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WO2019031754A1 - Ospémifène à sucre ajoute, procédé de préparation s'y rapportant et composition pharmaceutique contenant celui-ci en tant que principe actif - Google Patents

Ospémifène à sucre ajoute, procédé de préparation s'y rapportant et composition pharmaceutique contenant celui-ci en tant que principe actif Download PDF

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WO2019031754A1
WO2019031754A1 PCT/KR2018/008662 KR2018008662W WO2019031754A1 WO 2019031754 A1 WO2019031754 A1 WO 2019031754A1 KR 2018008662 W KR2018008662 W KR 2018008662W WO 2019031754 A1 WO2019031754 A1 WO 2019031754A1
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glucose
formula
ospemifene
compound
ospem
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PCT/KR2018/008662
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Korean (ko)
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박제원
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고려대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin

Definitions

  • the present invention relates to a novel ospemipene derivative compound to which a sugar is added to ospemifene, which is a selective estrogen receptor modulator (SERM), and a method for producing the compound.
  • SERM selective estrogen receptor modulator
  • Ospemiphen (or deaminohydroxy-toremifene) is one of the selective estrogen receptor modulators (SERMs) and has been known to provide mixed activities such as relatively low estrogenic and anti-estrogenic activities in the universal hormone test 1, 2, and 3), and anti-tumor activity in preclinical experiments on breast cancer animal models (Non-Patent Document 4).
  • SERMs selective estrogen receptor modulators
  • Non-Patent Document 4 With the approval of the US FDA for the purpose of treating certain menopausal dysfunctions such as dyspareunia and vaginal dryness among healthy women's menopausal syndromes in 2013, it is now being marketed as Ospena by Japanese pharmaceutical company Shinogi Co., Prescription medicine that sells.
  • Dyspareunia is a menopausal female disorder disease in which vaginal atrophy occurs due to a decrease in body estrogen levels after menopause and pain during sexual activity.
  • the administration of ospemipene stimulates the endometrium by providing an estrogenic effect on the vaginal tissue, thereby thickening the tissue, thereby alleviating the pain experienced by menopausal women during sexual activity.
  • Clinically reported side effects of ospemiphen were redness, increased vaginal discharge, muscle spasms and sweating, and boxed warnings suggest that they should be prescribed for the shortest period of time consistent with individual women's treatment goals and risks.
  • Non-Patent Document 5 The US market is expected to surpass US $ 900 million in 2012, expanding to more than US $ 1.9 billion in 2022, and the Japanese market is expected to reach US $ 20 million in 2022.
  • the FDA approval of osmempen was presented as a major growth driver for postmenopausal vaginal atrophy.
  • Ospemiphen is similar to the universal selective estrogen receptor (SERM), through metabolism of the cytochrome p450 enzyme system of hepatic microsomes after human administration, that is, by the action of CYP2D6 or CYP2C9, 4-hydroxysulfemiphen 4-hydroxyspemifene (hereinafter referred to as 4-OH-OSPEM) or 4'-hydroxyospemifene (hereinafter referred to as 4'-OH-OSPEM).
  • SERM universal selective estrogen receptor
  • Ospemiphen is a highly hydrophobic compound that provides excellent tolerability, but the resulting low solubility and hence markedly lower bioavailability means that the recommended dose of ospemiphene daily is 60 mg or less As a result, pharmaceutical improvement of the above medicament is necessary.
  • Patent Documents 1 and 2 The patent for the production method and use of ospemiphen is described as a chemical synthesis method through an organic synthesis method and for the treatment and prevention of a female disease atrophy-related disease or disorder after menopause.
  • Patent Documents 1 and 2 the production of compounds in which sugar is added to 4-OH-OSPEM and 4'-OH-OSPEM metabolites, the metabolites of ospemiphen (hereinafter referred to as OSPEM)
  • Korean Patent No. 10-1717212 (entitled “Method for preparing an estrogen receptor modulator added with a sugar-modified enzyme") and International Application No. PCT-KR2017-004022 (entitled “ A method for producing the same, and a pharmaceutical composition containing the same) have succeeded in the conversion of tamoxifen and necromphene derivatives, which can be used as a preventive and therapeutic agent for breast cancer, as a kind of SERM.
  • CYP2C9 which is a type of recombinant human cytochrome P450, firstly added hydroxyl groups to OSPEM -OH-OSPEM and 4'-OH-OSPEM, and at the same time a novel glucosyltransferase (MrGT2) derived from a microorganism monospora rhodozyma actinomycetus The utilization rate is increased and the pharmacological activity is improved, and the present invention is completed.
  • MrGT2 novel glucosyltransferase
  • Patent Document 1 WO 1996007402A
  • Patent Document 2 WO 2003103649A1
  • Non-Patent Document 1 Kangas L, Biochemical and pharmacological effects of toremifene metabolites. Cancer Chemother Pharmacol. 1990. 27: p. 8-12.
  • Non-Patent Document 2 Unkila M., et al., Vaginal effects of ospemifene in the ovariectomized rat preclinical model of menopause. J. Steroid Biochem, Mol. Biol. 2013. 138: p. 107-115.
  • Non-Patent Document 3 Kaur G., et al., Design, synthesis and evaluation of ospemifene analogs as anti-breast cancer agents. Eur. J. Med. Chem. 2014. 86: p. 211-218.
  • Non-Patent Document 4 Nappi R. E., et al., Advances in pharmacotherapy for treating female sexual dysfunction. Exp. Opin. Pharmacother. 2015, 16 (6): p. 875-887.
  • Non-Patent Document 5 GlobalData, PharmaPoint: Postmenopausal vaginal atrophy-Global drug forecast and market analysis to 2022. 2013.
  • Non-Patent Document 6 Tolonen A., et al., Ospemifene metabolism in human in vitro and in vivo: metabolite identification, quantification, and CYP assignment of major hydroxylations. Drug Metabol. Drug Interact. 2013. 28 (3): p. 153-161.
  • Another object of the present invention is to provide a method for converting ospemiphen to a glucosyl-omepemiphen derivative by a one-pot bioconversion method of a recombinant human cytochrome c enzyme and a microorganism-derived recombinant sugar transferase .
  • the present invention provides a compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof.
  • R1 is independently selected from the group consisting of glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose, Or 2'-deoxy-glucose.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and having a pharmacological activity similar to that of osfemiphen (OSPEM), a dyspareunia and vaginal dryness treatment drug, Or a pharmaceutically acceptable salt thereof.
  • OSPEM osfemiphen
  • a dyspareunia and vaginal dryness treatment drug Or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a process for preparing an osmemphene derivative represented by formula (1), comprising the steps of:
  • the one-pot reaction was carried out by mixing the ospemiphen solution with the glycosyltransferase, cytochrome P450, UDP-glucose or TDP-2-deoxy-glucose and glucose 6-phosphate, glucose 6-phosphate dehydrogenase and NADP + ;
  • R1 is independently selected from the group consisting of glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose, -Deoxy-glucose. ≪ / RTI >
  • the present invention also relates to a method of treating dyspareunia, vaginal dryness, osteoporosis, facial palsy, osteoarthritis, and osteoporosis of a postmenopausal woman, comprising administering a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
  • a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof Such as hyperglycemia or hot flashes, vaginal atrophy, gynecomastia, hypogonadal hypogonadism, non-insulin dependent diabetes mellitus, lipid dystrophy, infertility, male prostate hypertrophy, prostate cancer, ovarian cancer and breast cancer
  • the present invention provides a method for treating or preventing a disease selected from the group consisting of
  • the present invention also relates to the use of a compound of formula (I) for the treatment of menopausal women with dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy, gynecomastia,
  • a compound represented by Formula 1 for the treatment or prevention of a disease selected from the group consisting of hypogonadal hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer and breast cancer.
  • Lt; RTI ID 0.0 > acceptable < / RTI >
  • the present invention also relates to the use of a compound of formula (I) for the treatment of menopausal women with dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy, gynecomastia, (1) for the manufacture of a medicament for the treatment or prevention of diseases selected from the group consisting of hypogonadal hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer and breast cancer, Or a pharmaceutically acceptable salt thereof.
  • diseases selected from the group consisting of hypogonadal hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer and breast cancer, Or a pharmaceutically acceptable salt thereof.
  • FIG. 1 is a schematic diagram showing transformation of a recombinant CYP enzyme derived from human and a recombinant sugar transferase MrGT2 enzyme derived from actinomycetes into an osfempiren one pot.
  • FIG. 2 is a graph showing the results of a comparison between two kinds of mammalian cell lines, MCF-1 and MCF-2, which are two kinds of ospemipene derivatives (4-OSPEM-G and 4-OSPEM-DG), osfemimene (OSPEM) and tamoxifene 7 and MDA-MB-231 in half-life inhibitory concentration (IC50).
  • MCF-1 and MCF-2 which are two kinds of ospemipene derivatives (4-OSPEM-G and 4-OSPEM-DG), osfemimene (OSPEM) and tamoxifene 7 and MDA-MB-231 in half-life inhibitory concentration (IC50).
  • FIG. 3 is a graph showing the effect of the osmotic pressure on osmolysis of osmotic pressure in a dose of 10 mg / kg / day for 2 weeks using osfemiphen (OSPEM), 2 kinds of oselpemiphen (4-OSPEM-G, 4-OSPEM-DG) This is a bar graph comparing vaginal epithelial changes in ovariectomized rats administered orally.
  • OSPEM osfemiphen
  • the vaginal epithelium thickness and weight and uterine weight were 100% Relative epithelium thickness, relative quality of body mass, and relative uterine weight were calculated as percentages (%) of the above parameters in the derivative and the raloxifene treatment group.
  • Fig. 4 shows the results of ovariectomized rats administered orally at a dose of 10 mg / kg / day for 2 weeks with ospemiphen (OSPEM) and two different ospemipene derivatives (4-OSPEM-G and 4-OSPEM-DG)
  • OSPEM ospemiphen
  • 4-OSPEM-DG 4-OSPEM-DG
  • a GT encoding gene is isolated from the genome of a micro-monospore rhodolagenic strain to develop an ospemiphen derivative capable of overcoming the disadvantages of osupimpen which has a low bioavailability, , And transformed E. coli were prepared.
  • the present invention relates to a compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof:
  • R1 is independently selected from the group consisting of glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose, -Deoxy-glucose. ≪ / RTI >
  • the compound is selected from the group consisting of ospemifene glucoside, ospemiphenegalactoside, ospemiphenaloside, ospemiphentaloside, ospemipheniloside, ospemipene N-acetylglucosamine- 4-O-2'-N-acetylglucosaminide, ospemifene lactose, or ospemifene-4-O-2'-deoxyglucoside.
  • the compound of formula (I) according to the present invention may be used in the form of a pharmaceutically acceptable salt.
  • the salt an acid addition salt formed by a pharmaceutically acceptable free acid is useful.
  • free acid inorganic acid and organic acid can be used.
  • inorganic acid hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid and the like can be used.
  • organic acid citric acid, acetic acid, lactic acid, maleic acid, pumaric acid, gluconic acid, Succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid and the like can be used.
  • the compounds of formula (I) according to the present invention include not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.
  • the compound of Formula 1 according to the present invention may be prepared in crystalline form or amorphous form, and may be arbitrarily hydrated or solvated when the compound of Formula 1 is prepared in crystalline form.
  • the compound of formula (1) according to the present invention is an ospemipene derivative, and its sugar moiety exhibits relatively low in vitro breast cancer cell toxicity than ospemiphen, a former compound, It was confirmed that the bioavailability of the ovariectomized rats was improved, and it is possible to apply the present invention to a medicament for the treatment of diseases such as dyspareunia and vaginal dryness, It could be used as a new drug formulation that overcomes the disadvantages of low-bioavailability ospemiphen.
  • the present invention provides a method for treating menopausal female disorders, such as dyspareunia and vaginal dryness, comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, (Eg, dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, vaginal atrophy, gynecomastia, male secondary hypogonadism, non-insulin dependent Diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer or breast cancer.
  • menopausal female disorders such as dyspareunia and vaginal dryness
  • a pharmaceutically acceptable salt thereof comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, (Eg, dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, vaginal atrophy, gynecomastia, male secondary hypogonadism,
  • the present invention provides a method of treating dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy, gynecomastia, ), A compound of formula (1) for the treatment or prevention of diseases selected from the group consisting of male secondary hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male hyperplasia, prostate cancer, ovarian cancer and breast cancer, Or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating dyspareunia, vaginal dryness, osteoporosis, facial flushing or hot flashes, menopausal atrophy, gynecomastia, )
  • a medicament for the treatment or prophylaxis of diseases selected from the group consisting of male hypogonadism, non-insulin dependent diabetes mellitus, fat dystrophy, infertility, male prostatic hyperplasia, prostate cancer, ovarian cancer and breast cancer. ≪ / RTI > or a pharmaceutically acceptable salt thereof.
  • treatment means all the actions of improving or ameliorating the symptoms of the diseases by administration of the pharmaceutical composition comprising the compound represented by the above-mentioned formula 1 or a pharmaceutically acceptable salt thereof .
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof of the present invention exhibits some in vitro cytotoxic activity against breast cancer cell lines and does not show cytotoxicity in normal cells (Experimental Example 1)
  • the extracts of the mice showed activity against the vaginal epithelial tissue similar to that of the original compound ospemiphen (Experimental Example 2), and compared with administration of ospemipene And the bioavailability of the sugar portion is improved (Experimental Example 3). Therefore, it can be effectively used as an improved dosage form which can partially improve the low bioavailability of the existing ospemiphen.
  • compositions of the present invention may be formulated into oral or parenteral administration forms according to standard pharmaceutical practice. These formulations may contain, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, adjuvant or diluent.
  • an additive such as a pharmaceutically acceptable carrier, adjuvant or diluent.
  • composition of the present invention When the composition of the present invention is used as a medicine, a pharmaceutical composition containing at least one compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient can be administered orally or parenterally But are not limited thereto.
  • the formulations for oral administration may be, for example, tablets, pills, hard capsules, soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and the like, (E.g., silica, talc, magnesium salt of stearic acid, calcium salt of stearic acid, and / or polyethylene glycol), such as sodium chloride, dextrose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine have.
  • the tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt The same disintegrating or boiling mixture and / or absorbing agent, coloring agent, flavoring agent, and sweetening agent.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt
  • agar alginic acid or its sodium salt
  • Formulations for parenteral administration may include subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection.
  • the pharmaceutical composition of the present invention contains at least one compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof, which is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, Which can be prepared in an ampoule or vial unit dosage form.
  • the composition may be sterilized and / or contain preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances and may be mixed, granulated Or a coating method.
  • the dose of the compound of the present invention to the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity.
  • the patient is 70 kg in body weight, And may be 0.01-100 mg / day, preferably 0.01-500 mg / day.
  • it may be administered once to several times a day at intervals of a certain period of time.
  • the present invention provides a method for the production of a pharmaceutical composition for the treatment and / or prophylaxis of atherosclerosis, comprising the steps of: (a) contacting an osfemimene solution with a glycoconjugate, cytochrome P450, UDP- glucose or TDP-2-deoxy- glucose and glucose 6-phosphate, Mixing NADP + to perform a one-pot reaction; And (b) obtaining an osmemimene derivative represented by the formula (1).
  • the present invention relates to a process for obtaining a recombinant GT enzyme derived from actinomycetes in Escherichia coli and 2) obtaining a total of two saccharide-derived ospemiphene derivatives by a sequential enzymatic reaction of a recombinant CYP with human recombinant GT (i.e., MrGT2) .
  • the production method may further include a step of separating, purifying, or separating and purifying the enzyme reaction product obtained in the above step by Medium Pressure Liquid Chromatography (MPLC) have.
  • MPLC Medium Pressure Liquid Chromatography
  • the recombinant glycoprotein of step 1) can be isolated from micro-monospore rhododendron, but is not limited thereto.
  • the recombinant glycoprotein is preferably MrGT2, more preferably MrGT2 expressed in E. coli.
  • the stationary phase used in the intermediate-pressure liquid chromatography method in this step may be, but is not limited to, reversed phase C18.
  • the mobile phase used in the above intermediate-pressure liquid chromatography method may be, but not limited to, a mixed solution of methanol: water: formic acid 65: 35: 0.2 (v / v / v / v).
  • the medium pressure liquid chromatography retention time in this step may be 11 to 13 minutes, but is not limited thereto.
  • " vector &quot means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host.
  • the vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms " plasmid " and " vector " are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use a plasmid vector.
  • Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.
  • the vector should be transformed into the appropriate host cell.
  • the preferred host cells are prokaryotic cells.
  • Suitable prokaryotic host cells include E. coli DH5 ⁇ , E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, .
  • E. coli strains such as FMB101, NM522, NM538 and NM539, as well as the speices and genera of other prokaryotes, and the like, can also be used.
  • Strain such as Agrobacterium A4, bacilli such as Bacillus subtilis, Salmonella typhimurium or Serratia marcensis marcescens) and various strains of the genus Pseudomonas can be used as host cells.
  • Transformation of prokaryotic cells can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used to transform these cells.
  • the expression " expression control sequence " in the present invention means a DNA sequence essential for the expression of a coding sequence operably linked to a particular host organism.
  • regulatory sequences include promoters for conducting transcription, any operator sequences for modulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.
  • regulatory sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosome binding sites.
  • Eukaryotic cells include promoters, polyadenylation signals and enhancers. The most influential factor on the expression level of the gene in the plasmid is the promoter.
  • SR ⁇ promoter and cytomegalovirus-derived promoter are preferably used.
  • any of a wide variety of expression control sequences may be used in the vector.
  • Useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, The promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of the phosphatase, such as Pho5, the promoter of the yeast alpha-mating system and the gene expression of prokaryotic or eukaryotic cells or viruses And other combinations known in the art.
  • the T7 promoter may be useful for expressing the protein of the present invention in E. coli.
  • a nucleic acid is " operably linked " when placed in a functional relationship with another nucleic acid sequence.
  • This may be the gene and regulatory sequence (s) linked in such a way that the appropriate molecule (e. G., Transcriptional activator protein) is capable of gene expression when bound to the regulatory sequence (s).
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence;
  • the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence;
  • a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation.
  • " operably linked &quot means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame.
  • the enhancer need not be in contact.
  • the linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.
  • the term " expression vector " is usually a recombinant carrier into which a fragment of different DNA is inserted, and generally means a fragment of double-stranded DNA.
  • the heterologous DNA means a heterologous DNA that is not naturally found in the host cell.
  • the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in the selected expression host.
  • the expression control sequence and the gene are contained within an expression vector containing a bacterial selection marker and a replication origin.
  • Host cells transformed or transfected with the above expression vectors constitute another aspect of the present invention.
  • the term " transformation " means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration.
  • the term " transfection " means that an expression vector, whether or not any coding sequence is actually expressed, is accepted by the host cell.
  • PCR polymerase chain reaction
  • SEQ ID NO: 1 5'-GGCATATGATCCACGCGCACGACTTCCGGATG-3 '(NdeI restriction site)
  • SEQ ID NO: 2 5 '- CGCTCGAGATTCGGCCTGCGCCTCCCACGTCCA-3' (XhoI restriction site)
  • PCR product was purified and ligated to pGEMR-T easy vector (pGEMR-T easy vector, Promega, Madison, Wis., USA) and then ligated to E. coli XL1 blue (XL1 blue, Stratagene, La Jolla, Lt; 0 > C for 45 seconds, followed by transformation.
  • pGEMR-T easy vector Promega, Madison, Wis., USA
  • MrGT2 DNA fragment treated with NdeI and XhoI restriction enzymes was inserted into pET-28 (a +) (Novagen, Madison, WI, USA), a protein expression vector treated with the same restriction enzymes, Were transformed into E. coli BL21 (DE3) (Stratagene, La Jolla, CA, USA). At this time, 50 ppm of kanamycin antibiotics was used for selection of transformants.
  • the recombinant E. coli was inoculated into the LB medium (Luria Bertani) supplemented with 1 M of sorbitol and 2.5 mM betaine at a final concentration of 1%, and cultured at 30 ° C. D-thiogalactopyranoside (IPTG; Sigma-Aldrich) was added at a final concentration of 0.5 mM to induce protein expression when growth was observed between 0.6 and 0.8 optical density. , St. Louis, Mo., USA). The recombinant E. coli strain was further cultured at 22 DEG C for 18 hours.
  • IPTG D-thiogalactopyranoside
  • the culture was centrifuged at 2000 rpm for 10 minutes to collect the cells, and the cells were suspended in 50 mM sodium phosphate lysis buffer (300 nM NaCl, 10 mM imidazole, 10% glycerol, 1% Triton X-100) And sonication was carried out. After centrifugation at 12000 rpm for 20 minutes, the supernatant was collected separately and analyzed by 12% SDS-PAGE to confirm the expression level of the recombinant MrGT1 enzyme.
  • 50 mM sodium phosphate lysis buffer 300 nM NaCl, 10 mM imidazole, 10% glycerol, 1% Triton X-100
  • the supernatant was diluted with Talon-metal affinity resin (Clontech, Mountain View, Calif., USA) equilibrated with 50 mM phosphate buffer (0.3 M NaCl, 20 mM imidazole) CA, USA) and incubated at 4 ° C for one hour. After refrigerated centrifugation at 2000 rpm for 5 minutes, the resin was introduced into a disposable column and washed with phosphate buffer containing 50 mM imidazole in a volume of 10 times the resin. Finally, 3 ml of a phosphate buffer solution containing 150 mM imidazole was used to purify the recombinant glycoconjugate MrGT2 bound to the resin.
  • Ospemiphen (OSPEM, Sigma-Aldrich) used as a substrate was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM, and the resultant was dissolved in a reaction buffer (50 mM phosphate buffer, 10 mM magnesium chloride) (CYP2C9 20 ⁇ M level) purchased from BD Gentest (Woburn, MA, USA), 25 ⁇ M recombinant herpesvirus MrGT2, and UDP-Glc (10 mM, Sigma-Aldrich), NADP + (1 mM, Sigma-Aldrich) at a level of 2 mM each of TDP-2'-deoxy-Glc and for the continuous feeding of NADH coenzyme for CYP oxidation reaction.
  • DMSO dimethyl sulfoxide
  • CYP2C9 20 ⁇ M level purchased from BD Gentest (Woburn, MA, USA)
  • UDP-Glc 10 mM, Sigma-A
  • the mobile phase was Acquity CSH C18 (Waters, 50x1.0 mm, 1.7 ⁇ m; Milford, MA) as a stationary phase column at a rate of 140 ⁇ l / min in methanol: water: formic acid 65: , USA).
  • the individual fractions were again concentrated under reduced pressure and the mass spectra were analyzed with an ion trap mass spectrometer (LCQ ion-trap mass spectrometer, ThermoFinnigan, San Jose, Calif., USA) to isolate the desired sugar moieties.
  • LCQ ion-trap mass spectrometer As a substrate, ospemiphen was aliquoted at a retention time of about 15 to 16 minutes on the MPLC system, and the desired sugar moiety was detected in 11 to 13 minutes, which is a faster retention time than the substrate.
  • the yield of biotransformation using nucleic acid per UDP-Glc donor was about 48%, but the yield of biotransformation was lower than 30% , which suggests that the enzyme has some substrate flexibility for the sugar donor substrate and a difference in preference for the sugar donor substrates.
  • the fact that the sugar portion of the produced osfemiphen derivative is limited to the hydroxyl group at the 4-position rather than the 4'-position during the one-pot reaction of CYP and MrGT2 indicates that the specificity of the glycosyltransferase Respectively.
  • NMR samples were prepared by dissolving each derivative in 200 ⁇ l of DMSO-d6 followed by leaving the solvent in 5 mm Shigemi advanced NMR microtube (Sigma, St. Louis, Mo.). 13 C NMR spectra were acquired at 298 K using a Varian INOVA 500 spectrophotometer and chemical shifts were recorded in ppm using TMS as internal reference. All NMR data were calculated using Mnova Suite 5.3.2 software and compared to osmemphen using the 13C-NMR spectra of SERM derivatives as substrate.
  • DMSO-d6 [delta] 35.8, 42.7, 60.7, 62.2, 69.4, 71.5, 73.3, 76.7, 81.4, 109.2, 114.1, 126.3, 127.5, 127.9, 128.7, 129.1, 131.6, 139.2, 156.4, 161.5)
  • DMSO-d6 [delta] 35.8, 37.5, 42.7, 60.7, 62.2, 68.9, 69.4, 71.4, 81.2, 104.1, 114.1, 126.3, 127.5, 127.9, 128.6, 129.1, 131.6, 139.3, 156.4, 161.5)
  • MCF-7 ER positive, ATCC, Manassas, VA
  • MDA-MB-231 cell lines ER negative and ATCC
  • DMEM fetal bovine serum Serum
  • penicillin 100 units / ml level
  • streptomycin 100 ⁇ g / ml level
  • Each activated cell line (2 x 10 3) was dispensed into individual wells in a 96-well plate and OSPEM (Sigma-Aldrich) at various concentrations (0.1, 1, 3, 10, 30, The cells were incubated for 4 days (96 hours), MTT reagent was added to each well, and the cells were further cultured for an additional 3 hours. After addition of DMSO, the absorbance was measured with a Tecan microwell plate reader (Tecan AG, Mannedorf, Switzerland) set at a wavelength of 570 nm, and nonlinear regression analysis with Prism software revealed that the half-inhibition concentration ) Were measured.
  • OSPEM Sigma-Aldrich
  • the OSPEM and the OSPEM derivatives of the present invention were compared with the cytotoxicity of normal cells to confirm the cytotoxicity of breast cancer cell lines.
  • Mouse embryonic fibroblast (ATCC) was used as a normal cell and the cytotoxicity of the treated group was compared with the above MTT assay.
  • the half-inhibition concentration was about 52.7 ⁇ M and 100 ⁇ M for the breast cancer cell lines MCF-7 and MDA-MB-231, respectively, whereas the half-inhibition concentration in the control group, tamoxifen- 62.6 [mu] M and 77.7 [mu] M.
  • the MCF-7 cell line which is an ER positive breast cancer cell line, lt; / RTI >
  • MDA-MB-231 an ER-negative breast cancer cell line, showed that the half-inhibitory concentrations of two sugar derivatives were more than 100 ⁇ M as in the case of osmempen treatment.
  • the two derivatives of the glucosidase exhibit relatively low cytotoxicity against two types of breast cancer cell lines rather than the ospemipene control group, but they provide selective cytotoxicity to ER-positive breast cancer cell lines similar to ospemifene Showed the same tendency.
  • the cytotoxicity test for mouse fetal fibroblasts which are normal cells, the toxicity was not observed in all treatment groups up to 100 ⁇ M level, and osfemiphen and tamoxifen and glucoside were found to be the novel osmemiphen derivatives 2 All species have cytotoxicity specific to cancer cells.
  • mice Female Sprague Dawley rats (DBL, Chungbuk, Korea) were housed in an individual cage for 12 hours in an amnion cycle. Female rats from 9 to 11 weeks of age were used for oral administration and ovaries of experimental rats were harvested 10 to 14 days prior to administration. The following experiment was performed on ovariectomized rats. In each experiment, treatment groups (between 5 and 6) were randomly selected and the mean body weight of the treatment groups was the same.
  • raloxifene (Sigma-Aldrich) was first dissolved in ethanol and diluted to the appropriate concentration with the PEG-treated untreated group. The dose was 3 mL per kg body weight. In order to test the relationship between dose and effect, oral administration was performed once a day for a total of 2 weeks.
  • vaginal specimens except the cervix were weighed and the uterus was preserved in 10% buffered formalin. Each vaginal site was cut in half, immersed in 10% buffered formalin in a paraffin block, and cut into 4 ⁇ m sections for hematoxylin-eosin staining. Relative mass weight was converted to Equation 1 below.
  • vaginal epithelium thickness was measured by image analysis using Image Pro Plus software after capturing images using a microscope equipped with a CCD camera.
  • the prepared cross section was magnified 100 times and measured from 3 to 4 times. Extraction and histological analysis of all organs were performed after the blindfolding of the individual treatment groups.
  • the measurement average of the ethinyl estradiol treatment group as the positive control group was taken as 100, and the relative value, that is, the percentage of ethinylestradiol reaction In terms of percentage (%), the vaginal epithelium thickness, relative quality and relative uterine weight of the other treatment groups were expressed.
  • One-way ANOVA ANOVA with Dunnett's post-validation method and Kruskal-Wallis method as a nonparametric test method were used for the statistical analysis of the three parameters and SPSS version 14.0 was used as the statistical analysis program .
  • FIG. 3 The in vivo animal test results (FIG. 3) as described above show that in vitro breast cancer cytotoxicity, which is relatively low compared to the osmemphene derivative of the ospemipene sugar derivative shown in Experimental Example 1 2).
  • FIG. 2 in the case of two new oregus-free glucoside derivatives administered orally for 2 weeks in the ovariectomized rats, ≪ / RTI > activity.
  • the compound of formula (I) according to the present invention is a glucosoxifene derivative prepared by the conversion of one-pot, which shows lower cytotoxicity than ospemiphen, has improved bioavailability, It is possible to use it as a new drug formulation for existing diseases of ospemipen.

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Abstract

La présente invention concerne un nouveau composé dérivé d'ospémifène dans lequel un sucre est ajouté à l'ospémifène en tant qu'un type de modulateur sélectif du récepteur des oestrogènes (ci-après SERM) et un procédé de préparation pour le composé. Le composé dérivé d'ospémifène présente une biodisponibilité améliorée par comparaison avec l'ospémifène existant et présente également une biodisponibilité partiellement améliorée par comparaison avec le composé ospémifène d'origine, ce qui assure un effet de traitement de maladie pour la dyspareunie, la sécheresse vaginale et similaires apparaissant chez les femmes ménopausées, et il peut ainsi être avantageusement utilisé en tant que forme pharmaceutique modifiée.
PCT/KR2018/008662 2017-08-10 2018-07-31 Ospémifène à sucre ajoute, procédé de préparation s'y rapportant et composition pharmaceutique contenant celui-ci en tant que principe actif WO2019031754A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
WO1996007402A1 (fr) * 1994-09-07 1996-03-14 Orion-Yhtymä Oy Triphenylethylenes pour la prevention et le traitement de l'osteoporose
KR20080015924A (ko) * 1999-11-16 2008-02-20 호르모스 메디칼 리미티드 트리페닐알켄 유도체 및 그것의 선택적 에스트로겐 수용체조절자로서의 용도
KR20170011542A (ko) * 2015-07-23 2017-02-02 고려대학교 산학협력단 스테롤 당전이 효소를 이용한 스테롤 글루코사이드의 제조방법
KR20170011543A (ko) * 2015-07-23 2017-02-02 고려대학교 산학협력단 당전이 효소를 이용한 당 부가된 에스트로겐 수용체 조절제의 제조방법

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WO2003103649A1 (fr) 2002-06-06 2003-12-18 Hormos Medical Corporation Methode d'inhibition de l'atrophie ou de traitement ou de prevention de symptomes lies a l'atrophie chez les femmes

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WO1996007402A1 (fr) * 1994-09-07 1996-03-14 Orion-Yhtymä Oy Triphenylethylenes pour la prevention et le traitement de l'osteoporose
KR20080015924A (ko) * 1999-11-16 2008-02-20 호르모스 메디칼 리미티드 트리페닐알켄 유도체 및 그것의 선택적 에스트로겐 수용체조절자로서의 용도
KR20170011542A (ko) * 2015-07-23 2017-02-02 고려대학교 산학협력단 스테롤 당전이 효소를 이용한 스테롤 글루코사이드의 제조방법
KR20170011543A (ko) * 2015-07-23 2017-02-02 고려대학교 산학협력단 당전이 효소를 이용한 당 부가된 에스트로겐 수용체 조절제의 제조방법

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