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RNA complexes that inhibit melanin production

A complex, melanocyte technology, applied in the field of RNA complexes that inhibit melanin production, can solve problems such as ineffectiveness and significant side effects

Active Publication Date: 2018-06-08
OLIX PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such treatments are often ineffective and can have significant side effects

Method used

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  • RNA complexes that inhibit melanin production
  • RNA complexes that inhibit melanin production
  • RNA complexes that inhibit melanin production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: Screening of tyrosinase-specific asymmetric small interfering RNA

[0098] In order to efficiently identify asymmetric small interfering RNAs (asiRNAs) that inhibit tyrosinase, 62 asiRNAs were synthesized and screened. The nucleic acid sequences of the screened asiRNAs are provided in Table 1.

[0099] Table 1: Nucleic acid sequences of exemplary tyrosinase-targeting asiRNAs

[0100]

[0101]

[0102]

[0103]

[0104] The asiRNAs listed in Table 1 were incubated in 1 x siRNA duplex buffer (STpharm) at 95°C for 2 minutes and at 37°C for 1 hour. Proper strand annealing was confirmed via gel electrophoresis. For screening, place the 1.6 x 10 4 A375 cells (ATCC) were seeded in a 24-well plate, and the A375 cells had been cultured in Dulbecco's Modified Eagle's Medium (Gibco) in a 100 mm cell culture dish, and the Dulbecco's Modified Eagle's Medium (Gibco) Erl's medium (Gibco) contained 10% fetal bovine serum (Gibco) and 100 μg / ml penicillin / str...

Embodiment 2

[0111] Example 2: Chemical modification of asiRNA for self-delivery

[0112] Chemical modifications were applied to the six asiRNAs selected in Example 1, and cellular delivery of the modified asiRNAs was tested in the absence of other delivery vehicles. As described below, certain modifications improve the endocytosis and stability of asiRNAs. Such cell penetrating asiRNAs (cp-asiRNAs) can be delivered into cells in the absence of delivery vehicles.

[0113] Thirty-eight potential cp-asiRNAs (Table 2) were screened for tyrosinase mRNA inhibition in MNT-1 cells. Each potential cp-asiRNA was incubated with 1 μM of human melanoma cell line MNT-1 cells without delivery vehicle, and tyrosinase mRNA levels were measured by real-time PCR.

[0114] Table 2. Modified asiRNA sequences tested for self-delivery and tyrosinase inhibition. m = 2'-O-methyl RNA. * = phosphorothioate linkage.

[0115]

[0116]

[0117]

[0118] Minimal essential medium (Welgene) containing 20% ​...

Embodiment 3

[0122] Example 3: Inhibition of tyrosinase protein and melanin using tyrosine-specific cp-asiRNA

[0123] The efficacy of cp-asiTYR(4)-1 on inhibition of tyrosinase protein and inhibition of melanogenesis was tested. To test for non-specific effects, a mutated cp-asiTYR lacking sequence complementarity to the tyrosinase mRNA sequence (termed cp-asiTYR (seed mutation)) was also tested. The sequence of cp-asiTYR (seed mutation) is provided in Table 3.

[0124] Table 3. Sequence m = 2'-O-methyl RNA used in cp-asiRNA(4)-1 (seed mutation). * = phosphorothioate linkage.

[0125] cp-asiTYR(4)-1 (seed mutation) S:GCUGACAGGUCUAC*U*A*cholesterol

cp-asiTYR(4)-1 (seed mutation) AS:UAGUAGACCUGUCAGCU*U*C*U*G

[0126] The cp-asiRNA was incubated in OPTI-MEM buffer (Gibco) at 95°C for 2 minutes and at 37°C for 1 hour. Proper strand annealing of potential cp-asiRNAs was confirmed by gel electrophoresis.

[0127] MNT-1 cells were incubated in minimal essential medium...

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Abstract

In certain aspects, provided herein are RNA complexes (e.g., asymmetric RNA complexes, such as asiRNAs and lasiRNAs) that inhibit tyrosinase expression and are therefore useful for reducing melanin production and for treating pigmentation-related disorders associated with excessive melanin production, such as melasma and age spots.

Description

[0001] related application [0002] This application claims priority to US Provisional Application 62 / 197,370, filed July 27, 2015, which is hereby incorporated by reference in its entirety. Background technique [0003] Hypermelanin production by melanocytes is associated with a variety of skin pigmentation-related disorders, including melasma and age spots. In melasma, overproduction of melanin causes dark deposits in melanocytes present in the epidermal skin layer. Melasma is one of the main intractable diseases that occur in women's skin. Melasma occurs frequently in pregnant women and women who are taking oral or patch contraceptives or undergoing hormone replacement therapy. [0004] Tyrosinase is an oxidase that is the rate-limiting enzyme in the synthesis of melanin and is therefore an important therapeutic target for agents that reduce hyperpigmentation and treat skin pigmentation-related disorders. In humans, tyrosinase is encoded by the TYR gene. Mutations in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7105
CPCA61Q19/08C12N2310/14C12N2310/321C12N2310/344A61K8/606A61K31/713A61K45/06A61K2800/782A61Q19/02C12N15/1137C12N2310/315C12Y114/18001A61P17/00A61P43/00C12N2310/3521A61K31/7105C12N15/113C12N2310/3515C12N2310/11C12N2310/111C12N2320/31
Inventor S·W·弘I·弘J·H·金
Owner OLIX PHARMA
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