KR20170011543A - Method for Preparing Glycosylated Estrogen Receptor Modulators Using Glycosyltransferase - Google Patents

Abstract

The present invention relates to glycosyltransferase derived from Micromonospora rhodorangea actinomycetes, to a method for bio-converting a sugar-added selective estrogen receptor modulator (SERM) capable of improving the bioavailability of conventional tamoxifen-structured analogues by using the same, and to a use of the same. A sugar-added SERM enzymatically biosynthesized by using novel recombinant glycosyltransferase according to the present invention shows an in vitro antagonistic activity with respect to an ER, and simultaneously has improved in vivo bioavailability, thereby being useful as a novel medical product for preventing or treating breast cancer, wherein the medical product can decrease side effects occurring when conventional tamoxifen is administrated for a long time.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an estrogen receptor modulator,

The present invention relates to a method for preparing a selective estrogen receptor modulator (SERM), and more particularly, to a method for producing a selective estrogen receptor modulator ( Micromonospora The present invention relates to a method for bio-conversion of SERM compounds and its use, which can improve the bioavailability of existing tamoxifen structural analogues by using a sugar- derived enzyme derived from actinomycetes.

Selective estrogen receptor modulators act on the intracellular estrogen receptors (ER) and provide antagonist or agonist effects depending on the application tissue to inhibit or activate the estrogen-like role. Representative SERMs include tamoxifen, raloxifene, lasofoxifene, and toremifene (Ward et al., Br. Med . J. 1: 13-14, 1973; Draper et al, Pharmacology 50:. ... 209-217, 1995; Ke et al, J. Am Aging Assoc 25:. 87-99, 2002; Valavaara et al, Ann Clin Res 20:.... 380- 388, 1988). Of these, tamoxifen acts as an antagonist for breast tissue ER, as an agonist for bone and endometrial tissue ER, while for raloxifene and lasofoxifene as an antagonist for breast and uterine ER, It acts as an agonist only in tissue ER.

Tamoxifen is a type of prodrug that has a relatively low affinity for ER. It is a metabolite of 4-hydroxytamoxifen (aka afimoxifene) and N- N-desmethyl-4-hydroxytamoxifen (aka endoxifen) is an active metabolite (Desta et al., J. Pharmacol . Exp . Ther . 310: 1062-1075, 2004 ), And as an antagonist to tissue ER with a 30- to 100-fold higher affinity than tamoxifen (Muerdter et al., Clin . Pharmacol . Ther . 89: 708-717, 2011).

However, long-term prescription of tamoxifen may cause osteoporosis due to hypertension of bone tissue ER, and the possibility of uterine cancer may be caused by the hypertensive effect of endometrial tissue ER. and (Vehmanen et al, J. Clin Oncol 24:..... 675-680, 2006; Gallo et al, Semin Oncol 24: S1-71-S1-80, 1997). In addition, tamoxifen stimulates the proliferation of uterine epithelial cells, which are known to increase the risk of uterine cancer, and the American Cancer Society reports that tamoxifen reduces the risk of breast cancer recurrence, but increases the risk for some types of uterine cancer. It is therefore important to develop novel prodrugs that can reduce the risk of cervical cancer with low dose administration by providing novel derivatives of tamoxifen or bioavailability of existing tamoxifen bioavailability.

The development of new derivatives through structural modification of the SERM is a key technology for the development of new drug and drug delivery systems, one of which is the transfer of sugar molecules to hydroxy functions in the chemical structure Is an enzymatic production method using enzyme (GT). In particular, the transfer of tamoxifen and its structural analogues can improve the bioavailability by maintaining the concentration of the drug in the blood above a certain level during the first pass metabolism process compared with the original substance, Bio-conversion technology that is useful for the improvement of the formulation of a drug, the development of new formulations, and the prodrug of the drug.

Most of the reports related to the glycosylation of tamoxifen are activated by the metabolism of apimaciphen and endoxifene, which are hydroxylated at position 4 through hepatic metabolism after administration, and then glucuronic acid is added to the hydroxyl group. (McCague et al., Biochem . Pharmacol . 39: 1459-1465, 1990, Poon et al., Drug Metabol. Disposit. : 1119-1124,1993). Recently, the development of a quantitative monitoring technique for tamoxifen and its metabolites in human plasma using a high-sensitivity mass spectrometer has revealed new metabolites that have not been detected in the past, including glucuronic acid (Dahmane et al., Anal. Bioanal. Chem . 406: 2627-2640, 2014), in which glucose is added in addition to the metabolic process in which glucuronic acid is added. However, it has not been known until now to produce a compound that adds sugar to tamoxifen and its structural similar SERMs through enzymatic bioconversion.

Accordingly, the present inventors have made intensive efforts to develop a SERM derivative having significantly improved ER binding affinity and bioavailability compared to the original SERM before saccharification, and as a result, found that a novel glycosyltransferase capable of reacting with a hydroxyl group-added SERM as a substrate And the effect of increasing the antagonistic activity and bioavailability of the SERM derivative to the ER was confirmed.

It is an object of the present invention to provide a novel glycosyltransferase (MrGT2), a polynucleotide encoding the same, an expression vector comprising the polynucleotide, and a recombinant microorganism into which the polynucleotide or expression vector is introduced.

It is another object of the present invention to provide a method for producing a recombinant glycosidase (MrGT2) comprising culturing the microorganism to induce expression of the glycosyltransferase and recovering the same.

It is another object of the present invention to provide a method for preparing a selective glucose estrogens receptor modulator (SERM) using the sugar-modified enzyme (MrGT2) and a glucose selective estrogen receptor modulator (SERM).

It is still another object of the present invention to provide a pharmaceutical composition and food for preventing or treating breast cancer, wherein the sugar portion contains a selective estrogen receptor modulator (SERM) as an active ingredient.

In order to achieve the above object, the present invention provides a glycosyltransferase (MrGT2) represented by the amino acid sequence of SEQ ID NO: 4.

The present invention also provides a polynucleotide encoding said enzyme.

The present invention also provides an expression vector comprising the polynucleotide.

The present invention also provides a recombinant microorganism into which the polynucleotide or the expression vector has been introduced.

The present invention also provides a method for producing a recombinant herpesvirus (MrGT2) comprising culturing the microorganism to induce expression of the glycosyltransferase, and recovering the same.

The present invention also relates to a method for preparing a selective estrogen receptor modulator comprising the steps of: (a) reacting a selective estrogen receptor modulator (SERM) and a sugar donor in the presence of the glycoprotein (MrGT2) And (b) recovering the synthesized sugar portion of the selective estrogen receptor modulator. The method for producing a sugar ester selective estrogen receptor modulator (SERM) according to claim 1,

[Chemical Formula 1]

Wherein R ₁ is CH ₃ or H, R ₂ is H ₂ or Cl, and R ₃ is But are not limited to, glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose or 2'- Glucose (2'-deoxy-glucose).

The present invention also provides a sugar portion selective estrogen receptor modulator (SERM), or a pharmaceutically acceptable salt thereof, represented by Formula 1:

[Chemical Formula 1]

Wherein R ₁ is CH ₃ , R ₂ is Cl, and R ₃ is Glucose, galactose or 2'-deoxy-glucose.

The present invention also provides a pharmaceutical composition for preventing or treating breast cancer, wherein the sugar portion contains a selective estrogen receptor modulator (SERM) as an active ingredient.

The present invention also provides a food for preventing or improving breast cancer, wherein the sugar portion contains an optional estrogen receptor modulator (SERM) as an active ingredient.

A novel recombinant glycosyltransferase (MrGT2) in which a glycoprotein derived from a microorganism of Micromonospora rhodorangea according to the present invention is expressed in Escherichia coli is enzymatically biotransformed to a selective estrogen receptor modulator SERM) is useful as a new drug for the prevention or treatment of breast cancer, which can reduce side effects caused by long-term administration of existing tamoxifen, because it exhibits antagonistic activity against ER in vivo and improves in-vivo bioavailability.

FIG. 1 is a schematic diagram of bioconversion through a glycosyltransferase MrGT2 enzyme reaction. FIG.
Figure 2 is a graph showing the in vitro antagonistic activity of estrogen receptor alpha (ER < alpha >) of the SERM derivatives and the original SERM compounds before bioconversion.
FIG. 3 is a graph comparing the degree of bioavailability improvement of sugar-derived derivatives by analyzing the concentrations of SERM and its active metabolites in the serum of experimental animals. The SERM derivatives and the original SERM compounds before bioconversion were orally administered to the rats at a dose of 3 μmole once a day for a week, and then collected 24 hours after the last administration.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, a GT encoding gene was isolated from the genome of a microorganism, and a recombinant expression vector and a transformed E. coli containing the GT encoding gene were prepared and then a recombinant glycoprotein (MrGT2) was prepared. Thereafter, the SERM compound (4-hydroxytremeni pen, apimociphen, and endoxifene), to which the hydroxyl group is added at the 4-position of the recombinant glycoprotein, is used as a receptor and uridine diphosphate glucose (UDP-Glc), uridine diphosphate galactose (UDP-Gal) or thymidine diphosphate 2'-deoxy-glucose (hereinafter referred to as TDP-2 '-deoxy-Glc) was reacted with a sugar donor to convert the SERM derivative into glucose. Then, the structure was confirmed by mass spectrometry and nuclear magnetic resonance spectrometry instrumental analysis, and the structure of the original SERMs and their derivatives was measured in vitro ) Antagonistic activity and the enhancement of bioavailability in experimental animals were partially confirmed, thereby producing SERM derivatives to which certain sugars were enzymatically added.

Accordingly, in a first aspect, the present invention relates to a glycosyltransferase (MrGT2) represented by the amino acid sequence of SEQ ID NO: 4.

In a second aspect, the present invention relates to a polynucleotide encoding said enzyme.

In the present invention, the polynucleotide may be characterized by being represented by the nucleotide sequence of SEQ ID NO: 3, wherein the polynucleotide is derived from a strain of Micromonospora rhodorangea .

In a third aspect, the present invention relates to an expression vector comprising the polynucleotide.

In a fourth aspect, the present invention relates to the polynucleotide or a recombinant microorganism into which the expression vector is introduced.

In the present invention, the microorganism is preferably Escherichia coli, but is not limited thereto.

According to a fifth aspect of the present invention, there is provided a method for producing a recombinant herpesvirus (MrGT2) comprising culturing the microorganism to induce expression of the transgene, and recovering the expression.

As used herein, "vector" means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in the 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 purpose of the present invention, it is preferable 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 to several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector, (C) a restriction enzyme cleavage site into which a 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.

After ligation, the vector should be transformed into the appropriate host cell. Transformation can be readily accomplished using a calcium chloride method or electroporation (Neumann, et al., EMBO J., 1: 841, 1982). As a vector used for overexpression of the gene according to the present invention, an expression vector known in the art can be used. The framework vectors that may be used in the methods of the present invention include, but are not limited to, various vectors capable of transforming into E. coli selected from the group consisting of pT7, pET / Rb, pGEX, pET28a, pET-22b Can be used.

A nucleotide sequence 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 as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, 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, and the promoter or enhancer is a sequence Or the ribosome binding site is operably linked to the coding sequence if it affects the transcription of the sequence, or the ribosome binding site is arranged to facilitate translation, Lt; / RTI > sequence. Generally, "operably linked" 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. However, 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.

As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in a selected expression host. Preferably the expression control sequence and the gene are contained within a recombinant vector containing a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the recombinant vector should further comprise a useful expression marker in the eukaryotic expression host.

The host cells transformed with the recombinant vectors described above constitute another aspect of the present invention. As used herein, the term "transformation" means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration.

Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections among a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered.

It will be apparent to those skilled in the art that the gene has the same effect as that of introducing the recombinant vector into the host cell by inserting the gene into the genome chromosome of the host cell.

In the present invention, the gene may be inserted into the chromosome of the host cell by a commonly known gene manipulation method. Examples of the method include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector , A poxvirus vector, a lentiviral vector, or a nonviral vector.

(A) reacting a selective estrogen receptor modulator (SERM) and a sugar donor in the presence of the glycosyltransferase (MrGT2) to synthesize a sugar moiety selective estrogen receptor modulator represented by the following formula (1); And (b) recovering the synthesized sugar portion of the selective estrogen receptor modulator. The present invention also relates to a method for preparing a sugar ester selective estrogen receptor modulator represented by the following formula (1).

[Chemical Formula 1]

Wherein R ₁ is CH ₃ or H, R ₂ is H ₂ or Cl, and R ₃ is But are not limited to, glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose or 2'- Glucose (2'-deoxy-glucose).

In the present invention, the selective estrogen receptor modulator (SERM) may be selected from the group consisting of tamoxifen, afimoxifene, endoxifene, raloxifene, lasofoxifene, but is not limited to, one selected from the group consisting of toremifene and 4-hydroxytoremifene.

In the present invention, the saccharide donor may be selected from the group consisting of UDP-glucose (Glc), UDP-galactose (Gal), TDP-allose, UDP- N-acetylglucosamine and TDP- Glucose (deoxy-Glc), but the present invention is not limited thereto. Examples of the added sugars include monosaccharides such as glucose, mannose, galactose, altrose, idose, fructose, and arabinose and the amino sugars may be N-acetyl-galactosamine, N-acetyl-neuraminic acid, glucosamine or galactosamine, galactosamine and in the case of di-saccharide it may be sucrose, cellobiose, maltose, trehalose, gentiobiose or melibiose, In the case of tri-saccharide, it may be raffinose or gentianose, more preferably monosaccharide such as allose, talose or xylose, ), Amino sugars such as N-acetyl-glucosamine, disaccharides May be, but is not limited to, lactose.

In the present invention, the assignment of the formula (1) a selective estrogen receptor modulator is Bahia neck when pen 4- O - glucoside, yen idoxifene 4- O - glucoside, toremifene 4- O - glucoside, Bahia neck when pen 4 - O - galactosyl side, yen idoxifene 4- O - galactosyl side, toremifene 4- O - galactosidase when side, Bahia neck pen 4- O -2'- deoxy glucoside, yen idoxifene 4- O - 2'-deoxyglucoside, 2'-deoxyglucoside and 4'- O- 2'-deoxyglucoside, but is not limited thereto.

In the present invention, step (b) may be carried out using a reversed phase C18 stationary phase, a mobile phase of a mixture of methanol: water: formic acid 65: 35: 0.2 (v / v / v / v) and a retention time It is preferable to use Medium Pressure Liquid Chromatography (MPLC), but the present invention is not limited thereto.

In a seventh aspect, the present invention relates to a selective estrogen receptor modulator (SERM) or a pharmaceutically acceptable salt thereof, wherein the sugar moiety represented by the formula (1)

[Chemical Formula 1]

Wherein R ₁ is CH ₃ , R ₂ is Cl, and R ₃ is Glucose, galactose or 2'-deoxy-glucose.

In the present invention, it is preferable that the glucose-selective estrogen receptor modulator (SERM) is a tremiphene 4- O -glucoside, a toremifene 4- O -galactoside or a toremifene 4- O- 2'-dioxy glucoside , But is not limited thereto.

In a eighth aspect, the present invention relates to a pharmaceutical composition for preventing or treating breast cancer, wherein the sugar portion contains a selective estrogen receptor modulating agent as an active ingredient, and a food for preventing or improving breast cancer.

In the present invention, the compound of Chemical Formula 1 may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid and the like can be used. As the 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.

In addition, 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 term "prophylactic " used in the present invention means any action that inhibits or delays a cancerous disease by the administration of a pharmaceutical composition comprising the compound represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof. The term "treatment ", as used herein, refers to any action that improves or ameliorates symptoms of breast cancer by administering a pharmaceutical composition comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof do.

The compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention exhibits antagonistic activity against some of the in vivo ERs, and the bioavailability of the SERM derivatives can be lowered compared to conventional SERMs, Tamoxifen, and the like, which can improve side effects such as osteoporosis and the risk of developing uterine cancer.

The pharmaceutical 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.

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.

Formulations for parenteral administration may include subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection. In this case, in order to formulate the composition for parenteral administration, 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, weight, sex, dosage form, health condition, and disease severity. In general, when referring to an adult patient weighing 70 kg, Day, preferably 0.01 to 500 mg / day, and may be administered once to several times a day at a predetermined time interval according to the judgment of a doctor or pharmacist.

[Example]

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.

Example  One: Micro Monospore Rhodolajia  From the dielectric GT  Isolation of the coding gene

In order to isolate the glycosyltransferase from the microorganism Monospora rhodorans strain, first, a genome isolated from the microorganism monospora rhodorania strain was used as a template, The polymerase chain reaction (PCR) was carried out using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, which were prepared based on the sequence, as follows. The N-terminal primer is the sequence of SEQ ID NO: 1 below and the C-terminal primer is the sequence of SEQ ID NO: 2 below.

[SEQ ID NO: 1] 5'-GG CATATG ATCCACGCGCACGACTTCCGGATG-3 '( Nde I restriction site)

[SEQ ID NO: 2] 5'-CG CTCGAG ATTCGGCCTGCGCCTCCCACGTCCA-3 '( Xho I restriction site)

The genomic DNA of the strain and the primers were mixed together with Taq DNA polymerase to perform a total of 32 cycles of PCR (initial inactivation was performed at 98 [deg.] C for 5 min, at 52.8 [deg.] C to 69.3 [ ≪ / RTI > for 5 minutes). 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. After isolating and purifying the T-iso vector from the selected transformed E. coli, the nucleotide sequence thereof (SEQ ID NO: 3) and the amino acid sequence deduced therefrom (SEQ ID NO: 4) were determined. The base sequence of the whole ORF is 1146 bp, and the translated protein is composed of 381 amino acids (total 41.1 kDa).

Example  2: Recombination in E. coli The party  enzyme MrGT2's  Expression

The MrGT2 DNA fragment obtained by treating the T-isovector of Example 1 with Nde I and Xho I restriction enzymes was subjected to restriction enzyme digestion with pET-28 (a +) (Novagen, Madison, WI, USA ), And then 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 in the LB medium (Luria Bertani) supplemented with the above-mentioned antibiotic and 1 M sorbitol and 2.5 mM betaine at a final concentration of 1% by volume, and then cultured at 30 ° C. Then, an optical density of 0.6 to 0.8 was obtained by adding isopropyl-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo., USA) When growth was confirmed, the protein expression was induced and the recombinant E. coli strain was further cultured at 22 DEG C for 18 hours. After the culture, the culture was centrifuged at 2000 rpm for 10 minutes to collect the cells, and then the cells were dissolved in 50 mM sodium phosphate lysis buffer (300 nM NaCl, 10 mM imidazole, 10% glycerol, 1% Triton X-100) Sonication was performed. After centrifugation at 12000 rpm for 20 minutes, the supernatant was collected separately, and some samples were analyzed by 12% SDS-PAGE to confirm the expression level of recombinant MrGT2.

The purified supernatant was diluted with a Talon-metal affinity resin (Clontech, Mountain View, CA, USA) equilibrated with 50 mM phosphate buffer (0.3 M NaCl, 20 mM imidazole) to purify the expressed recombinant protein. 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 solution containing 20 mM imidazole in a volume of 10 times the resin. Finally, the recombinant glycoprotein MrGT2 bound to the resin was purified with 3 ml of phosphate buffer containing 180 mM imidazole.

Example  3: MrGT2  By recombinant enzyme reaction SERM  Of the compounds The party  As a derivative Live conversion , Separation and purification

In this Example, a sugar derivative of SERM compounds was prepared by an enzymatic bioconversion method using MrGT2 instead of the conventional chemical organic synthesis method.

([Z] -afimoxifene, Sigma-Aldrich, St. Louis, Mo., USA), endoxifen hydrochloride hydrate (Sigma-Aldrich) (E) -4-hydroxy toremifene, ~ 5% Z-isomer, Toronto Research Chemicals Inc., North York, ON, Canada) was dissolved in dimethyl sulfoxide (DMSO) UDP-Glc, UDP-Gal and TDP-2'-deoxy-Glc were added to 25 μM of the recombinant MrGT2, and the nucleic acid was added to the reaction mixture. The reaction mixture was diluted with a reaction buffer (50 mM phosphate buffer, 10 mM magnesium chloride) Glc was also added at a level of 2 mM each, and the enzyme reaction was performed for 3 hours at 37 ° C (FIG. 1). That is, the reaction was stopped by adding 3 equivalents of hydroxyl group-containing SERM to the sugar receptor and 3 equivalents per nucleic acid with the sugar donor. After the enzymatic bioconversion, the reaction was stopped by addition of the same amount of ethyl acetate, followed by centrifugation at 6000 rpm for 10 minutes Only the upper organic solvent layer was collected and dried under reduced pressure. HPLC-ESI-MS analysis was performed to confirm the biosynthesis of the target compound in the extract. (Waters, 50 x 1.0 mm, 1.7 쨉 m; Milford, MA) as a mobile phase at a flow rate of 120 분 / min in methanol: water: formic acid 65: 35: 0.2 (v / v / v / v) , USA).

On the other hand, the following Combi Flash Rf MPLC system was applied to isolate and purify desired sugar-derived SERM derivatives from the crude extract. To a MPLC system in which a mixture of methanol: water: formic acid 65: 35: 0.2 (v / v / v / v) was flowed to a mobile phase at a rate of 12 ml per minute in a reverse-phased C18 cartridge, After injecting a crude extract, the peak detected in the chromatogram was automatically collected. 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. The apimoxiphenes, enoxyspenes and 4-hydroxytremeni pens used as substrates were aliquoted at a retention time of about 13 to 15 minutes on the MPLC system, and the desired sugar moieties were 10 to 12 Min. These purified sugar components are shown in FIG. 1, and the yield of the bioconversion reaction using a donor per UDP-Glc per nucleic acid was at least 50%, while in the case of UDP-Gal, the yield was 30% (TDP-2'-deoxy-Glc) was found to be 18% (4-hydroxytremeni pen as receptor) and 47% Pen) to 28% (apitoxiphen with its receptor). Particularly, in the case of toremifene having a halogen functional group as a sugar acceptor, the yield of the biodegradation was generally low, and the yield of TDP-2'-deoxy-Glc as a sugar donor was relatively lower than that of other sugar donors Which indicated a difference in specificity for the sugar receptor and sugar donor substrates of the enzyme.

NMR samples were prepared by dissolving the respective derivatives in 200 μl of DMSO-d6 and then leaving the solvent in a 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 an internal standard. All NMR data were calculated using Mnova Suite 5.3.2 software and compared to SERMs using the ¹³ C-NMR spectra of SERM derivatives as substrates.

Bahia neck when pen 4- O - glucoside (AG; 26mg; lifetime rates ^{66%; 13 C NMR [125} MHz, DMSO-d6] δ 13.3, 27.2, 47.2, 60.5, 62.0, 66.1, 71.5, 73.1, 76.7, 81.3 , 108.9, 114.2, 126.6, 127.6, 127.9, 128.5, 129.2, 131.4, 139.5, 156.4, 158.5)

Yen idoxifene 4- O - glucoside (EG, 24.7mg; lifetime rates ^{59%; 13 C NMR [125} MHz, DMSO-d6] δ 13.3, 27.0, 36.1, 51.8, 62.1, 68.6, 71.4, 73.2, 76.7, 81.3 , 190.0, 114.2, 126.6, 127.6, 127.9, 128.5, 129.2, 131.4, 139.4, 156.2, 158.3)

Toremifene 4- O - glucoside (TG, 20.4mg; lifetime rates ^{51%; 13 C NMR [125} MHz, DMSO-d6] δ 35.7, 42.7, 47.1, 60.5, 62.1, 66.2, 71.3, 73.3, 76.7, 81.4, 190.1, 114.3, 126.5, 127.8, 128.3, 129.1, 131.5, 139.4, 156.4, 158.2)

Bahia neck when pen 4- O - galactosyl side (AGL, 16.8mg; lifetime rates ^{44%; 13 C NMR [125} MHz, DMSO-d6] δ 13.3, 27.2, 47.2, 60.5, 62.3, 66.1, 71.6, 73.1, 76.9, 81.4, 108.9, 114.2, 126.6, 127.6, 127.9, 128.5, 129.2, 131.4, 139.5, 156.4, 158.5)

Yen idoxifene 4- O - galactosyl side (EGL, 18.7mg; lifetime rates ^{47%; 13 C NMR [125} MHz, DMSO-d6] δ 13.3, 27.0, 36.1, 51.8, 62.4, 68.6, 71.6, 73.2, 76.9 , 81.5, 190.0, 114.2, 126.6, 127.6, 127.9, 128.5, 129.2, 131.4, 139.4, 156.2, 158.3)

Toremifene 4- O - galactosyl side (TGL, 11.9mg; lifetime rates ^{30%; 13 C NMR [125} MHz, DMSO-d6] δ 35.7, 42.7, 47.1, 60.5, 62.1, 66.2, 71.3, 73.3, 76.7, 81.4, 190.1, 114.3, 126.5, 127.8, 128.3, 129.1, 131.5, 139.4, 156.4, 158.2)

Bahia neck when pen 4- O -2'- deoxy glucoside (ADG, 11.1mg; lifetime rates ^{28%; 13 C NMR [125} MHz, DMSO-d6] δ 13.3, 27.2, 37.4, 47.1, 60.5, 62.2, 68.9 , 71.2, 81.1, 104.5, 114.2, 126.6, 127.6, 127.9, 128.5, 129.2, 131.4, 139.5, 156.4, 158.7)

Yen idoxifene 4- O -2'- deoxy glucoside (EDG, 8.9 mg; lifetime rates ^{23%; 13 C NMR [125} MHz, DMSO-d6] δ 13.3, 27.0, 36.2, 37.5, 51.8, 62.2, 68.8, 71.6, 81.2, 104.2, 114.2, 126.6, 127.6, 127.9, 128.5, 129.2, 131.4, 139.4, 156.2, 158.6)

Toremifene 4- O -2'- deoxy glucoside (TDG, 6.8mg; lifetime rates ^{18%; 13 C NMR [125} MHz, DMSO-d6] δ 35.8, 37.4, 42.7, 47.0, 60.5, 62.1, 66.2, 68.8 , 71.3, 81.3, 104.2, 114.3, 126.5, 127.8, 128.3, 129.1, 131.5, 139.4, 156.6, 158.5)

Example  4: The party SERM  Estrogens of derivatives Receptor alpha (ER alpha)  Binding affinity test for

An NR peptide ERα ELISA kit (ActiveMotif, Carlsbad, Calif., USA) was used to test the binding affinity of the SERM derivatives to the estrogen receptor α (ERα), and the agonist activity Estradiol and tamoxifen were used as the active and antagonist active controls, respectively. The detailed experimental method was performed according to the protocol of the manufacturer. Each control dissolved in DMSO was diluted with the buffer solution contained in the kit to a level of 25 μM and the nuclear extracts were prepared according to the protocol of the kit manufacturer per well in 96-well microplates ) Was added at a level of 15 占 퐂. On the other hand, the samples were prepared at 25 μM level even for the sugar derivatives. Activity against estrogen receptor α (ERα) was quantified with a microplate reader with a final absorbance of 450 nm, and all assays were expressed as means and standard deviations of a minimum of three iterations.

As a result, antagonistic activity against ERα was evident at 25 μM level of tamoxifen, which is a positive antagonistic control, and 6 kinds of SERM derivatives (AG, AGL, ADG, EG, EGL and EDG) showed statistically significant antagonistic activity at the level of 25 μM and the tremiphene sugar fraction had relatively low antagonistic activity in the three derivatives (TG, TGL and TDG) as compared to tamoxifen (Fig. 2). These results indicate that the glucosidase and galactosidase, which are present in the nucleus extract and in addition to ERα, are present in the glucosidase and galactosidase, 4-HYBROXY SERMs to provide antagonistic activity. On the other hand, the antagonistic activity of 4-hydroxy-tramiphene, which is a metabolite, is lower than the antagonistic activity of apimociphen and endoxifene, which are the metabolism-active metabolites of tamoxifen, Respectively.

Thus, SERM derivatives of the present invention exhibit a certain level of antagonistic activity against the estrogen receptor alpha (ERa), while the triple derivatives of toremifene have lower antagonistic activity than the positive control tamoxifen alone It was confirmed that the derivatives of apimoxifen and endoxifene were similar to tamoxifen.

Example  5: The party SERM  Comparison of bioavailability of derivatives

To compare the bioavailability of the saccharide derivatives produced in Example 3 with tamoxifen and apimociphene in a clinical trial using the experimental rat model, Detailed experiments were carried out as follows. One week after the uterine incision was performed on female rats weighing about 150 g, they were used in this experiment. The four groups were designed as one treatment group, giving a total of ten groups (i.e., one group is a placebo, three groups are tamoxifen, apimociphen and endoxifene treatment control groups, AG, EG, AGL, EGL, ADG and EDG treatment groups). After one week of oral administration at 3 μmole level, 24 hours after the last administration, the body weight of each group of rats was measured and blood was collected. After collecting blood, whole blood was treated with the same volume of ethyl acetate, mixed vigorously for 30 seconds, and then centrifuged at 4000 rpm for 5 minutes. Then, the organic solvent upper layer was separated, dried under reduced pressure, and stored at 70 ° C until analysis. Quantification of the blood active metabolites apimothiphen and endoxifene was carried out by HPLC-ESI-MS instrumental analysis as described in the present invention.

As a result, the body weight of rats before administration was the same in all groups, but the average body weight of each group was measured 24 hours after the last administration, and the increase in body weight (about 30 to 40 g) Respectively. On the other hand, the blood levels of the active metabolites of tamoxifen, apimoxifene and endoxifene, were compared with those of the tamoxifen control group, with two control groups treated with apimoxifene and endoxifene and the remaining six groups A high blood level of apimoxifene and endoxifene was observed in a total of eight groups (Fig. 3). In addition, in the AG, AGL and ADG treatment groups, the concentration of apoptocopen in the blood was higher than that of the control group, apithoxifene treatment group. Similarly, in the group treated with EG, EGL and EDG, The concentration of enoxiphan in the blood was higher than that of the group treated with. This shows that, as in the result of Example 4 above, the glucoside derivatives are able to function as prodrugs metabolized by the active metabolites apimothiophen and endoxifene, and in the case of sugar derivatives, Suggesting that it may remain in the blood circulation system for a relatively long period of time rather than oral administration. As a result, it is expected that some side effects of long-term administration of tamoxifen may be partially improved.

As shown in the examples 4 and 5, the sugar moieties of the saccharide-converted saccharides in Example 3 exhibited some in vitro antagonistic activity against ER, It was confirmed that the bioavailability was improved in animals, and it could be applied as a medicine for prevention and treatment of breast cancer, and at the same time, it would reduce side effects such as osteoporosis caused by long-term administration of SERM, especially tamoxifen and the risk of developing some uterine cancer Which can be used as a new drug formulation. Therefore, it is possible to improve existing compounds through structural modification by sugar transfer of existing health functional and pharmacologically active compounds, and as such improvement includes possibility of substitution of existing drugs into prodrugs and application to new dosage forms And the potential for use as a variety of health functional foods or pharmaceuticals.

Thus, the compounds produced in the above Examples were formulated as follows.

Formulation Example 1: Tablets (Direct Pressurization)

After 5.0 mg of the compound was sieved, 14.1 mg of lactose, 0.8 mg of crospovidone (USNF), and 0.1 mg of magnesium stearate were mixed and pressed to prepare tablets.

Formulation Example 2: Tablet (wet assembly)

After 5.0 mg of the compound was sieved, 16.0 mg of lactose and 4.0 mg of starch were mixed. After 800.3 mg of polysorbate was dissolved in pure water, an appropriate amount of this solution was added to make it finely pulverized. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The mixture was pressurized to prepare tablets.

Formulation Example 3: Powder and Capsule

5.0 mg of the compound was sieved, followed by mixing with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was filtered through a hard No. 5 gelatin capsules.

Formulation Example 4: Injection

180 mg of mannitol, 26 mg of Na ₂ HPO _{4 12} H ₂ O and 2974 mg of distilled water were added to 100 mg of the active ingredient to prepare an injection.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea University Research and Business Foundation <120> Method for Preparing Glycosylated Estrogen Receptor Modulators          Using Glycosyltransferase <130> P15-B199 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> P1 <400> 1 ggcatatgat ccacgcgcac gacttccgga tg 32 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> P2 <400> 2 cgctcgagat tcggcctgcg cctcccacgt cca 33 <210> 3 <211> 1146 <212> DNA <213> Micromonospora rhodorangea <400> 3 atgcgcaccc tcagcgccac cgccggcccg cggggcgacg gggccccgta caccggccgc 60 ggcgcggccc ggcggcgcgc ggggcacgac gtggccgtcg ccacgaccga cacgtcggcc 120 cgagtggtcc gggagcccgg gccagggttc cgggccctcc cggccgacca ccgcgcgcac 180 gcgggctggc ggccccaccg cgaggtcgtg cgcgcggccg gcgggcaccg cggaactccg 240 ccagccgttc gccgacgcgc cgtctcggag ggcacggacc tccggccgcg gccgacgacc 300 accgcgccgc tgcggtggct cagcggcgag gcgacggccc cccgggacct ccggcgcgac 360 caccagccca ccgccaccac cgccgacggc ccgccggtcg tcaccggcgc gcgctccctg 420 gggcggcccg gcaaccgcac ggccggccgc ctggccctgc gcatggccga ccggatctac 480 gccgaggccg tcgcgcgcct gcgggagcgg ctgtgcctgc cgccgctgtc cgcggccgcg 540 atgccggcgc ggcaggagcg ggccggctgg cccgtcctgc acggcttcgg cacggccctc 600 gtcccgcggc cggccgactg gcgccccggc ctcgaggtcg tcgggccgtg gtggccgcac 660 ccgaggccg gcgagcgcct gccctcggaa ctcgaggact tcctggacgc gggaccgcgg 720 ccggtcctgg tgcgcttcgg cagcatggcc gcccggcacg gcgagcggct cagcgaactg 780 gcggtgcgcg cgctgcgccg cgccggcctc cgcggcatcc tccagtccgg caacgcctgc 840 ctcgcggcgg agggcgacga cgtcctgacg gtcggcgacg tcccccacgc cctcctgttc 900 ccccggctcg cggcggtggt gcaccacgcg cgccggcacc agcgccgcga ccctgcgggc 960 ggccgtaccc tcggtggcgg tcccggtgac cgccgaccag agccgttctg ggccggccgg 1020 ctggcgcgga tccgggccgc cccagccccg gtccccttca cgtcgtcggc gacgaggcgt 1080 cgccgcgcgc gtgtga 1146 <210> 4 <211> 381 <212> PRT <213> Micromonospora rhodorangea <400> 4 Met Arg Thr Leu Ser Ala Thr Ala Gly Pro Arg Gly Asp Gly Ala Pro   1 5 10 15 Tyr Thr Gly Arg Gly Ala Ala Arg Arg Ala Gly His Asp Val Ala              20 25 30 Val Ala Thr Thr Asp Thr Ser Ala Arg Val Val Arg Glu Pro Gly Pro          35 40 45 Gly Phe Arg Ala Leu Pro Ala Asp His Arg Ala His Ala Gly Trp Arg      50 55 60 Pro His Arg Glu Val Val Arg Ala Gly Gly His Arg Gly Thr Pro  65 70 75 80 Pro Ala Val Arg Arg Ala Val Ser Glu Gly Thr Asp Leu Arg Pro                  85 90 95 Arg Pro Thr Thr Thr Ala Pro Leu Arg Trp Leu Ser Gly Glu Ala Thr             100 105 110 Ala Pro Arg Asp Leu Arg Arg Asp His Gln Pro Thr Ala Thr Thr Ala         115 120 125 Asp Gly Pro Pro Val Val Thr Gly Ala Arg Ser Leu Gly Arg Pro Gly     130 135 140 Asn Arg Thr Ala Gly Arg Leu Ala Leu Arg Met Ala Asp Arg Ile Tyr 145 150 155 160 Ala Glu Ala Val Ala Arg Leu Arg Glu Arg Leu Cys Leu Pro Pro Leu                 165 170 175 Ser Ala Ala Met Pro Ala Arg Gln Glu Arg Ala Gly Trp Pro Val             180 185 190 Leu His Gly Phe Gly Thr Ala Leu Val Pro Arg Pro Ala Asp Trp Arg         195 200 205 Pro Gly Leu Glu Val Val Gly Pro Trp Trp Pro His His Hisp Ala Gly     210 215 220 Glu Arg Leu Pro Ser Glu Leu Glu Asp Phe Leu Asp Ala Gly Pro Arg 225 230 235 240 Pro Val Leu Val Arg Phe Gly Ser Met Ala Ala Arg His Gly Glu Arg                 245 250 255 Leu Ser Glu Leu Ala Val Arg Ala Leu Arg Arg Ala Gly Leu Arg Gly             260 265 270 Ile Leu Gln Ser Gly Asn Ala Cys Leu Ala Ala Glu Gly Asp Asp Val         275 280 285 Leu Thr Val Gly Asp Val Pro His Ala Leu Leu Phe Pro Arg Leu Ala     290 295 300 Ala Val Val His His Ala Arg Arg His Gln Arg Arg Asp Pro Ala Gly 305 310 315 320 Gly Arg Thr Leu Gly Gly Gly Pro Gly Asp Arg Arg Pro Glu Pro Phe                 325 330 335 Trp Ala Gly Arg Leu Ala Arg Ile Arg Ala Ala Pro Ala Pro Val Pro             340 345 350 Phe Thr Ser Ser Ala Thr Arg Arg Thr Arg Gly Pro Leu Gly Arg Arg         355 360 365 Arg Pro Asp Leu Ala Ala Glu Asp Gly Ala Ala Arg Val     370 375 380

Claims (16)

(MrGT2) represented by the amino acid sequence of SEQ ID NO: 4.
A polynucleotide encoding the enzyme of claim 1.
3. The polynucleotide according to claim 2, wherein the polynucleotide is represented by the nucleotide sequence of SEQ ID NO: 3.
3. The polynucleotide according to claim 2, wherein the polynucleotide is derived from a strain of Micromonospora rhodorangea .
An expression vector comprising the polynucleotide of claim 2.
A recombinant microorganism into which the polynucleotide of claim 2 or the expression vector of claim 5 has been introduced.
The microorganism according to claim 5, wherein the microorganism is E. coli.
A method for producing recombinant herpes zoster (MrGT2) comprising culturing the microorganism of claim 6 to induce the expression of the glycosyltransferase and recovering the enzyme.
A method for preparing a sugar-free selective estrogen receptor modulator (SERM) represented by the following formula (1)
(a) reacting a selective estrogen receptor modulator and a sugar donor in the presence of the glycoprotein (MrGT2) of claim 1 to synthesize a sugar moiety selective estrogen receptor modulator represented by the following formula (1); And
(b) recovering the synthesized sugar portion of the selective estrogen receptor modulator;
[Chemical Formula 1]

Wherein R ₁ is CH ₃ or H, R ₂ is H ₂ or Cl, and R ₃ is But are not limited to, glucose, galactose, allose, tallose, xylose, N-acetyl-glucosamine, lactose or 2'- Glucose (2'-deoxy-glucose).
10. The method of claim 9, wherein the selective estrogen receptor modulator (SERM) is selected from the group consisting of tamoxifen, afimoxifene, endoxifene, raloxifene, lasofoxifene, Wherein the selective regulator is any one selected from the group consisting of toremifene and 4-hydroxytoremifene.
10. The method of claim 9, wherein the sugar donor is selected from the group consisting of UDP-glucose (Glc), UDP-galactose (Gal), TDP-allose, UDP-N-acetylglucosamine and TDP- And deoxy-Glc. &Lt; / RTI &gt;&lt; Desc / Clms Page number 19 &gt;
The method of claim 9, wherein the assignment is a selective estrogen receptor modulator of the formula (1) is Bahia neck when pen 4- O - glucoside, yen idoxifene 4- O - glucoside, toremifene 4- O - glucoside, Bahia neck when pen 4- O - galactosyl side, yen idoxifene 4- O - galactosyl side, toremifene 4- O - galactosyl side, Bahia pen when neck 4- O -2'- deoxy glucoside, yen idoxifene 4- O process for producing a 2'-deoxy-glucoside and toremifene 4- O-2'-deoxy-glucose tolerance is a selective estrogen receptor modulator, characterized in that any one of selected from the group consisting of glucosides.
10. The method of claim 9, wherein step (b) is performed using a reversed phase C18 stationary phase, a mobile phase of a mixture of methanol: water: formic acid 65: 35: 0.2 (v / v / v / v) and a retention time of 15-19 min. Wherein the method comprises using Medium Pressure Liquid Chromatography (MPLC).
Wherein the sugar moiety represented by Formula 1 is a selective estrogen receptor modulator (SERM) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

Wherein R ₁ is CH ₃ , R ₂ is Cl, and R ₃ is Glucose, galactose or 2'-deoxy-glucose.
The pharmaceutical composition for preventing or treating breast cancer according to claim 14, wherein the sugar moiety contains a selective estrogen receptor modulating agent as an active ingredient.
14. The food for preventing or improving breast cancer according to claim 14, wherein the sugar portion contains a selective estrogen receptor modulating agent as an active ingredient.

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