KR101835108B1 - Pharmaceutical Composition for Preventing or Treating Diabetes Comprising Recombinant Adenovirus Expressing PREB - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the prevention or treatment of diabetes comprising as an active ingredient a recombinant adenovirus comprising a nucleotide sequence encoding a prolactin regulatory element-binding protein (PREB). The composition of the present invention can effectively prevent and treat diabetes, especially type 2 diabetes, by reducing the resistance of glucose and pyruvic acid and inhibiting your biosynthesis.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating diabetes mellitus comprising recombinant adenovirus expressing PREB as an active ingredient.

The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetes comprising recombinant adenovirus expressing PREB as an active ingredient.

Diabetes mellitus is an increasing worldwide trend as one of representative metabolic diseases with hypertension, obesity, atherosclerosis and hyperlipidemia due to over consumption, stress and lack of exercise due to an increase in life expectancy and improvement of living standards of a person, The global market for diabetes treatments is also on the rise. The fundamental pathogenesis of type 2 diabetes is the lack of understanding of the molecular level of insulin resistance, making it difficult to develop new drugs. In addition, one of the characteristics of type 2 diabetes is that the amount of glucose in the liver is increased. It is very important to control the amount of glucose in the blood to treat diabetes. Recently, a method of treating diabetes by controlling the biosynthesis process has been suggested. However, there has been no report on the role of PREB in your biosynthesis process in relation to diabetes. Thus, it is required to develop a therapeutic agent for diabetes and diabetic complications using the target protein, in view of the fact that it can be a target of diabetic and diabetic therapeutic agents effectively through quantitative regulation of PREB.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Mol Endocrinol. 1999 Apr; 13 (4): 644-57 Atherosclerosis. 2009 Nov; 207 (1): 45-50 J Cell Mol Med. 2009 Aug; 13 (8B): 2386-95 Diabetes. 2016 Jan; 65 (1): 62-73

As a result of in-depth study on a method for effectively preventing or treating diabetes, the present inventors have found that recombinant adenovirus having the nucleotide sequence of PREB (Prolactin regulatory element-binding protein) reduces the resistance of glucose and pyruvic acid, The present inventors have confirmed that diabetes can be effectively prevented and treated, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diabetes.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a recombinant adenovirus comprising a nucleotide sequence of Sequence Listing No. 1 sequence encoding a prolactin regulatory element-binding protein (PREB) and transferring the nucleotide to liver tissue And a pharmaceutical composition for preventing or treating diabetes mellitus, which is contained as an active ingredient.

As a result of in-depth study on a method for effectively preventing or treating diabetes, the present inventors have found that recombinant adenovirus having a PREB coding nucleotide sequence reduces the resistance of glucose and pyruvic acid and effectively inhibits and cures diabetes by inhibiting biosynthesis And it was confirmed experimentally.

According to the present invention, the PREB to be delivered into cells in the present invention is overexpressed in liver tissues by the recombinant adenovirus of the present invention and increases glucose and pyruvate tolerance.

According to one embodiment of the present invention, the increase in glucose and pyruvic acid resistance by administration of the composition of the present invention is induced by a decrease in the expression of your biosynthetic genes by overexpression of PREB. Your biosynthetic-related genes whose expression is reduced by the composition of the present invention are Pck (phosphoenolpyruvate carboxykinase) and G6pc (Glucose-6-phosphatase).

Insulin is a hormone that maintains balance in the body by storing glucose in the form of glycogen in the blood or by inhibiting the biosynthesis of the liver. When insulin secretion is abnormal, glucose is not properly removed or stored, and it is excreted in the urine to cause diabetes. Insulin is known to modulate glucose levels in the blood by regulating the gene expression of enzymes necessary for your biosynthesis in the liver. In addition, it is known that glucose is not inhibited in rats or obese model rats that are genetically impaired by insulin receptor, and blood glucose is increased (Diabetes. 2016 Jan; 65 (1): 62-73). Therefore, the composition of the present invention for inhibiting glycation in a diabetic mouse model has a preventive and therapeutic effect on diabetes, particularly, type 2 diabetes.

According to one embodiment of the present invention, the recombinant adenovirus in the composition of the present invention is produced by a recombinant adenovirus vector in which the viral gene El region and the E3 region have been deleted.

The cell line for producing the recombinant adenovirus of the present invention may be a cell line for producing adenovirus, and it is most preferable to use 293 cells which is an adenovirus producing cell line, but the present invention is not limited thereto.

In the present invention, the nucleotide sequence encoding the PREB to be delivered into the cell is preferably inserted into the deleted E1 region or E3 region of the recombinant adenovirus vector. The term " deletion " as used herein in connection with a viral genome sequence has the meaning not only of a complete deletion of the sequence but also of a partial deletion thereof.

The nucleotide sequence encoding the PREB of the present invention is preferably present in a suitable expression construct.

As used herein, the term " expression construct " refers to the minimal elements for expression, including the nucleotide sequence of interest for expression and an expression sequence (e.g., a promoter) that directs expression of this sequence. Such an expression construct preferably comprises a nucleotide sequence-polyadenylation sequence for transcriptional control sequence-expression purposes. In the present invention, a vector may be constructed such that the expression of the sequence encoding the PREB is carried out in an individual expression construct. For example, a recombinant adenovirus vector comprising the expression construct of " promoter-PREB coding nucleotide sequence-polyadenylation sequence operable in eukaryotic cells " can be produced.

As used herein, the term " a promoter operable in eukaryotic cells " means a transcriptional control sequence capable of inducing the transcription of a desired gene in eukaryotic cells. Each subunit coding nucleotide sequence is operatively linked to a promoter. As used herein, the term " operably linked " means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The regulatory sequence will regulate transcription and / or translation of the other nucleic acid sequences.

In the present invention, the promoter bound to the PREB coding nucleotide sequence is preferably capable of regulating the transcription of the PREB coding nucleotide sequence by working in an animal cell, more preferably a mammalian cell, and comprises a promoter derived from a mammalian virus Promoter, cytomegalo virus (CMV) promoter, late adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV (promoter of HSV), and promoter derived from the genome of mammalian cells. Promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of EF1 alpha promoter, metallothionein promoter, beta -actin promoter, human IL- Gene promoter, an inducible promoter, a cancer cell specific A promoter (e.g., the TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, and the AFP promoter) and a tissue specific promoter, including, (e. G., Albumin promoter), and the like. Most preferably, it is a CMV promoter.

A gene map of a recombinant adenovirus comprising the nucleotide sequence encoding the PREB of the present invention is disclosed in detail in FIG.

The method of introducing the recombinant adenovirus of the present invention into cells can be carried out through various methods known in the art.

In the present invention, the recombinant adenovirus was produced based on a viral vector, and thus can be carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the above cited documents.

22: 479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094 (1980)) when the gene delivery system is an naked recombinant DNA molecule or plasmid in the present invention. 7: 2745-2752 (1987)), calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456 (1973), and Chen and Okayama, Mol. Cell. Biol. (1986)), liposome-mediated trafficking of the liposome-mediated trait (Neumann, E. et al., EMBO J. 1: 841 Methods: Enzymol., 149: 282 (1982), and Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1987)), DEAE-dextran treatment (Gopal, MoI. Cell Biol., 5: 1188-1190 (1985)), and gene bend buddhite (Yang et al., Proc. Natl. , ≪ / RTI > 87: 9568-9572 (1990)).

The pharmaceutically acceptable carriers to be contained in the composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition of the present invention is preferably parenterally administered, and can be administered, for example, by intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and responsiveness of the patient Ordinarily skilled physicians can easily determine and prescribe dosages effective for the desired treatment. In general, the pharmaceutical composition of the present invention comprises 1 x 10 < ⁵ > - 1 x 10 < ¹⁵ > pfu / ml of recombinant adenovirus.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a pharmaceutical composition for preventing or treating diabetes comprising as an active ingredient a recombinant adenovirus comprising a nucleotide sequence encoding a prolactin regulatory element-binding protein (PREB).

(Ii) The composition of the present invention can effectively prevent and treat diabetes, especially type 2 diabetes, by reducing the resistance of glucose and pyruvic acid and inhibiting your biosynthesis.

Figure 1 is a schematic diagram of Ad-PREB (a) and Ad-GFP vectors.
FIG. 2 is a graph showing the results of immunoprecipitation of recombinant adenovirus expressing Ad-PREB or Ad-GFP in normal mice (C57BL / 6J) by intravenous injection, followed by a glucose tolerance test (GTT) (Pyruvate Tolerance Test, PTT).
The X axis is expressed in hours (minutes), and the Y axis is the result of measuring the amount of blood glucose measured by the blood glucose meter.
Figure 3 shows that recombinant adenovirus expressing Ad-PREB or Ad-GFP is transfected into a diabetic mouse (db / db) by intravenous injection followed by a glucose tolerance test (GTT) and a fibrinolytic (Pyruvate Tolerance Test, PTT).
The X axis is expressed in hours (minutes), and the Y axis is the result of measuring the amount of blood glucose measured by the blood glucose meter. In the case of the blood glucose level, the maximum of 600 mg / ml is measured and higher than that, so high is predicted to be 600 mg / ml.
FIG. 4 shows the results of transfection of normal venous (C57BL / 6J) and diabetic mice (db / db) with recombinant adenovirus expressing Ad-PREB or Ad-GFP, The mice were sacrificed one day later in the afternoon, and plasma obtained from the whole blood collection was assayed for the amount of insulin using a Mouse Ultrasensitive ELISA.
The X-axis represents the recombinant adenovirus, and the Y-axis represents the amount of insulin.
FIG. 5 shows the results of transfection of recombinant adenovirus expressing Ad-PREB or Ad-GFP into the normal vein of normal mice (C57BL / 6J) and diabetic mice (db / db) The mouse was sacrificed in the afternoon and the obtained liver tissue was extracted with Tri-zol, and cDNA samples were prepared using a reverse-transcription system kit. The primers were used to express each gene. Real- MRNA expression was measured by Time PCR.
Each gene was standardized using 18S.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and methods

Production, production and potency of recombinant adenoviral vectors

To prepare recombinant adenovirus expressing mouse PREB (Prolactin Regulatory Element Binding; NCBI Accesstion No. BC017527), adenovirus vector provided in Takara's Adenovirus Expression Vector Kit (Cat. # 6170) was cut through SwaI and PCR The PREB gene was inserted into the cell line. Adenovirus vectors deficient in E1 and E3 were used to prevent the adenovirus itself from growing. The adenovirus vector into which the PREB gene had been inserted was digested with PacI and the linearized DNA fragment was transfected into an AD-293 cell line (Cat # 240085) containing E1 of Stratagene, and then concentrated with a CsCl concentration gradient to purely isolate the adenovirus And the plaque forming unit (PFU) was calculated using Clontech's Adenovirus Titration Kit. As a control, a recombinant adenovirus vector (Ad-GFP) expressing green fluorescent protein (GFP) without an expression cassette was used.

Animal experiment

Seven male male C57BL / 6J and C57BLKS / J-db / db mice were received from the central laboratory animals, which allowed them to ingest standard rodent diets and water as desired, and with rigorous accreditation by the Yonsei University Medical Center Ethical Laboratory Ethics Committee The experiment was carried out.

Glucose Tolerance Test (GTT) was performed by adding each recombinant adenovirus, and fasting was performed from the evening after 3 days. After the oral administration, glucose was administered at 0, 10, 20 , 30, 45, 60, 90, and 120 minutes, and blood glucose was measured using a glucose meter.

The Pyruvate Tolerance Test (PTT) was performed by infusing each recombinant adenovirus 3 days after the fasting and intraperitoneal injection (Pyruvate Tolerance Test, PTT) was performed so that pyruvate was 1.5 g / kg the following morning Blood was obtained from tail cut with scissors at 0, 10, 20, 30, 45, 60, 90, and 120 minutes after injection and blood glucose was measured using a glucose meter. After the completion of all the experiments, the mice were killed by CO ₂ inhalation anesthesia the following afternoon, and whole blood collection was performed using a 1 ml syringe to obtain liver and adipose tissue.

Recombinant adenovirus treatment in animals

The mice were fixed in an intravenous holder and recombinant adenoviruses expressing Ad-PREB (2 * 10 ¹⁰ pfu / ml) and Ad-GFP (2 * 10 ¹⁰ pfu / ml) Tail Vein) were injected with 100 μl each.

Plasma insulin measurement

Plasma insulin levels were measured using an insulin measurement ELISA (ALPCO).

Real-Time PCR for gene expression confirmation

A portion of the liver tissue obtained from the mouse was cut out, the liver tissue was finely ground using a Qiagen Tissue Lyzer, and RNA was obtained from each tissue using the Tri-Zol (Invitrogen) Protocol. A cDNA sample was prepared using the reverse transcription kit with the obtained RNA, and then oligos (18s-for; agtccctgccctttgtacaca, 18s-rev; cgatccgagggcctcacta, Preb-for; gactcttcacagtgcagata, Preb-rev; gaaatgacctcatggccaca, Real-Time PCR was performed using SYBG and pgcaggaggaccaaggaagc) and SYBG for pck-for; tggccaggatcgaaagcaaga, pck-rev; gccccatgctgaatgggatg, g6pc-for; gccagaatgggtccaccttg, g6pc-rev; The quantification was quantified using 18s and each was statistically treated (* p≤0.05, ** p≤0.01).

Experiment result

When the PREB gene was transfected into a normal mouse (C57BL / 6J) using an intravenous injection using the recombinant adenovirus vector of the present invention, PREB was overexpressed in mouse liver, and glucose tolerance test and pyruvic acid resistance test were performed 2). It can be confirmed that glucose and pyruvic acid resistance are improved in a mouse in which PREB is overexpressed.

In addition, when PREB gene was transfected into a diabetic mouse (db / db) using an intravenous injection using the recombinant adenovirus vector of the present invention, PREB was overexpressed in mouse liver, and glucose tolerance test and pyruvic acid resistance test were conducted (Fig. 3).

As above, it can be confirmed that the glucose and pyruvic acid resistance of the PREB-overexpressed mouse is significantly improved as compared with the green fluorescent protein recombinant adenovirus lacking the expression cassette.

As a result of measuring the amount of insulin in plasma after induction of PREB overexpression, it was confirmed that the amount of insulin between green fluorescent protein without expression cassette and recombinant adenovirus of PREB was not statistically changed (FIG. 4).

In addition, mRNA levels were measured to determine if the results of the experiments (FIGS. 2 and 3) through the resistance of glucose and pyruvic acid were due to a decrease in Pck and G6pc, which are important genes involved in the biosynthesis process (Gluconeogenesis) ). The overexpression of PREB suggests that Pck and G6pc are involved in the biosynthesis process, and overexpression of PREB induces a decrease in the expression of your biosynthetic genes, thereby reducing sugar resistance and pyruvic acid resistance.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Pharmaceutical Composition for Preventing or Treating Diabetes          Comprising Recombinant Adenovirus Expressing PREB <130> PN160112 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 2211 <212> DNA <213> Prolactin Regulatory Element Binding <400> 1 cctgcgcgga gggagccgcg agacaggtgc gcatgcgcag tgcgcgtctg cgagaccgac 60 ttggacggag ccgagctgag gctcggcttc ctgctgatgg tcagggtttt ggcaactccc 120 cggtgtgaga ggggtaggga gtgctcccgg cggcgacggg gccgagttca ccagccgccg 180 gggcagtagt cgaaggcccg gcgcggcatg tcctgggtgc cgcggtgcgg gcagtgaacg 240 cgcgccgggc gggatgggcc ggcgccgggc gccagagctg taccgggctc cgttcccgtt 300 gtacgcgctt caggtcgacc ccagcactgg gctgctcatc gctgcgggcg gaggaggcgc 360 cgccaagaca ggcataaaga atggcgtgca ctttctgcag ctagagctga ttaatgggcg 420 cttgagtgcc tccttgctgc actcccatga cacagagaca cgggccacca tgaacttggc 480 actggctggt gacatccttg ctgcagggca ggatgcccac tgtcagctcc tgcgcttcca 540 ggcacatcaa cagcagggca acaaggcaga gaaggccggt tccaaggagc aggggcctcg 600 acaaaggaag ggagcagccc cagcagagaa gaaatgtgga gcggaaaccc agcacgaggg 660 gctagaactc agggtagaga atttgcaggc ggtgcagaca gactttagct ccgatccact 720 gcagaaagtt gtgtgcttca accacgataa taccctgctt gccactggag gaacagatgg 780 ctacgtccgt gtctggaagg tgcccagcct ggagaaggtt ctggagttca aagcccacga 840 aggggagatt gaagacctgg ctttagggcc tgatggcaag ttggtaaccg tgggccggga 900 ccttaaggcc tctgtgtggc agaaggatca gctggtgaca cagctgcact ggcaagaaaa 960 tggacccacc ttttccagca caccttaccg ctaccaggcc tgcaggtttg ggcaggttcc 1020 agaccagcct gctggcctgc gactcttcac agtgcaaatt ccccacaagc gcctgcgcca 1080 gccccctccc tgctacctca cagcctggga tggctccaac ttcttgcccc ttcggaccaa 1140 gtcctgtggc catgaagtcg tctcctgcct cgatgtcagt gaatccggca ccttcctagg 1200 cctgggcaca gtcactggct ctgttgccat ctacatagct ttctctctcc agtgcctcta 1260 ctacgtgagg gaggcccatg gcattgtggt gacggatgtg gcctttctac ctgagaaggg 1320 tcgtggtcca gagctccttg ggtcccatga aactgccctg ttctctgtgg ctgtggacag 1380 tcgttgccag ctgcatctgt tgccctcacg gcggagtgtt cctgtgtggc tcctgctcct 1440 gctgtgtgtc gggcttatta ttgtgaccat cctgctgctc cagagtgcct ttccaggttt 1500 cctttagctt ccctgcttcc tgggaatcag gagcctggac actgccatct ctagagcaga 1560 gtggaggcct ggactccctt tgctcactcc attcgggtcc acagctgagg ttgcctctga 1620 caagatgaat gggcactgcc tgcccttcta gtgaaaaggc ttggctatgg ccctgtgtga 1680 ctccaggtcc caggaacctt gccttcgtca tctgtggatc catccagaac agcggtatct 1740 gaagcccagg ccatactccc tgcctccttt cttctgccta ccagaggctc cagagttgag 1800 cttgtcctta tctagaaaca tgtgaagatg cccaagagcc tggaggcact gctgtccttc 1860 ctgcagaaac agtttctcct cctcccctca gccttgtggc cagttcctct tcacatgaag 1920 cccctggcat ttgctgggga agggactggc ctggtacttg ctgttagggc aggaaggggc 1980 aaaaggaaga cttgggtagt aatctggggg ttcagatggg tagcactaag ccagctggcc 2040 taaagatgca ataagttcct aggtagtcta cccttacctt gaggaatggg aaaatgaacc 2100 tcagcccatt aggcaggaaa agttgatatt taataaacaa ggaaagagtg aactgagacc 2160 ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2211 <210> 2 <211> 21 <212> DNA <213> 18s Forward Primer <400> 2 agtccctgcc ctttgtacac a 21 <210> 3 <211> 19 <212> DNA <213> 18s Reverse Primer <400> 3 cgatccgagg gcctcacta 19 <210> 4 <211> 20 <212> DNA <213> Preb Forward Primer <400> 4 gactcttcac agtgcagata 20 <210> 5 <211> 20 <212> DNA <213> Preb Reverse Primer <400> 5 gaaatgacct catggccaca 20 <210> 6 <211> 21 <212> DNA <213> pck Forward Primer <400> 6 tggccaggat cgaaagcaag a 21 <210> 7 <211> 20 <212> DNA <213> pck Reverse Primer <400> 7 gccccatgct gaatgggatg 20 <210> 8 <211> 20 <212> DNA <213> g6pc Forward Primer <400> 8 gccagaatgg gtccaccttg 20 <210> 9 <211> 20 <212> DNA <213> g6pc Reverse Primer <400> 9 tgcaggagga ccaaggaagc 20

Claims (7)

Type 2 diabetes preventive or therapeutic method comprising recombinant adenovirus comprising the nucleotide sequence of Sequence Listing 1 sequence encoding PREB (Prolactin regulatory element-binding protein) and transferring the nucleotide to liver tissue as an active ingredient, A pharmaceutical composition for therapeutic use. The pharmaceutical composition for the prevention or treatment of type 2 diabetes according to claim 1, wherein the recombinant adenovirus is produced by a recombinant adenovirus vector in which the viral gene E1 region and the E3 region are deleted. The pharmaceutical composition for the prevention or treatment of type 2 diabetes according to claim 1, wherein the PREB coding nucleotide sequence is inserted into the gene E1 region of the recombinant adenovirus vector. The pharmaceutical composition for the prevention or treatment of type 2 diabetes according to claim 1, wherein the PREB is overexpressed in liver tissue. The pharmaceutical composition for the prevention or treatment of type 2 diabetes according to claim 1, wherein the composition reduces glucose and pyruvate tolerance. The pharmaceutical composition according to claim 1, wherein the composition inhibits gluconeogenesis. The pharmaceutical composition for the prevention or treatment of type 2 diabetes according to claim 1, wherein the composition reduces the expression of genes Pck (phosphoenolpyruvate carboxykinase) and G6pc (Glucose-6-phosphatase) related to your biosynthesis.

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