WO2009039743A1 - Arnsi utiles pour l'inhibition de gene tyrosinase, compositions comportant les arnsi et leurs utilisations - Google Patents

Arnsi utiles pour l'inhibition de gene tyrosinase, compositions comportant les arnsi et leurs utilisations Download PDF

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WO2009039743A1
WO2009039743A1 PCT/CN2008/072142 CN2008072142W WO2009039743A1 WO 2009039743 A1 WO2009039743 A1 WO 2009039743A1 CN 2008072142 W CN2008072142 W CN 2008072142W WO 2009039743 A1 WO2009039743 A1 WO 2009039743A1
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tyro
sirna
cosmetic composition
nucleotide sequence
chain
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PCT/CN2008/072142
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Chinese (zh)
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WO2009039743A8 (fr
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Zicai Liang
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Suzhou Ribo Life Science Co., Ltd
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Publication of WO2009039743A1 publication Critical patent/WO2009039743A1/fr
Publication of WO2009039743A8 publication Critical patent/WO2009039743A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/18—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with another compound as one donor, and incorporation of one atom of oxygen (1.14.18)
    • C12Y114/18001—Tyrosinase (1.14.18.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • siRNA composition and application thereof for inhibiting tyrosinase gene expression
  • the present invention relates to an siRNA for inhibiting tyrosinase gene expression and a cosmetic composition containing the same and use of the siRNA for preparing a cosmetic composition for inhibiting or eliminating melanin production and deposition. Background technique
  • Melanin can be divided into two categories, one is true melanin, contains no sulfur atom, is brown or black; the second is demelamine, containing sulfur atoms, yellow or reddish brown.
  • Animal and human skin and hair color are directly related to the number, size, and distribution of melanosomes. Excessive melanin darkens the skin, and non-uniform distribution of melanin causes chloasma and ecchymoses.
  • RNA interference is a process in which double-stranded RNA (dsRNA) molecules shut down the expression of the corresponding gene at the mRNA level or silence the gene.
  • dsRNA double-stranded RNA
  • RNA interference technology also known as knock-down or gene silencing
  • PTGS post-transcriptional gene silencing
  • RNA interference is directed to gene silencing in the post-transcriptional phase, the design of the entire process is simpler and more rapid, and the effect is obvious compared to traditional gene therapy methods such as antisense nucleic acid technology or the transfer of non-functional mutants to compete with the gene. .
  • This feature of RNA interference opens up new and powerful tools for gene therapy.
  • the siRNA has a problem in that the inhibitory activity against tyrosinase gene expression is low. Therefore, development of a siRNA having a high inhibitory activity on tyrosinase gene expression has become an urgent problem to be solved. Summary of the invention
  • Antisense chain 5 '-UUGAAUCCCAUGAAGUUGCdTdT-3,;
  • Antisense strand 5 '-AAGAGUCGUCUCUCUGUGCdTdT-3,;
  • TYRO-4 justice chain 5 ' -GCCAUCAGUCUUUAUGCAAdTdT-3 '
  • TYRO-5 justice chain 5 ' -CCAUCAGUCUUUAUGCAAUdTdT-3 '
  • Antisense chain 5 '-AUUGCAUAAAGACUGAUGGdTdT-3,;
  • TYRO-6 justice chain 5 ' -GCGUAAUCCUGGAAACCAUdTdT-3 '
  • TYRO-8 justice chain 5 ' -GGUAAUGAGGAACUGUUAUdTdT-3 '
  • Antisense strand 5 '-AUAACAGUUCCUCAUUACCdTdT-3 '.
  • the present invention provides a cosmetic composition comprising the siRNA provided by the present invention as an active ingredient.
  • the present invention provides the use of an siRNA for the preparation of a cosmetic composition for inhibiting melanin production and deposition or removal of black pigment.
  • the present invention provides an siRNA capable of inhibiting the expression of a tyrosinase gene, which has high inhibitory activity against tyrosinase gene expression and is capable of effectively inhibiting melanin production and deposition.
  • the siRNAs TYRO-1 to TYRO-8 provided by the present invention can efficiently inhibit the expression of the tyrosinase gene, in particular, the siRNA TYRO-1, TYRO-4 and TYRO-5 inhibit the tyrosinase gene, respectively. 81%, 82%, and 87%, while the reference siRNA C1-C3 inhibited the tyrosinase gene by 65%, 58%, and 52%, respectively, indicating that it is used to inhibit cheese.
  • the siRNA provided by the present invention is used for inhibiting the expression of the tyrosinase gene, the inhibitory activity against tyrosinase gene expression is greatly improved.
  • the present invention provides an siRNA for inhibiting tyrosinase gene expression, wherein the siRNA has TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 Or a nucleotide sequence as shown by TYRO-8, or having a nucleus as shown for TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 or TYRO-8 a nucleotide sequence obtained by chemically modifying a nucleotide sequence, wherein
  • TYRO-1 justice chain 5 '-GCAACUUCAUGGGAUUCAAdTdT-3 '
  • Antisense chain 5 '-UUGAAUCCCAUGAAGUUGCdTdT-3,;
  • TYRO-2 justice chain 5 '-GCACAGAGACGACUCUUdTdT-3 '
  • TYRO-3 justice chain 5 '-GGAGGAGUACAACAGCCAUdTdT-3 '
  • Antisense chain 5 '-AUGGCUGUUGUACUCCUCCdTdT-3,;
  • TYRO-4 justice chain 5 '-GCCAUCAGUCUUUAUGCAAdTdT-3 '
  • Antisense chain 5 '-UUGCAUAAAGACUGAUGGCdTdT-3,;
  • TYRO-5 justice chain 5 ' -CCAUCAGUCUUUAUGCAAUdTdT-3 '
  • TYRO-6 justice chain 5 ' -GCGUAAUCCUGGAAACCAUdTdT-3 '
  • Antisense strand 5 '-AUGGUUUCCAGGAUUACGCdTdT-3,;
  • TYRO-7 justice chain 5 ' -CCAGGUUCCCAGAGAAUAUdTdT-3 '
  • Antisense chain 5 '-AUAUUCUCUGGGAACCUGGdTdT-3,;
  • TYRO-8 justice chain 5 ' -GGUAAUGAGGAACUGUUAUdTdT-3 '
  • Antisense strand 5 '-AUAACAGUUCCUCAUUACCdTdT-3 '.
  • the siRNA has a nucleotide sequence represented by TYRO-1, TYRO-4 or TYRO-5; more preferably, the siRNA has TYRO-5 shown Nucleotide sequence.
  • the tyrosinase is a copper-containing oxidoreductase widely present in plants, animals and microorganisms. In the synthesis of melanin, tyrosinase is required in two steps, therefore, tyrosine Enzymes play a pivotal role in the production of melanin.
  • the tyrosinase gene refers to a nucleotide sequence encoding a tyrosinase.
  • the human tyrosinase gene is about 35kb-70kb long and is located on chromosome 11, which contains 5 exons and 4 introns. The lengths of 5 exons are 919bp, 135bp, 150bp, 183bp and 219bp, respectively.
  • the chemical modification is well known to those skilled in the art, and the modification of the phosphodiester bond refers to modification of oxygen in the phosphodiester bond, including phosphorothioate modification, as shown in Formula 1; and boron bismuth phosphate Salt modification, as shown in Formula 2. Both modifications stabilize the siRNA structure, maintaining high specificity and high affinity for base pairing.
  • the ribose modification refers to a modification of 2'-OH in a nucleotide pentose, gp, which introduces a certain substituent at the hydroxyl position of the ribose, for example, a 2'-fluoro modification, as shown in Formula 3; - oxymethyl modification, as shown in Formula 4; 2,-oxyethylene methoxy modification, as shown in Formula 5; 2,4,-dinitrophenol modification, as shown in Formula 6; LNA), as shown in Formula 7; 2'-amino modification, as shown in Formula 8; 2'-deoxy modification, as shown in Formula 9.
  • the base modification refers to modification of a base of a nucleotide, for example, 5'-bromouracil modification, as shown in Formula 10; 5'-iodouracil modification, as shown in Formula 11; N-A The quinolidine modification, as shown in Formula 12; 2,6-diaminopurine modification, as shown in Formula 13.
  • the modification enhances the ability of the modified siRNA to resist nuclease hydrolysis in the cell.
  • a lipophilic group such as cholesterol may be introduced at the end of the siRNA sense strand on the basis of the above modification to facilitate interaction with the intracellular mRNA by the cell membrane composed of the lipid bilayer.
  • the design of the siRNA sequence means a 19 bp nucleotide sequence is selected within the range of l-2082 bp of the tyrosinase gene cDNA sequence (Genbank accession number NM- 000372).
  • the principle of selecting the 19 bp nucleotide sequence is: (1) the total siRNA double bond energy ⁇ 1; (2) the 5' end binding energy of the antisense strand 3-9; (3) the 5' end binding energy of the sense strand 5-9; (4) GC content is between 36-53%; (5) The energy difference between the 5' end of the antisense strand and the sense strand 5' is between -1 and 0.
  • the 3' end of the obtained 19 bp nucleotide sequence was added with two deoxythymidine nucleotides as the sense strand of the siRNA sequence, and two deoxythymidines were added to the 3' end of the complementary sequence of the 19 bp nucleotide sequence. Nucleotides serve as the antisense strand of the siRNA sequence.
  • the synthesis of the siRNA sequence can be carried out by chemical synthesis or by a biotechnology company specializing in nucleic acid synthesis, such as commissioning by Shanghai GenePharma.
  • the method of chemical synthesis includes the following four processes: (1) synthesis of oligoribonucleoside acid; (2) deprotection; (3) purification separation; (4) desalting.
  • RNA 1 millimolar RNA was synthesized on an automated DNA/RNA synthesizer (for example, Applied Biosystems EXPEDITE 8909), and the coupling time per cycle was set to 10-15. Minutes, the starting material is a solid phase-linked 5'-0-p-dimethoxy-thymidine support, the first cycle is linked to a base on the solid support, and then at the nth (19 ⁇ n ⁇ 2) In the cycle, one base is ligated to the base to which the n-1th cycle is ligated, and this cycle is repeated until the synthesis of all nucleic acid sequences is completed.
  • an automated DNA/RNA synthesizer for example, Applied Biosystems EXPEDITE 8909
  • the solid support to which the siRNA was attached was added to the test tube, and 1 ml of ethanol/ethylamine (1:3 by volume) was added to the test tube, which was then sealed and placed in a 55-70 ° C incubator. After incubation for 2-30 hours, the solid support to which siRNA was attached was taken out and rinsed twice with double distilled water (1 ml each time), the eluate was collected, and dried at room temperature for 30 minutes.
  • the obtained crude product of siRNA was dissolved in 2 ml of an aqueous solution of ammonium acetate having a concentration of 1 mol/ml, and then separated by C18 high pressure liquid chromatography to obtain a purified siRNA product.
  • a cosmetic composition for inhibiting melanin production and depositing or removing melanin according to the present invention which comprises the siRNA provided by the present invention as an active ingredient.
  • the cosmetic composition further comprises a carrier.
  • the content of the siRNA and the carrier may vary within a wide range.
  • the carrier is contained in an amount of from 1000 to 10,000,000 parts by weight relative to 100 parts by weight of the siRNA; further preferably, relative to 100
  • the siRNA is contained in an amount of from 2,000 to 100,000 parts by weight.
  • the carrier is not particularly limited and may be any carrier for a cosmetic composition, and may be, for example, one or more of animal and vegetable oils, hydrocarbon oils, esters, higher fatty acids, and higher fatty alcohols;
  • the animal and vegetable oil may be one or more of eucalyptus oil, eucalyptus oil, soybean oil, sesame oil, corn oil, rapeseed oil, cottonseed oil, olive oil and castor oil;
  • the hydrocarbon oil may be paraffin oil, One or more of isotrimidine and petrolatum;
  • the ester may be isopropyl palmitate, butyl stearate, hexyl laurate, isodecyl isononanoate, 2-ethylhexyl palm One or more of an acid ester, 2-hexyldecyl laurate, and 2-octyldodedodecyl myristate;
  • the higher fatty acid may be palmitic acid, stea
  • the cosmetic composition further contains other ingredients for the cosmetic composition.
  • the content of the siRNA and other ingredients for the cosmetic composition may vary within a wide range, and preferably, the content of the other ingredients for the cosmetic composition is relative to 100 parts by weight of the siRNA. It is preferably from 1000 to 10,000,000 parts by weight; more preferably, the content of the other component for the cosmetic composition is from 2,000 to 100,000 parts by weight with respect to 100 parts by weight of the siRNA.
  • the other ingredients for the cosmetic composition are not particularly limited, and for example, may be one of a moisturizing agent, a coloring agent, a preservative, a thickener, an antioxidant, a surfactant, and an adjuvant or Several.
  • the humectant is not particularly limited, and may be various humectants for use in a cosmetic composition.
  • the humectant may be propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol, or sorbitol. , one of hexanediol, 1,3-butanediol, glycerin, seaweed extract and hyaluronic acidkind or several.
  • the coloring agent is not particularly limited, and may be various coloring agents for use in a cosmetic composition.
  • the coloring agent may be carmine, talc, mica, magnesium carbonate, calcium carbonate, silicic acid.
  • magnesium, silica, zinc oxide, and ultramarine may be carmine, talc, mica, magnesium carbonate, calcium carbonate, silicic acid.
  • magnesium, silica, zinc oxide, and ultramarine may be carmine, talc, mica, magnesium carbonate, calcium carbonate, silicic acid.
  • the thickener is not particularly limited and may be various thickeners for use in a cosmetic composition.
  • the thickener may be beeswax, candelilla wax, cetyl wax, or microcrystals.
  • the preservative is not particularly limited, and may be various preservatives for use in a cosmetic composition.
  • the preservative may be benzoic acid, salicylic acid, methyl paraben, p-hydroxyl
  • the preservative may be benzoic acid, salicylic acid, methyl paraben, p-hydroxyl
  • propyl benzoate, resorcinol and diimidazolium urea may be benzoic acid, salicylic acid, methyl paraben, p-hydroxyl
  • propyl benzoate propyl benzoate, resorcinol and diimidazolium urea.
  • the kind of the adjuvant is not particularly limited and may be various adjuvants which can be used in a cosmetic composition, for example, the adjuvant may be mannitol, glucose, albumin, arginine, azone and trehalose. One or several of them.
  • the present invention also provides the use of an siRNA for the preparation of a cosmetic composition for inhibiting melanin production and deposition or for removing melanin, wherein the siRNA is an siRNA provided by the present invention.
  • Example 1 The present invention will be further illustrated by the following examples, and the reagents and culture media used in the present invention are commercially available unless otherwise specified.
  • Example 1 The reagents and culture media used in the present invention are commercially available unless otherwise specified.
  • the designed siRNA was chemically synthesized by GenePharma, and the siRNAs TYRO-1 to TYRO-8 were obtained.
  • the nucleotide sequences of the siRNAs TYRO-1 to TYRO-8 are shown in Table 1.
  • a reference siRNA Cl-C3 having the nucleotide sequence shown in Table 2 was chemically synthesized by Shanghai GenePharma. Table 2
  • the range of attack refers to the corresponding position of the reference siRNA in the sequence No. 1 of the sequence listing.
  • siRNA TYRO- 1 to TYRO-8 were transfected siRNA as control without adding.
  • the specific steps are as follows: The siRNA was dissolved in RNase-free sterile water to prepare a solution having a concentration of 20 mol/L.
  • SK-MEL-1 melanoma cells were seeded into 24-well plates and diluted to a concentration of 8 ⁇ 10 5 cells/ml with 500 ⁇ l per well using OptiMEM I low serum medium (Invitrogen, 31985-062).
  • tyrosinase mRNA in SK-MEL-1 melanoma cells transfected with siRNA TYR0-1 to TYR0-8 was detected by RT-PCR. The specific steps were as follows:
  • the SK-MEL-1 melanoma cells transfected with siRNA were lysed with 1 ml of Trizol (GIBCOL), and total RNA was extracted.
  • the specific steps for extracting total RNA were as follows: The transfected cells were at a temperature of 37 ° C and a C 2 2 content.
  • the cells were cultured for 24 hours in a 5% incubator, and then the cells were collected by centrifugation and washed once with pre-cooled 2 ml of PBS; the composition of the PBS was: NaCl 137 mmol/L, KC1 2.7 mmol/L, Na 2 HP0 4 4.3 mmol/ L, KH 2 P0 4 1.4mmol / L; Add 1ml Trizol per well, let stand for 5 minutes at room temperature, lyse the cells; transfer the lysate to 1.5ml EP tube; add 200 ⁇ 1 chloroform, shake vigorously for 15 seconds by hand, place at room temperature 3 Minutes; 14000 rpm, centrifuge at 15 °C for 15 minutes; take liquid phase supernatant to about 500 ⁇ l and put in a new tube, add 500 ⁇ l of isopropanol, let stand for 10 minutes at room temperature; centrifuge at 10 °C for 10 minutes at 12000 rpm, remove the supernatant.
  • the precipitate was washed once with 1 ml of 75% ethanol; centrifuged at 7600 rpm for 5 minutes at 4 ° C; the supernatant was removed, and the RNA was dried at room temperature for 10 minutes; 20 ⁇ l of ddH 2 0 was added to dissolve the RNA.
  • RNA-free Two units of DNase I (RNase-free) (TakaRa) were added to the above-mentioned RNA-dissolved DEPC water, and allowed to stand at 37 ° C for 30 minutes to remove residual DNA in the total RNA. After treatment with DNase I, the total RNA was purified by Invitrogen PureLink Micro-to-Midi Total RNA Purification Kit. The specific steps of purification were as follows: 20 ⁇ l of 70% ethanol was added to the total RNA, and the mixture was shaken evenly.
  • the mixture was transferred to a purification column, centrifuged at 12,000 rpm for 15 seconds at room temperature, the filtrate was discarded, 700 ⁇ l of Wash Buffer I (TakaRa) was added, and the mixture was centrifuged at 12,000 rpm for 15 seconds at room temperature, and the filtrate was discarded, and 500 ⁇ l of Wash Buffer II (TakaRa) was added. Centrifuge at 12000 rpm for 15 seconds at room temperature, discard the filtrate, add 500 ⁇ l of Wash Buffer II (TakaRa), centrifuge at 12,000 rpm for 15 seconds at room temperature, discard the filtrate, centrifuge at 12000 rpm for 1 minute at room temperature, and transfer the purification column to the RNA collection tube. 30 ⁇ l of DEPC water was added, allowed to stand at room temperature for 1 minute, centrifuged at 13,000 rpm for 2 minutes at room temperature, and the RNA sample was stored at -80 ° C.
  • the reverse transcription reaction is carried out on the total RNA obtained after purification.
  • the reverse transcriptase used is Promega's M-MLV reverse transcriptase.
  • the specific steps of the reverse transcription reaction are: the total RNA after purification of lug and 0.5ug of Oligo dT is mixed in test tubes with DEPC water The total volume was made up to 16.25 ⁇ 1, and the tube was heated under heating conditions of 70 ° C for 5 minutes; then the tube was rapidly cooled to 0 ° C and buffer (5 x MLV buffer 5 ⁇ , lOmMDntp 1.25 1, RNasin0.5 l, M-MLV 1 ⁇ 1), incubated at 42 °C for 1 hour to obtain cDNA.
  • the obtained cDNA was used as a template for a PCR reaction, and a Real-time PCR reaction was carried out.
  • the Real-time PCR reaction system is: ddH 2 017.5 ⁇ 1, 10mM Dntp 0.5 ⁇ 1, lO Taq buffer 2 ⁇ 5 ⁇ 1, Taq 0.5 1, F primer 0.5 ⁇ , R primer 0.5 1, Syber Green I 1 ⁇ 1, ⁇ 2 1; PCR reaction
  • the conditions were: 94 ° C for 2 minutes, 94 ° C for 15 seconds, and 60 ° C for 30 seconds for a total of 40 cycles.
  • the inhibitory activity of siRNA was calculated according to the following formula, and the results are shown in Table 3.
  • siRNA against tyrosinase mRNA expression was examined by the method shown in Example 2, except that the siRNA used was the reference siRNACl-C3 obtained in Comparative Example 1, and the results are shown in Table 3.
  • the siRNAs TYRO-1 to TYRO-8 provided by the present invention can efficiently inhibit the expression of tyrosinase genes, particularly siRNAs TYRO-1, TYRO-4 and TYRO-5 to tyrosinase.
  • the inhibitory activity of the gene reached 81%, 82% and 87%, while the inhibitory activity of the reference siRNA C1-C3 on the tyrosinase gene was 65%, 58% and 52%, respectively, indicating that it is used to inhibit cheese.
  • the siRNA provided by the present invention When the siRNA provided by the present invention is used for inhibiting the expression of the tyrosinase gene, the siRNA for expressing the tyrosinase gene has a higher inhibitory activity against tyrosinase gene expression.
  • Example 3
  • siRNA TYRO-1 to TYRO-8 on tyrosinase protein were detected, and SK-MEL-1 melanoma cell suspension (8 ⁇ 10 5 cells/ml) was added to a 96-well culture plate at a volume of lOOul per well.
  • siRNA was then added and the liposome Lipofectamine TM 2000 (Invitrogen) were transfected as a control siRNA without adding.
  • the final concentration of siRNA was 50 nM.
  • the cells were collected by centrifugation 72 hours after transfection and washed twice with PBS of pH 7.4.
  • the composition of the PBS was NaCl 137 mmol/L, KC1 2.7 mmol/L, Na 2 HP0 4 4.3 mmol/L, KH 2 P0 4 1.4 Methyl/L; 50 L of 1% by weight of Triton X-100 solution per well, the composition of the Triton X-100 solution is 99 ml of water and 1 ml of Triton X-100; quickly placed at -80 ° C, frozen After 30 minutes; then the cells were completely ruptured by melting at room temperature. After pre-warming at 37 °C, L-dopa solution with a concentration of 2 mg/ml was added, and reacted at 37 ° C for 2 hours to measure the absorbance at 475 nm. As shown in Table 4.
  • the siRNAs TYRO-1 to TYRO-8 provided by the present invention are capable of efficiently inhibiting the activity of tyrosinase proteins, particularly tyrosinase by TYRO-1, TYRO-4 and TYRO-5.
  • the inhibitory activities of the proteins were 46%, 42%, and 56%, respectively, while the inhibitory activities of the reference siRNAs C1-C3 on tyrosinase proteins were 21%, 18%, and 20%, respectively, indicating that the siRNA provided by the present invention is effective. Inhibition of tyrosinase activity.
  • TritonX-100 solution is 99ml water, 1ml TritonX-100; quickly placed at -80 ° In C, it was frozen for 30 minutes; then the cells were completely ruptured by melting at room temperature, centrifuged, and the precipitate was washed once with 10% by weight of trichloroacetic acid (2 ml) and absolute ethanol (2 ml), and centrifuged at 12,000 rpm for 5 minutes.
  • siRNA was used to inhibit the melanin content in SK-MEL-1 melanoma tumor cells according to the method described in Example 4, except that the siRNA used was the reference siRNA C1-C3 obtained in Comparative Example 1, and the results are shown in Table 5.
  • the siRNAs TYRO-1 to TYRO-8 provided by the present invention can significantly increase the inhibition rate of melanin content, particularly by inhibition of TYRO-1, TYRO-4 and TYRO-5, in melanocytes.
  • the inhibition rate of melanin content reached more than 35%, and the inhibition rate of melanin content in melanocytes was 19%, 18% and 21% by the inhibition of reference siRNA C1-C3, respectively, indicating that the siRNA provided by the present invention can be remarkable. Increase the inhibition rate of melanin content.
  • siRNA TYRO-1 to siRNA TYRO-8 was entrusted, among which, chemically modified siRNA TYRO-1 to siRNA TYRO-8 all strands of U and C nucleotides
  • the 2'-OH of the pentose sugar was subjected to 2'-fluoro modification, and the 2'-OH of all U and C nucleotide pentose sugars of the antisense strand was subjected to 2'-oxymethyl modification.
  • the components were mixed according to the composition shown in Table 6, and then uniformly stirred to obtain Compositions 1 to 8 to evaluate the clinical whitening effect of the composition containing the siRNA provided by the present invention as an active ingredient.
  • the composition containing the siRNAs TYRO-1 to TYRO-8 provided by the present invention has a remarkable whitening effect on the blackened skin after 5 months of use, particularly by containing TYRO-K TYRO-4.
  • the composition with TYRO-5 has a slight whitening effect on blackened skin after 1 month of use, and has a significant whitening effect on blackened skin after 3 months of use, and a reference containing reference siRNA Cl-C3.
  • Compositions D1-D3 showed a slight whitening effect after four months of use, and there was no significant increase in whitening effect after six months of use, indicating that the whitening effect of the composition containing the siRNA provided by the present invention was more remarkable.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des ARNsi utiles pour inhiber l'expression du gène tyrosinase, les ARNsi ayant la séquence nucléotidique représentée dans TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 ou TYRO-8, ou la séquence nucléotidique chimiquement modifiée dérivée de la séquence nucléotidique représentée dans TYRO-1, TYRO-2, TYRO-3, TYRO-4, TYRO-5, TYRO-6, TYRO-7 ou TYRO-8. L'invention concerne également des compositions cosmétiques et les utilisations des ARNsi dans la préparation d'une composition cosmétique permettant d'inhiber la production et le dépôt de mélanine ou d'éliminer la mélanine. Les ARNsi présentent une activité élevée pour l''inhibition de l'expression du gène tyrosinase, et peuvent efficacement inhiber la production et le dépôt de mélanine.
PCT/CN2008/072142 2007-09-20 2008-08-26 Arnsi utiles pour l'inhibition de gene tyrosinase, compositions comportant les arnsi et leurs utilisations WO2009039743A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102793639A (zh) * 2011-12-16 2012-11-28 百奥迈科生物技术有限公司 一种植物提取物介导的药物透皮导入系统及其透皮方法
CN108138181A (zh) * 2015-07-27 2018-06-08 奥利克斯医药有限公司 抑制黑色素生成的rna复合物
CN110066800A (zh) * 2019-04-30 2019-07-30 厦门甘宝利生物医药有限公司 一种新化合物及其组合物的应用
CN113667669A (zh) * 2021-08-06 2021-11-19 暨南大学 抑制酪氨酸酶表达的反义寡核苷酸及其应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180025701A (ko) * 2016-09-01 2018-03-09 (주)아모레퍼시픽 특정 siRNA을 포함하는 멜라닌 증진용 조성물
CN110643596A (zh) * 2018-06-26 2020-01-03 煌鼎科技有限公司 一种美白酵母dna原料的制法
CN117070583B (zh) * 2023-10-16 2024-05-14 吉林凯莱英制药有限公司 抑制PCSK9基因表达的siRNA的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005060536A2 (fr) * 2003-12-17 2005-07-07 Avon Products, Inc. Technique de silençage genique mediee par si-rna en vue d'inhiber la tyrosinase et reduire la pigmentation
CN1966084A (zh) * 2005-09-21 2007-05-23 莱雅公司 抑制酪氨酸酶表达的双链rna寡核苷酸

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1506041A (zh) * 2002-12-12 2004-06-23 殷冬生 基因皮肤美白化妆品的配方和制造方法
CN101601635A (zh) * 2008-06-13 2009-12-16 殷勤伟 小rna/dna美白产品的配方和制造方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005060536A2 (fr) * 2003-12-17 2005-07-07 Avon Products, Inc. Technique de silençage genique mediee par si-rna en vue d'inhiber la tyrosinase et reduire la pigmentation
CN1966084A (zh) * 2005-09-21 2007-05-23 莱雅公司 抑制酪氨酸酶表达的双链rna寡核苷酸

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102793639A (zh) * 2011-12-16 2012-11-28 百奥迈科生物技术有限公司 一种植物提取物介导的药物透皮导入系统及其透皮方法
WO2014029143A1 (fr) * 2012-08-23 2014-02-27 百奥迈科生物技术有限公司 Système d'introduction transdermique de médicament à base d'extrait de plante et procédé transdermique associé
CN108138181A (zh) * 2015-07-27 2018-06-08 奥利克斯医药有限公司 抑制黑色素生成的rna复合物
CN108138181B (zh) * 2015-07-27 2022-05-24 奥利克斯医药有限公司 抑制黑色素生成的rna复合物
CN110066800A (zh) * 2019-04-30 2019-07-30 厦门甘宝利生物医药有限公司 一种新化合物及其组合物的应用
CN113667669A (zh) * 2021-08-06 2021-11-19 暨南大学 抑制酪氨酸酶表达的反义寡核苷酸及其应用
CN113667669B (zh) * 2021-08-06 2023-12-22 暨南大学 抑制酪氨酸酶表达的反义寡核苷酸及其应用

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