WO2020118843A1 - Mutant de glp-1, procédé de préparation et application associés - Google Patents

Mutant de glp-1, procédé de préparation et application associés Download PDF

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WO2020118843A1
WO2020118843A1 PCT/CN2019/071736 CN2019071736W WO2020118843A1 WO 2020118843 A1 WO2020118843 A1 WO 2020118843A1 CN 2019071736 W CN2019071736 W CN 2019071736W WO 2020118843 A1 WO2020118843 A1 WO 2020118843A1
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solution
recombinant
seq
bacteria
nucleotide sequence
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PCT/CN2019/071736
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Chinese (zh)
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顾建文
马婕
马永平
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四川利通科创生物医药科技有限公司
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Priority claimed from CN201811521016.1A external-priority patent/CN110251662B/zh
Priority claimed from CN201811519742.XA external-priority patent/CN110256553B/zh
Priority claimed from CN201811520996.3A external-priority patent/CN110251661B/zh
Application filed by 四川利通科创生物医药科技有限公司 filed Critical 四川利通科创生物医药科技有限公司
Publication of WO2020118843A1 publication Critical patent/WO2020118843A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a GLP-1 mutant and its preparation method and use.
  • T2DM Type 2 Diabetes Mellitus
  • overweight and obesity will further aggravate the insulin resistance of T2DM patients, and greatly increase the risk of cardiovascular disease and other complications.
  • Strengthening weight loss can bring clear benefits to patients with T2DM: reducing insulin resistance, repairing damaged islet ⁇ -cell function, optimizing blood sugar control, and improving risk factors related to cardiovascular disease.
  • the vicious circle caused by obesity and T2DM makes the traditional treatment mode with "hypoglycemic" as the core into a dilemma, which also makes more and more scholars pay attention to the importance of "weight loss” treatment.
  • weight loss drugs bring good weight loss efficacy, they gradually withdraw from the market due to serious safety problems.
  • the weight loss drugs currently in clinical use are mainly orlistat capsules approved in 1999, lorcaserin and phentermine and topiramate capsules approved in 2012, and naltrexone hydrochloride and bupropion compound sustained release approved in 2014. Tablets and liraglutide injection.
  • Liraglutide is a glucagon-like peptide-1 (Glucagon-likepeptide-1, GLP-1)-like peptide and is a new type of drug based on the action of incretin.
  • GLP-1 analogue peptides mainly reduce the energy intake of patients by inhibiting central appetite for appetite and inhibiting gastrointestinal motility and increasing satiety, thereby exerting the effect of weight loss.
  • Linaclutide has a glucose concentration-dependent hypoglycemic effect, and monotherapy does not cause hypoglycemia.
  • Liraglutide was launched in the United States and the European Union in 2009 and 2010 for the treatment of type 2 diabetes (T2DM).
  • T2DM type 2 diabetes
  • the FDA and the European Medicines Agency approved it as a supplement to diet control and physical exercise for the treatment of chronic obesity.
  • the effect is better than the mainstream weight loss drug on the market-orlistat.
  • the weight loss effect is clear.
  • Liraglutide was approved for listing in China in 2011. Long-term trials have shown that liraglutide can effectively reduce the weight of patients.
  • the suitable population is adults with a body mass index (BMI) greater than or equal to 30, or a BMI of 27 or more, accompanied by T2DM, hypertension, or high cholesterol. An adult with complications of obesity.
  • BMI body mass index
  • GLP-1 like peptide polypeptide drugs including liraglutide need to be administered by injection.
  • Direct oral administration is ineffective, which has the disadvantages of inconvenience.
  • the treatment of diabetes usually requires long-term, continuous medication.
  • the method of injection administration is inconvenient to use and carry, and the medication compliance is poor.
  • oral preparations are most in line with people's habit of "taking" medicines, and the preparation process is mature and simple. Therefore, it is of great significance to start by changing the route of administration of GLP-1 analogue peptides, to develop medicines and to carry them conveniently, and to be suitable for long-term use of GLP-1 analogue peptides.
  • the purpose of the present invention is to provide a GLP-1 mutant and its preparation method and use.
  • the present invention provides a GLP-1 mutant, which is obtained on the basis of wild-type GLP-1 through amino acid site mutation, and its amino acid sequence is shown in SEQ ID NO: 1.
  • the invention also provides a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1.
  • the present invention provides a recombinant vector comprising the above nucleotide sequence; further, the recombinant vector is a recombinant prokaryotic vector; further, the prokaryotic vector is a pGEX plasmid or a pMG36e vector.
  • the present invention provides a recombinant bacterium, characterized in that it contains the above-mentioned recombinant vector; further, the recombinant bacterium is recombinant E. coli Nissle 1917 or recombinant lactic acid bacterium.
  • the present invention provides a method for preparing the above-mentioned GLP-1 mutant, which includes the following steps:
  • the medium is LB medium
  • the medium is MRS medium.
  • the present invention provides the use of the above mutants, nucleotide sequences, recombinant vectors, and recombinant bacteria in the preparation of a medicament for treating diabetes.
  • the present invention provides a medicament for treating diabetes, which contains the above mutant, nucleotide sequence, recombinant vector and/or recombinant bacteria.
  • the present invention provides the use of the above mutants, nucleotide sequences, recombinant vectors, and recombinant bacteria in the preparation of drugs for treating obesity.
  • the invention also provides a medicine for weight loss, which has an amino acid sequence as shown in SEQ ID NO: 1 GLP-1 mutant, SEQ ID NO: 2 nucleotide sequence, including SEQ ID NO: 2 Recombinant vectors of nucleotide sequences and/or recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2 are effective ingredients.
  • the invention also provides a health food for weight loss, which has an amino acid sequence as shown in SEQ ID NO: 1 GLP-1 mutant, SEQ ID NO: 2 nucleotide sequence, including SEQ ID NO: 2
  • SEQ ID NO: 2 nucleotide sequence, including SEQ ID NO: 2
  • the recombinant vector of nucleotide sequence and/or the recombinant bacterium containing the nucleotide sequence shown in SEQ ID NO: 2 is the active ingredient.
  • the invention provides a medicament for treating diabetes or weight loss, which has an amino acid sequence as shown in SEQ ID NO: 1 GLP-1 mutant, SEQ ID NO: 2 nucleotide sequence, including SEQ ID NO:
  • the preparation is an oral preparation.
  • the oral preparation is a tablet, capsule, powder, granule or oral liquid.
  • the powder is a lyophilized powder.
  • the lyophilized powder is prepared according to the following method: take the bacterial precipitate of the recombinant bacterium containing the nucleotide sequence shown in SEQ ID NO: 2, and resuspend it with a 20% (w/v) oligofructose solution Bacteria, then add 15% (w/v) glycerol solution, 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and 3% (w /v) trehalose solution, prepared into a mixed liquid of 10 10 ⁇ 10 12 cfu live bacteria per gram, lyophilized after filling, and the mixed liquid contains fructooligosaccharide solution, glycerin solution, and skimmed milk powder
  • the volume ratio of the solution, lactose solution and trehalose solution is (1-10): (1-10): (1-10): (1-10), preferably 2:3:5: 6:1; preferably, the number of viable bacteria per gram of mixed liquid
  • the powder is a dry powder
  • the dry powder according to the following method: take the bacterial precipitate of recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2, add a low concentration of 20% (w/v) Resuspend the bacteria in the polyfructose solution, then add 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and 3% (w/v) trehalose solution.
  • the preparation is a granule
  • the granule is prepared according to the following method: the bacterial precipitate of the recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2 is added at a low concentration of 20% (w/v) Resuspend bacteria in polyfructose solution, then add 15% (w/v) glycerin solution, 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and concentration It is a 3% (w/v) trehalose solution, prepared as a mixed liquid of 10 10 ⁇ 10 12 cfu live bacteria per gram, dried to obtain a powder, and added 0.5 to 2 times the weight of flavoring agent, 0.5 to 2 times The weight of magnesium stearate is mixed and granulated to obtain granules; in the mixed liquid, the volume ratio of fructooligosaccharide solution, glycerin solution, skimmed milk powder solution, lactose solution and
  • the preparation is a tablet, and the tablet is as follows: take the bacterial precipitate of recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2, and add a low concentration of 20% (w/v) Resuspend bacteria in polyfructose solution, then add 15% (w/v) glycerin solution, 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and concentration It is a 3% (w/v) trehalose solution, prepared as a mixed liquid of 10 10 ⁇ 10 12 cfu live bacteria per gram, dried to obtain a powder, and added 0.5 to 2 times the weight of flavoring agent, 0.5 to 2 times The weight of magnesium stearate is mixed and compressed to obtain tablets.
  • the volume ratio of fructooligosaccharide solution, glycerin solution, skimmed milk powder solution, lactose solution and trehalose solution is (1-10): (1-10): (1-10): ( 1-10): (1-10), preferably 2:3:5:6:1; preferably, the number of viable bacteria per gram of mixed liquid is 10 10 cfu.
  • the preparation is a capsule
  • the capsule is prepared according to the following method: a bacterial precipitate of a recombinant bacterium containing the nucleotide sequence shown in SEQ ID NO: 2 is added at a low concentration of 20% (w/v) Resuspend bacteria in polyfructose solution, then add 15% (w/v) glycerin solution, 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and concentration It is a 3% (w/v) trehalose solution, prepared as a mixed liquid of 10 10 ⁇ 10 12 cfu live bacteria per gram, dried to obtain a powder, and added 0.5 to 2 times the weight of flavoring agent, 0.5 to 2 times Weight magnesium stearate, mixed, granulated to obtain granules, filled into capsules, obtained capsules; volume ratio of fructooligosaccharide solution, glycerin solution, skimmed milk powder solution, lactose solution and tre
  • the preparation is an oral liquid
  • the oral liquid is taken as follows: the bacterial precipitate of recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2 is added at a low concentration of 20% (w/v) Resuspend bacteria in polyfructose solution or L-arabinose solution with a concentration of 2% (w/v), prepare a fresh bacterial solution of not less than 10 10 ⁇ 10 12 cfu of live bacteria per milliliter, fill it, preferably; The number of viable bacteria per gram of mixed liquid is 10 10 cfu.
  • the GLP-1 mutant of the present invention reduces the degradation of DPP-IV, and has a half-life of about 3 hours; and the recombinant probiotic bacteria containing the GLP-1 mutant constructed by the present invention can be prepared It can be administered into various types of solid and liquid preparations to achieve oral administration to avoid the pain of long-term injection of patients.
  • the genetically engineered probiotic bacteria can survive and colonize the human intestine and become a functional in vivo bioreactor.
  • the continuous production and secretion of GLP-1 mutant polypeptides can play a role in continuous hypoglycemic, and the clinical application prospects are good.
  • FIG. 1 Screening results of GLP-1GM mutant protein in the present invention.
  • FIG. 1 SDS-PAGE and WB detection results of the GLP-1GM mutant of the present invention.
  • FIG. 3 Glucose-lowering effect of GLP-1GM mutant expressed in E. coli in the present invention by intragastric administration of T2DM rat model.
  • FIG. 4 Glucose-lowering effect of GLP-1GM mutant expressed by lactic acid bacteria in the present invention by intragastric administration of T2DM rat model.
  • Fig. 5 The effect of mutant GM transformation of E. coli Nissle1917 on the body weight and weight gain value of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 6 The effect of mutant GM transformation of Escherichia coli Nissle1917 on the food intake of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 7 The effect of mutant GM transformed Escherichia coli Nissle1917 on the area under the fasting blood glucose and blood glucose concentration change curve of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 8 The effect of GM transformation of mutant E. coli Nissle1917 on the weight of adipose tissue around the kidney and the ratio of liposomes in mice, compared with the normal group, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 9 The effect of GM transformation of mutant E. coli Nissle1917 on the weight of adipose tissue and the ratio of liposomes around the testes (ovaries) of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 10 The effect of mutant GM transformation of E. coli Nissle1917 on the liver weight of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • pGEX-4T-1, pMG36e, pET32a, E.coli TOP10 and E.coli BL21 (DE3), E. coli Nissle 1917, Lactobacillus, Lactococcus and other strains and plasmids are from Chengdu Lilai Biotechnology Co., Ltd. and Chongqing Medical University Preserved by the Department of Biochemistry and Molecular Biology; male rats are provided by the Laboratory Animal Center of Chongqing Medical University.
  • T4 ligase Taq common enzyme, SalI, BamHI, protein Marker, DNA Marker, Plasmid Mini Kit I, Cycle-Pure Kit, gel recovery kit, IPTG, erythromycin, ampicillin, urethromycin, BCA protein concentration Reagents such as measurement kits and PMSF (all commercially available).
  • GM is based on the original sequence of GLP-1, mutating the 8th alanine of GLP-1 to histidine, the 27th glutamic acid to lysine, and the 34th lysine to arginine. Acid, arginine at position 36 was mutated to glycine, glycine at position 37 was mutated to lysine, and a sequence was added at the end.
  • C33 is based on the original sequence of GLP-1, mutating glutamic acid at position 9 of GLP-1 to cysteine and valine at position 33 to cysteine.
  • K34 is based on the original sequence of GLP-1, mutating GLP-1 glycine at position 35 to lysine, arginine at position 36 to lysine, and glycine at position 37 to lysine.
  • R34 is based on the original GLP-1 sequence, mutating the 34th lysine to arginine, the 35th glycine to arginine, and the 37th glycine to arginine.
  • Natural GLP-1 is easily hydrolyzed and inactivated by dipeptidyl peptidase IV (DPP-IV), and its half-life is less than 5 minutes, which is not suitable for clinical application, so this animal experiment was not conducted.
  • DPP-IV dipeptidyl peptidase IV
  • GLP-1GM GLP-1 mutant numbered GM
  • Example 2 Expression and activity verification of recombinant E. coli of the mutant of the present invention
  • the pGEX-GLP-1GM expression vector was transformed into E. coli Nissle 1917, and PCR and sequencing confirmed that pGEX-GLP-1GM was successfully transformed.
  • Recombinant Escherichia coli group Nissle 1917 was inoculated onto LB medium supplemented with ampicillin at a final concentration of 100 ⁇ g/ml, and cultured on a shaker at 37°C and 250 r/min until the OD 600 value reached 0.8 to 1.0. Discard the supernatant and adjust the OD 600 value to 1.2 with PBS.
  • the GLP-1GM mutant expressed in E. coli Nissle 1917 has a hypoglycemic effect 2 hours after intragastric administration (compared to the control group without intragastric administration, P ⁇ 0.01). After 8 hours of intragastric administration, the blood glucose slightly increased.
  • Escherichia coli Nissle1917 (prepared in Example 2) stably transformed with genes identified by PCR verification and induced expression experiments was inoculated at 37°C, 250r /min shaker culture until the OD600 value reaches 0.8 ⁇ 1.0, centrifuge for 5min to harvest bacteria, wash twice with physiological saline, centrifuge to collect bacterial pellets, add 2% (w/v) concentration of L-arabinose solution or The 20% oligofructose solution was used to resuspend the bacteria, and it was prepared into a fresh bacterial solution of 10 10 cfu live bacteria per ml. After filling, it was stored in a refrigerator at 4°C.
  • the flavoring agent includes sweeteners or fruit flavoring aromatics, mix evenly for 20-30 minutes, put into dry granulation mechanism and collect After the 10-100 mesh particles were packed, the transformed E. coli Nissle 1917 granules were obtained.
  • the prepared E. coli Nissle1917 granules are filled into hollow hard capsules according to the corresponding specifications, and the resulting capsules are placed in a polishing machine to throw away excess dust to obtain transformed E. coli
  • Mutant GM transforms lactic acid bacteria
  • MRS liquid culture medium supplemented with erythromycin at a concentration of 20 ⁇ g/ml was steam sterilized, and lactic acid bacteria (prepared in Example 5) stably transformed with genes identified by PCR verification and induced expression experiments were inoculated, and incubated at 30°C until the OD600 value When it reaches 0.8 ⁇ 1.0, centrifuge, discard the supernatant, take the bacterial pellet and add 2% (w/v) L-arabinose solution or 20% fructooligosaccharide solution to resuspend the bacteria, and make it not lower per ml Fresh bacterial solution at 10 10 viable bacteria was refrigerated and stored in a refrigerator at 4°C.
  • the GLP-1GM mutant expressed in recombinant lactic acid bacteria has a certain hypoglycemic effect. Similar to the results in E. coli, the half-life of GLP-1GM is still short, and the blood glucose concentration decreases after 2 hours of intragastric administration (P ⁇ 0.01), After 5-7 hours, the blood glucose increased slightly (P ⁇ 0.05), and the hypoglycemic effect was weakened at 24h. If the same amount of recombinant lactic acid bacteria was immediately gavaged twice, the hypoglycemic effect was obtained again after 2h (ie 26h) (P ⁇ 0.01).
  • the GLP-1GM mutant expressed in recombinant lactic acid bacteria also has an oral hypoglycemic effect.
  • the GLP-1GM mutant expressed in recombinant lactic acid bacteria also has an oral hypoglycemic effect.
  • the GLP-1 mutant of the present invention reduces the degradation of DPP-IV, and the half-life is about 3h; and the recombinant probiotic bacteria containing the GLP-1 mutant constructed by the present invention has a continuous decrease Sugar effect.
  • SPF grade Kunming mice weighing 20-25g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. Certificate number: SCXK (Chuan) 2015-030. Animals were inspected for quarantine and adaptability for 3 days after acceptance, and the experiment was started after passing. Animals can drink water and eat freely, temperature (20 ⁇ 2)°C, humidity (60 ⁇ 5)%, 12h light period.
  • the experimental animals were randomly divided into: normal group, model group, recombinant E. coli Nissle 1917 (prepared in Example 5) high, middle and low dose groups, 10 animals in each group.
  • the normal group was fed with ordinary feed, and the remaining four groups were fed with high-fat and high-sugar feed (feed formula: 67% for ordinary feed, 20% for sucrose, 10% for lard, 2% for cholesterol, and 1% for sodium cholate).
  • the test animals eat and drink freely every day.
  • the recombinant Escherichia coli Nissle1917 high-medium-low-dose group was gavaged with 10 10 , 10 9 , and 10 8 CFU bacterial fluids, respectively, and the model group was gavaged with an equal volume of sterile saline, once a day, for the fourth week. And the eighth week of material determination.
  • Body weight Determine the body weight of experimental animals at a fixed time every week, and calculate the weight gain value.
  • the body weight and weight gain values of the model group were significantly higher than those of the normal group (p ⁇ 0.05).
  • the high-medium and low-dose groups of bacterial solution can significantly reduce the body weight and weight gain of experimental animals at four weeks (p ⁇ 0.05), and the high-medium-dose bacterial group at eight weeks can significantly reduce the weight and weight gain of experimental animals. Value (p ⁇ 0.05).
  • the high-medium-low-dose group of bacteria solution can significantly reduce the food intake of experimental animals (p ⁇ 0.05). Compared with the normal group, there was no significant difference in food intake in the high-medium-low-dose group (p>0.05).
  • the fatty tissue and liposomes around the kidneys in the model group were significantly higher than those in the normal group (p ⁇ 0.05).
  • the high-dose bacterial liquid group can significantly reduce the fatty tissue and liposome ratio around the kidneys of experimental animals (p ⁇ 0.05); at the eighth week, the high-dose bacterial liquid group can be significantly reduced Adipose tissue and liposome ratio around kidneys of experimental animals (p ⁇ 0.05).
  • the adipose tissue and liposomes around the testes (ovaries) in the model group were significantly higher than those in the normal group (p ⁇ 0.05).
  • the high-dose bacterial liquid group at the fourth week can significantly reduce the ratio of adipose tissue and liposomes around the testicles (ovaries) of experimental animals (p ⁇ 0.05); at the eighth week, the high-dose bacterial liquid group can be significantly Reduce the ratio of adipose tissue and liposomes around the testis (ovaries) of experimental animals (p ⁇ 0.05).
  • the liver weight of the model group was significantly higher than that of the normal group (p ⁇ 0.05).
  • the high-dose bacterial liquid group can significantly reduce the liver weight of experimental animals (p ⁇ 0.05).
  • the mutant GM transformed into E. coli Nissle1917 can inhibit the appetite of experimental mice and reduce energy absorption; significantly reduce the body weight, body fat content, liposome ratio and liver weight of experimental mice, with very obvious weight loss and lipid-lowering The role.
  • the transformed Escherichia coli Nissle1917 can also significantly reduce the area under the curve of blood glucose changes in the oral glucose tolerance test, and has a certain tendency to lower blood glucose.

Abstract

L'invention concerne un mutant de GLP -1. L'invention concerne également une séquence nucléotidique codant pour le mutant de GLP-1, un vecteur recombinant et une bactérie recombinante comprenant la séquence nucléotidique, un procédé de préparation de et une utilisation du mutant de GLP-1. La présente invention concerne également un médicament destiné au traitement du diabète ou à la régulation du poids. Le médicament contenant le mutant de GLP -1 peut réduire efficacement la teneur du sang en glucose ou réduire le poids et la graisse, peut être utilisé pour traiter le diabète de type II ou l'obésité, et présente de bonnes perspectives d'application clinique.
PCT/CN2019/071736 2018-12-12 2019-01-15 Mutant de glp-1, procédé de préparation et application associés WO2020118843A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN201811521016.1A CN110251662B (zh) 2018-12-12 2018-12-12 一种具有减肥作用的药物
CN201811519742.XA CN110256553B (zh) 2018-12-12 2018-12-12 一种glp-1突变体及其制备方法和用途
CN201811520996.3 2018-12-12
CN201811519742.X 2018-12-12
CN201811520996.3A CN110251661B (zh) 2018-12-12 2018-12-12 一种用于治疗糖尿病或者减肥的药物制剂
CN201811521016.1 2018-12-12

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