 2 mg, or 3 mg of tacrolimus treatment per day, respectively (see Table 3). The clinical pregnancy rate and live birth rate in patients receiving 1 mg of tacrolimus treatment per day were 83.3% and 83.3%, respectively. Clinical pregnancy and live birth rates in patients receiving daily tacrolimus treatment doses of 2 mg or 3 mg were 50.0% and 37.5%, and 40.0% and 40.0%, respectively (Table 3).

[0128] Only one pregnant patient in the treatment group (6.3%) miscarried at 10 weeks of gestation. A total of 15 healthy babies were born with tacrolimus treatment, and the live birth rate in the treatment group was 60.0%. Information on the newborns at birth was as follows: birth weight (mean ± SD) 2995 ± 400 g, mean gestational age at birth 279.5 ± 10.6 days, APGAR scores at 1 and 5 minutes after birth were 8.3 ± 0.5 and 9.2 ± 0.5, respectively. Six babies were delivered by Caesarean section and nine were delivered via vaginal delivery (Table 4).

[0129] In patients with RIF treated with tacrolimus, 12 of 16 women (75%) who underwent frozen-thawed ET became pregnant, and 4 of 9 women (44.4%) who underwent fresh ET became pregnant. Frozen-thawed ET cycles were more likely to be successful than fresh IVF-ET cycles, but this did not reach statistical significance. Twelve of 16 women (75%) who underwent FET and 3 of 9 women (33.3%) who underwent fresh ET delivered live newborns. There was a trend toward increased success with FET cycles compared with fresh ET cycles in patients with RIF treated with tacrolimus (P<0.1).

[0130] <Discussion> Recurrent implantation failure (RIF) is often defined by the failure of good quality embryos to implant in at least three consecutive IVF attempts, with one or two high quality embryos transferred each time (Ref. 15). However, there is still no general consensus on the definition. Implantation involves the maternal immune response and a number of mediators, such as embryo- and endometrial-derived cytokines, chemokines, and various growth factors. Thus, categorizing patients solely on the basis of infertility history, such as RIF, may not be appropriate for further evaluation and treatment. A recent review of endometrial receptivity reported that there is no single biomarker specific for endometrial receptivity (Ref. 16). Although the inventors of this study measured endometrial thickness as a standard infertility test item, there is a gap on the surface for an objective diagnosis of endometrial receptivity. The same may be true for immunological factors, which in this study focused only on the Th1 / Th2 cell ratio.

[0131] There are already some reports on treatment modalities for RIF patients with elevated Th1 / Th2 ratios. Intravenous gamma globulin G (IVIG) has been reported to be effective in women with repeated IVF and implantation failure (Refs. 17-19). IVIg reduces the Th1 / Th2 lymphocyte ratio in peripheral blood, supporting the importance of Th1 / Th2 immune control in implantation and continued pregnancy (Ref. 17). Etanercept (Enbrel®), a recombinant human TNF receptor [p75]:Fc fusion protein, has been reported to be effective in RIF patients with elevated Th1 / Th2 ratios in peripheral blood (Ref. 20). A recent paper detailing the use of IVIg and adalimumab (Humira®) has shown that it resulted in improved pregnancy rates in RIF patients with elevated Th1 / Th2 ratios (Ref. 21). Adalimumab is a recombinant human IgG1 monoclonal antibody specific for TNF. However, the safety of anti-TNF drugs such as etanercept and adalimumab in infertile and pregnant women has not been established (Reference 22). As tacrolimus has accumulated pregnancy safety data from transplantation subjects, in this study, we investigated whether tacrolimus is suitable for controlling immune responses and improving birth outcomes in women who have experienced a significant number (five or more) of RIFs after IVF cycles.

[0132] Previous reports have shown that a significant proportion of RIF patients have dysregulated cellular immune effector mechanisms (Refs. 4, 23), and that women with RIF showed enhanced Th1 cytokine responses during failed ART cycles (Ref. 24). In the present study, 51.9% of women with RIF (>5) showed elevated Th1 / Th2 ratios, especially when IL-4 and IFN-γ producing T cells were examined, which was consistent with previous studies (Refs. 4, 23). The presence of Th1 immunity against semi-allogeneic embryos reflects the process of graft rejection in tissue transplantation. Immunosuppressants such as tacrolimus are often prescribed to recipients of allogeneic tissue transplants to reduce the risk of tissue rejection.

[0133] Tacrolimus inhibits both T lymphocyte signaling and IL-2 signaling (Ref. 20). In this study, a Th1 shift was detected by measuring the ratios of CD4+ / IFN-γ positive cells and CD4+ / IL-4 positive cells. It has been reported that supraphysiological doses of IFN-γ induce pre- and post-implantation failure in animal models by locally decreasing Treg and Th17 cells at the implantation site (Ref. 25). Furthermore, insulin-like growth factor binding protein 7 (IGFBP7) leads to upregulation of IFN-γ accompanied by downregulation of IL-4 and IL-10, which results in implantation failure in a mouse model (Ref. 26). Conversely, IFN-γ induces appropriate arterial modifications at the implantation site (Ref. 27). Thus, appropriate expression and regulation of IFN-γ plays an important role in successful implantation and pregnancy. Considering the dose-dependent opposing biological effects of IFN-γ, the dose of tacrolimus in this study was determined based on the Th1 / Th2 cell ratio in women with RIF. Moreover, the dose of tacrolimus in the study group was 20-60% of the dose in the prophylactic kidney transplantation schedule (0.1 mg / kg / day). All patients tolerated tacrolimus treatment, and no side effects were reported during tacrolimus treatment. However, due to the small size of the study, this observation should be interpreted with caution.

[0134] Tacrolimus has been used throughout pregnancy in women receiving allogeneic tissue transplants, and many transplant recipients give birth while taking tacrolimus (Ref. 28). Tacrolimus reduces the activity of peptidyl prolyl isomerase by binding to the immunophilin FKBP12 (FK506 binding protein) to form a new complex. This FKBP12-FK506 complex interacts with and inhibits calcineurin, thereby inhibiting both T lymphocyte signaling and IL-2 signaling (Ref. 29). Although its activity is similar to that of cyclosporine, studies have shown that in acute rejection cases, the use of tacrolimus is more effective at reducing rejection than cyclosporine (Ref. 30). Although the short-term immunosuppression in patients and the effect on graft survival appear to be similar for the two drugs, tacrolimus results in a more favorable lipid profile, which may have important long-term implications for graft survival when considering the prognostic impact of rejection (Ref. 31). Tacrolimus is classified as a Class C drug by the FDA pregnancy category. Given that RIF is not a life-threatening condition, the administration of Class C drugs to women undergoing IVF cycles should be decided upon careful benefit-risk studies. The safety of tacrolimus for the mother and fetus / baby has been established in reports of female transplant recipients who became pregnant after transplantation (Refs. 8, 32). In 236 babies born to kidney transplant recipients exposed to tacrolimus during pregnancy, there was a 2% stillbirth rate and a 2% neonatal mortality rate (Ref. 33). Other reported obstetric complications included preeclampsia in 32%, diabetes mellitus during pregnancy in 8%, hypertension in 56%, infection in 22%, and spontaneous abortion in 26%. (Ref. 33) In this study, 60% (n=15) of RIF patients with elevated Th1 / Th2 ratio became pregnant and delivered a live neonate. None had obstetric complications, and only one baby had transient tachypnea of ​​the newborn.The differences in birth outcomes between our study and the study of kidney transplant recipients may be due to differences in the initial health status of each study group, the dose of tacrolimus, and the duration of exposure to the drug. These patients were unable to conceive even after undergoing ET with 5 or more MGEs.

[0135] Tacrolimus treatment significantly improved implantation, medical pregnancy, and live birth rates in women with RIF with elevated Th1 / Th2 ratio. Our data suggest that the benefits of tacrolimus treatment during the implantation period outweigh the risks to fetal development. Evaluation of the Th1 / Th2 cell ratio may be used as a biomarker to select women with RIF who may respond to immunosuppressive treatment with tacrolimus and achieve a favorable birth outcome. In this study, patients who underwent FET (75% success rate) had a higher success rate with tacrolimus treatment than those who underwent fresh ET (33.3% success rate). In recent years, the freeze-all strategy has been rapidly adopted in IVF centers due to the expected more receptive endometrium in FET cycles, the healthier offspring of frozen-thawed embryos compared to those from fresh ET cycles, and the increasing success rate of FET, now almost equivalent to that of fresh ET (Ref. 27). According to a 2011 CDC report, the live birth rate for fresh ET cycles in women aged 35 years or younger was 40%, while the live birth rate for FET cycles was 39% (Ref. 33). In the present study, despite the older age of the study group (mean age 36 years), the success rate of the FET cycle was 75%, 1.9 times higher than the reported success rate (39%) for women aged 35 years or younger (Ref. 27). Thus, FET cycles with tacrolimus treatment are applicable to older patient groups (>35 years) with RIF. The high medical pregnancy rate in the FET cycles may have been due to the combined effect of tacrolimus on the control of Th1 immunity and a more receptive endometrium in the FET cycle. However, the present study is limited in terms of not being a randomized controlled trial and the small sample size. Furthermore, we did not evaluate the changes in the endometrium or the peripheral immune response after tacrolimus treatment. Further studies are needed regarding the effect of tacrolimus on the systematic immune response and endometrial receptivity. In conclusion, immunosuppressive treatment with tacrolimus improved pregnancy outcomes in RIF patients with an elevated Th1 / Th2 ratio.Further studies taking into account Th17 and T regulatory cells, as well as endometrial receptivity, are required in due detail. In conclusion, this study shows that tacrolimus treatment significantly improves medical pregnancy and live birth rates in RIF patients with a shifted Th1 immune response, without increasing serious maternal complications.

[0136] [Table 1] TIFF2025029039000005.tif68166

[0137] Table 1 shows the age, parity, infertility history, and clinical characteristics of women treated with tacrolimus (treatment group) and untreated women (control group) with recurrent implantation failure (5 or more times) and elevated Th1 / Th2 cell ratio. In the table, "Body mass index" refers to BMI, "Unexplained infertility" refers to infertility of unknown cause, "Male infertility" refers to male infertility, and "Tubal infertility" refers to tubal infertility. "Previous pregnancy history" refers to whether or not a previous pregnancy history was present (Yes / No), "Obstetrical history" refers to the parity history (Gravidity, Parity, SAB = spontaneous abortion), "Previous embryo transfer history" refers to the previous embryo transfer history, "Number of failed embryo transfer cycle (n)" refers to the number of failed embryo transfer cycles, and "Total number of transferred embryos (n)" refers to the total number of transferred embryos. "MGEs (n)" refers to the total number of transplanted MGEs. Th1 / Th2 ratio refers to the Th1 / Th2 cell ratio.

[0138] [Table 2]

[0139] Table 2 shows the birth outcomes for women with recurrent implantation failure (RIF) treated with tacrolimus (treatment group) and not treated with tacrolimus (control group). In the table, "Number of transferred cycles" is the number of transfer cycles, "Number of transferred embryos per cycle" is the number of embryos transferred per cycle, "Frozen-thawed embryo transfer, n" is the number of frozen-thawed embryo transfers, "Fresh embryo transfer, n" is the number of fresh embryo transfers, "Percentage of MGEs" is the percentage of MGEs (%), "Endometrial lining" is the thickness of the endometrium, "Positive hCG (%)" is the percentage of hCG positives (%), "Implantation rate (%)" is the implantation rate (%), "Biochemical pregnancy rate (%)" is the biochemical pregnancy rate (%), "Clinical pregnancy (n)" is the number of individuals who achieved clinical pregnancy, "Clinical pregnancy of fresh ET (%)" is the clinical pregnancy rate in fresh ET (%), "Clinical pregnancy rate per ET (%)" is the clinical pregnancy rate per ET (%), "Spontaneous abortion rate" is the spontaneous abortion rate (%), "Live birth "(n)" is the number of live births, "Live birth rate of fresh ET (%)" is the live birth rate (%) in fresh ET, and "Live birth rate of "Thawed ET (%)" refers to the live birth rate (%) after freeze-thawed ET, and "Live birth rate (%)" refers to the live birth rate (%).

[0140] [Table 3]

[0141] Table 3 shows the birth outcomes of women (treatment groups) with recurrent implantation failure (RIF) treated with various doses of tacrolimus. In the table, "Number of patients treated with" indicates the number of patients treated with a given dose, and "Clinical pregnancy" indicates the number of patients treated with a given dose. "Live birth (n)" refers to the number of individuals who achieved clinical pregnancy, and "Live birth (n)" refers to the number of live births. The dose of tacrolimus was 1 mg, 2 mg, or 3 mg.

[0142] [Table 4]

[0143] Table 4 shows the outcomes of delivery and newborn birth in women with multiple implantation failures (5 or more) treated with tacrolimus. In the table, "Number of newborns (n)" is the number of newborns born, "Mode of delivery" is whether the delivery mode was vaginal delivery or cesarean section, "Gestational days to delivery (days)" is gestational age, "Birth weight" is birth weight, "Height" is birth height, "Sex of newborn" is the sex of the newborn (boy or girl), "APGAR score at 1 (or 5) minutes after birth" is the APGAR score 1 minute (5 minutes) after birth, "Umbilical cord gas analysis (pH)" is the umbilical cord gas analysis (pH), "Umbilical cord length" is the umbilical cord length, "Obstetrical complications (n)" is the number of individuals who developed pregnancy complications, and "Neonatal complications (n)" is the number of individuals who developed pregnancy complications. "(n)" indicates one case of transient tachypnea of ​​the newborn (TNN).

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[0145] Example 2 Materials and Methods (Research Subjects) The study subjects were 11 women who were eligible for IVF / ET and had a history of three or more consecutive miscarriages, stillbirths, or intrauterine growth retardation, but had not been able to conceive despite one or more of the existing treatments. These patients had no obvious abnormal findings related to infertility in prior serological tests.

[0146] Patients were evaluated by transvaginal ultrasound, tubal patency tests, and hysteroscopy before the index ART cycle. None of the participants had submucosal fibroids, endometrial polyps, uterine adhesions, congenital uterine anomalies, or tubal hydrops. Pregnant women or women with chronic medical or inflammatory conditions were excluded. Women who had a miscarriage in a previous IVF cycle, had undergone an IVF cycle, or had received a vaccination within 3 months were excluded. Women with acquired or congenital thrombophilia were excluded.

[0147] (Analysis of Th1 and Th2 cells) The method described in Example 1 was followed, except that the Th1 / Th2 cell ratio in the patient was determined immediately after the establishment of pregnancy (the day the establishment of pregnancy was confirmed). The level of the Th1 / Th2 cell ratio was determined by the mean + 1 standard deviation method as in Example 1, and a Th1 / Th2 cell ratio equal to or exceeding 10.3 was classified as an elevated Th1 / Th2 cell ratio.

[0148] (Treatment with tacrolimus) All 11 patients were treated with tacrolimus in the same manner as in Example 1, except for the period of administration. The dose of tacrolimus was 1 mg per day for 4 patients with a Th1 / Th2 cell ratio of 10.3 or more and less than 13.0, 2 mg per day for 3 patients with a Th1 / Th2 cell ratio of more than 13.0 and less than 15.8, and 3 mg per day for 4 patients with a Th1 / Th2 cell ratio of 15.8 or more. The period of administration of tacrolimus in all patients was from immediately after pregnancy was confirmed by a pregnancy test and the Th1 / Th2 cell ratio was confirmed until delivery.

[0149] (IVF-ET treatment) The same procedure as in Example 1 was followed.

[0150] (statistical analysis) The same procedure as in Example 1 was followed.

[0151] <Result> The characteristics of treated patients are summarized in Table 5 .

[0152] The treatment results are summarized in Table 6. Eight of the 11 patients who received treatment delivered healthy babies at term. The one case of premature birth shown here was a patient who was resistant to all currently available treatments, as described in the background, and had a history of more than 10 miscarriages. She gave birth with tacrolimus treatment alone, without any other treatments.

[0153] [Table 5]

[0154] [Table 6]

[0155] Example 3 Materials and Methods (Research Subjects) The study subjects were women who were eligible for IVF / ET and had a history of pregnancy-induced hypertension in a previous pregnancy or had clinical signs that predisposed them to developing pregnancy-induced hypertension. Once pregnancy-induced hypertension has developed, there is a very high possibility that it will reoccur in subsequent pregnancies.

[0156] Patients were evaluated by transvaginal ultrasound, tubal patency tests, and hysteroscopy before the index ART cycle. Women who were pregnant or had chronic medical or inflammatory conditions were excluded. Women who had a miscarriage in a previous IVF cycle, had undergone an IVF cycle, or had received a vaccination within 3 months were excluded. Women with acquired or congenital thrombophilia were excluded.

[0157] (Treatment with tacrolimus) All patients were treated with tacrolimus in the same manner as in Example 2. The dosage of tacrolimus was 1 mg to 3 mg per day. The period of administration of tacrolimus was from immediately after the establishment of pregnancy was confirmed by a pregnancy test until delivery for all patients. In addition, in Cases 1 and 3 shown below, 3 mg of tacrolimus was administered daily from immediately after the establishment of pregnancy was confirmed until mid-pregnancy, and 2 mg of tacrolimus was administered daily from late pregnancy until delivery. In Case 2 shown below, 2 mg of tacrolimus was administered daily from immediately after the establishment of pregnancy was confirmed until delivery.

[0158] (IVF-ET treatment) The same procedure as in Example 2 was followed.

[0159] (statistical analysis) The same procedure as in Example 2 was followed.

[0160] <Check the results> The effectiveness of tacrolimus administration is evaluated based on whether or not pregnancy is maintained in a good condition without developing pregnancy-induced hypertension at a specific timing between the middle and late stages of pregnancy. The following are some cases where the evaluation has been roughly completed.

[0161] A case of intrauterine fetal death at 38 weeks of pregnancy due to pregnancy-induced hypertension in a previous pregnancy (Case 1) gave birth to a healthy baby at 39 weeks without an increase in blood pressure after administration of tacrolimus. In addition, a case of a twin pregnancy with recurrent pregnancy loss and positive antiphospholipid antibodies, who is highly likely to develop pregnancy-induced hypertension (Case 2), also gave birth to a healthy baby at 35 weeks without an increase in blood pressure after administration of tacrolimus (premature rupture of membranes due to coughing, premature birth of twins). Furthermore, a case of intrauterine fetal death at 32 weeks (Case 3) in a previous pregnancy in which edema appeared from the 25th week, blood pressure rose from the 28th week, and she was diagnosed with pregnancy-induced hypertension, and is now 34 weeks into her pregnancy, but is progressing smoothly with no edema or tendency for blood pressure to rise.

[0162] The growth of both babies was good throughout the pregnancy, and no abnormalities were observed in either mother or child.

[0163] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in the different embodiments and examples. Furthermore, new technical features can be formed by combining the technical means disclosed in the respective embodiments and examples.

Claims

1. A drug for improving a pregnancy condition of a human subject with a poor pregnancy condition, comprising tacrolimus or a pharma- ceutical acceptable salt thereof as an active ingredient, The human subject has a history of recurrent implantation failure (RIF), is pregnant or has a history of pregnancy-induced hypertension. An agent for improving the pregnancy condition of a human subject.

2. The method of claim 1 , wherein the human subject is experiencing infertility or has a history of pregnancy-induced hypertension.

3. The method of claim 2, wherein the improvement of the pregnancy condition is to prevent the human subject with a history of pregnancy-induced hypertension from developing pregnancy-induced hypertension.

4. The drug according to any one of claims 1 to 3, wherein the human subject is a human subject in which the Th1 / Th2 cell ratio in peripheral blood during the menstrual cycle or after pregnancy is confirmed is elevated compared to a healthy subject.

5. The method according to any one of claims 1 to 3, wherein the human subject has a Th1 / Th2 cell ratio of less than 10.3 in peripheral blood during the menstrual cycle or after confirmed pregnancy.

6. The method according to any one of claims 1 to 5, wherein the amount of the active ingredient administered to the subject of treatment is 5 mg or less per day.

7. 7. The method of claim 6, wherein the amount of the active ingredient is in the range of 1 mg to 3 mg per day.

8. The method according to any one of claims 1 to 7, which is administered to a subject undergoing the implantation of an embryo obtained by in vitro fertilization.

9. The method according to any one of claims 1 to 8, wherein the subject is a human subject, and the human subject is not currently receiving tacrolimus or a derivative thereof for any medical purpose other than improving a pregnancy condition.

10. A drug for improving the pregnancy condition of a human subject with a poor pregnancy condition, comprising tacrolimus or a pharma- ceutically acceptable salt thereof as an active ingredient.

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