Lipophilic modified small nucleic acids for transdermal formulations, methods of making and using the same

Abstract

The application discloses a lipophilic modified small nucleic acid for transdermal preparation and a preparation method and application thereof, and belongs to the technical field of biotechnology. The lipophilic modified small nucleic acid is a compound shown in a general formula, stereoisomers, tautomers, crystal hydrates, solvates or pharmaceutically acceptable salts thereof, and the general formula is Nuc-L-Cer. In the formula, Nuc is a small nucleic acid; L is a linking group; and Cer is a lipophilic modification group, and the lipophilic modification group is selected from cholesterol, a fatty acid, a ceramide or choline. By utilizing the natural and high-affinity "molecular compatibility" of ceramides and the lipid matrix of the stratum corneum, the modified small nucleic acid molecules can be "disguised" as the lipid components of the skin, and are actively "integrated" into the lipid bilayer structure of the stratum corneum, so that the skin irritation and biological safety problems of the existing non-natural lipid modifiers are solved. The lipophilic modified small nucleic acid has the advantages of higher biocompatibility, better safety and significantly improved transdermal efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a lipophilic modified small nucleic acid for transdermal formulations, its preparation method, and its application. Background Technology

[0002] Small nucleic acid drugs, especially small interfering RNA (siRNA), have shown great potential in the treatment of various skin diseases (such as psoriasis, atopic dermatitis, skin tumors, and hereditary skin diseases) because they can specifically silence the expression of pathogenic genes through RNA interference mechanisms. However, the practical clinical application of siRNA molecules faces significant challenges, including their inherent characteristics such as large molecular weight (approximately 13-15 kDa), strong negative charge, high hydrophilicity, and susceptibility to degradation by nucleases. These characteristics make it difficult for them to effectively penetrate the stratum corneum, the main barrier of the skin, thus severely limiting transdermal delivery efficiency.

[0003] Researchers have explored various strategies to improve the transdermal penetration of siRNA. One approach involves lipophilic modification of siRNA, such as covalently linking hydrophobic groups like cholesterol, tocopherol (vitamin E), long-chain fatty acids, or alkyl chains, to increase its affinity for the stratum corneum lipid bilayer and its transmembrane capacity. However, existing modification strategies still have several limitations: while cholesterol modification enhances the interaction between siRNA and the lipid membrane, the improvement in transdermal efficiency is limited and may affect the silencing activity of siRNA; the synthetic process for tocopherol modification is complex, and its biocompatibility and long-term safety require further verification; and common long-chain fatty acid or alkyl chain modifications exhibit poor in vivo stability and are easily metabolized, losing their delivery efficiency. More importantly, existing lipophilic modifiers are not natural components of the stratum corneum, and their accumulation in the skin may cause irritation or inflammatory reactions.

[0004] Ceramides, as key components of the intercellular lipids in the stratum corneum (accounting for approximately 40-50% of the total lipids in the stratum corneum), play an irreplaceable role in maintaining skin barrier function, regulating epidermal cell signaling pathways, and hydration. Utilizing different carbon chain lengths (such as C8, C98, C10, C2 ... 16 C 18 C 24 Ceramides (such as ceramides) can be used as lipophilic modification carriers for siRNA. Theoretically, their natural high affinity with the lipid matrix of the stratum corneum can promote the "integration" of modified small nucleic acid molecules into the skin barrier and achieve efficient penetration.

[0005] Relevant non-patent literature retrieved: Preparation of Galactose-Modified siRNA Nanomedicines and Treatment of Alzheimer's Disease (Zhu Feiyan, Preparation of Galactose-Modified siRNA Nanomedicines and Treatment of Alzheimer's Disease [D], Henan University, June 2020) discloses a galactose-modified polymeric delivery carrier. This carrier forms highly blood-stable siRNA nanomedicines with siRNA through multiple interactions. Simultaneously, the galactose on the surface of the nanomedicine effectively mediates the nanomedicine's passage across the blood-brain barrier (BBB) ​​by recognizing glucose transporter 1 (Glut1). After loading siRNA specifically targeting β-secretase 1 (BACE1), this nanomedicine can be used for the treatment of Alzheimer's disease.

[0006] Preparation and Characterization of Ceramide-Modified Liposomes (Jia Liping, Yang Cheng, et al., Preparation and Characterization of Ceramide-Modified Liposomes [J], Journal of Food and Biotechnology, Vol. 42, No. 4, 2023, pp. 48-55) disclosed the use of ceramide III (Cer3) to modify liposomes, and the loading of retinyl palmitate (RP) as a lipophilic active ingredient into Cer3 liposomes. The stability, stabilization mechanism and in vitro release performance of RP-Cer3 liposomes were studied. The results showed that Cer3 reduces the fluidity of liposomes, thereby improving their stability, and RP-Cer3 liposomes have good in vitro release performance.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: There is a lack of systematic research on the effect of ceramide modification with different carbon chain lengths on the transdermal delivery efficiency of siRNA in the current technology, and there are no reports on the design and preparation process of ceramide-modified small nucleic acid structures optimized for transdermal formulations. Summary of the Invention

[0008] The purpose of this invention is to provide: A lipophilic modified small nucleic acid for transdermal formulations and related technologies, addressing technical issues such as developing a class of lipophilic modified small nucleic acids with both high transdermal efficiency and good biocompatibility, or combinations thereof.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent inventions, and publications cited throughout this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions provided in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Definitions of standard chemical terms can be found in the references “Medicinal Chemistry, edited by Bai Renren, Science Press, 2024” and “Pharmacopoeia of the People’s Republic of China (2020 edition)”.

[0012] Unless otherwise stated, conventional methods within the scope of the art shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] All numerical values ​​or expressions relating to component amounts, process conditions, etc., used in this invention shall be understood to be modified by the word "about" in all cases. When referring to a quantity or range of values, the quantity or range is an approximation within experimental variability (or within statistical experimental error). In this invention, the term "about" shall have the meaning of being within 10%, preferably within 5%, of a specified value or range. Unless otherwise specified, percentages are mass percentages.

[0016] In this invention, "room temperature" refers to ambient temperature, ranging from about 15°C to about 37°C. In some embodiments, "room temperature" refers to a temperature ranging from about 20°C to about 30°C; in other embodiments, "room temperature" refers to a temperature ranging from about 25°C to about 30°C; and in still other embodiments, "room temperature" refers to 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, etc.

[0017] The “compositions” described in this invention can be prepared by any method known in pharmaceutical science. Generally, these preparation methods involve associating the active ingredient with a carrier and / or one or more other auxiliary ingredients, and then, if desired and / or expected, shaping and / or packaging the product into desired single-dose or multi-dose units.

[0018] The “composition” of the present invention can be prepared according to known methods, such as those described in the General Rules for Preparation of the Chinese Pharmacopoeia 2025, the Japanese Pharmacopoeia 16th Edition, the United States Pharmacopoeia, and the European Pharmacopoeia 9th Edition. The specific preparation method depends on the dosage form.

[0019] The active ingredient and pharmaceutically acceptable excipients in the “composition” described in this invention will vary depending on the identity, body type and / or condition of the treated subject and further on the route of administration of the active ingredient. The composition may contain between 0.1% and 100% (w / w) of the active ingredient.

[0020] The term "stereoisomer" as used in this article refers to isomers formed by atoms in a molecule having the same connection order but different spatial arrangements, including enantiomers, diastereomers, and conformational isomers.

[0021] The term "stereoisomer" as used in this article refers to isomers formed by atoms in a molecule having the same connection order but different spatial arrangements. These include enantiomers, diastereomers, and conformational isomers. Their stereostructural differences can be characterized by methods such as chiral resolution, chiral chromatography, or nuclear magnetic resonance.

[0022] The term "tautomer" used in this article refers to the interconversion between two or more isomers with different functional group structures that undergo rapid proton migration under certain conditions (such as changes in temperature and pH). These are mainly keto-enol tautomers and amide-imine tautomers, and their existing forms do not change the core skeleton and pharmacological activity of the compound.

[0023] The term "crystalline hydrate" used in this article refers to a hydrate formed when water molecules combine with the crystal lattice through non-covalent bonds such as hydrogen bonds and van der Waals forces during the crystallization process of a compound. The ratio of water molecules to compound molecules can be an integer ratio (such as 1:1, 2:1) or a non-integer ratio. Its crystal morphology and physicochemical properties can be characterized by methods such as X-ray diffraction and thermogravimetric analysis.

[0024] The term "solvent" as used in this article refers to a stable substance formed by the combination of a compound and one or more solvent molecules (such as ethanol, dichloromethane, acetone, etc.) through intermolecular forces (hydrogen bonds, hydrophobic interactions, etc.). Its formation does not change the chemical structure of the compound, but only affects its physicochemical properties such as solubility and stability.

[0025] The term "pharmaceutically acceptable salt" as used herein refers to salts formed by the compounds of this invention with inorganic acids (such as hydrochloric acid, sulfuric acid, phosphoric acid, etc.), organic acids (such as acetic acid, citric acid, benzenesulfonic acid, etc.), inorganic bases (such as sodium hydroxide, potassium hydroxide, etc.), or organic bases (such as triethylamine, DIPEA, etc.). Such salts have no obvious toxic side effects, good water solubility, stability, and bioavailability, and are suitable for pharmaceutical use.

[0026] As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Therefore, the term "modified nucleotide" encompasses substitutions, additions, or removals of internucleotide bonds, sugar moieties, or nucleobases, such as functional groups or atoms. Modifications applicable to this disclosure include all types of modifications disclosed herein or known in the art. In this document, PS modification involves the substitution of a hydroxyl group on the phosphate backbone with a thiol group; MP modification involves the substitution of a hydroxyl group on the phosphate backbone with a methyl group; and MOP modification involves the substitution of a hydroxyl group on the phosphate backbone with a substituted methoxypropyl group.

[0027] As used herein, the term "oligonucleotide" refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length. Oligonucleotides may contain ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region. As a set of non-limiting examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotides (ASO), CpG oligodeoxynucleotides, aptamers, or single-stranded siRNAs.

[0028] As used in this article, the term "aptamer" refers to a single-stranded oligonucleotide that can specifically bind to a target through its three-dimensional spatial conformation.

[0029] In this article, "lipophilic modified small nucleic acid", "oligonucleotide conjugate" and "siRNA conjugate" have the same meaning.

[0030] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides: a lipophilic modified small nucleic acid for transdermal formulations, which is a compound of general formula (I), its stereoisomer, its tautomer, its crystalline hydrate, its solvate, or a pharmaceutically acceptable salt thereof. Nuc–L–Cer(I); In the formula, Nuc is a small nucleic acid; L is a linker group; and Cer is a lipophilic modifying group.

[0031] In some embodiments, the small nucleic acid is an oligonucleotide selected from small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), antisense oligonucleotide (ASO), CpG oligodeoxynucleotide, aptamer, Dicer substrate interfering RNA (dsiRNA), or single-stranded siRNA.

[0032] In some embodiments, the linking group is selected from amide bonds, ester bonds, disulfide bonds, carbonate bonds, hydrazone bonds, phosphate ester bonds, triazole groups, or carbamate bonds.

[0033] Preferably, the linking group is selected from phosphate ester bonds.

[0034] In some embodiments, the lipophilic modifying group is selected from cholesterol, fatty acids, ceramides, or choline, preferably a ceramide group.

[0035] In some embodiments, the ceramide group has the structure shown in formula (II): (II); Where R1 is C7-C 25 Saturated or unsaturated alkyl chains (ceramide fatty acid side chains); R2 is C 13 -C 17 Saturated or unsaturated alkyl chains (sphingosine skeleton alkyl).

[0036] Preferably, R1 is C7-C 25 saturated alkyl chains.

[0037] More preferably, R1 is C7-C 15 saturated alkyl chain, C 15 -C 17 saturated alkyl chain or C 17 -C 23 saturated alkyl chains.

[0038] More preferably, R1 is a C7 saturated alkyl chain, C 15 saturated alkyl chain, C 17 saturated alkyl chain or C 23 saturated alkyl chains.

[0039] Preferably, R2 is C 14 -C 16 Saturated or unsaturated alkyl chains.

[0040] More preferably, R2 is C 15 Saturated or unsaturated alkyl chains.

[0041] In some embodiments, the lipophilic modifying group is covalently linked to at least one site of the oligonucleotide via the linking group.

[0042] In some embodiments, the site is at least one of the following: 5' end, 3' end, ribose 2'-OH site, phosphate backbone site, pyrimidine base 5 position, and purine base 8 position.

[0043] In some embodiments, each nucleotide in the oligonucleotide is independently a modified or unmodified nucleotide.

[0044] In some embodiments, the modified nucleotide is selected from at least one of the following: 2'-fluorine modified nucleotide, 2'-O-methyl modified nucleotide, glycerol nucleic acid modified nucleotide, open-ring nucleic acid modified nucleotide, peptide nucleic acid modified nucleotide, 5′-(E)-vinylphosphonic acid modified nucleotide, 2'-amino modified nucleotide, 2'-hydroxy modified nucleotide, 2'-O-allyl modified nucleotide, 2′-O-methoxyethyl modified nucleotide, and 5'-phosphate modified nucleotide.

[0045] Preferably, the modified nucleotide is selected from at least one of the following: 2′-fluoro (2′-F) modified nucleotide, 2′-O-methyl (2′-OMe) modified nucleotide, 2′-O-methoxyethyl (2′-MOE) modified nucleotide, glycerol nucleic acid (GNA) modified nucleotide, open-ring nucleic acid (UNA) modified nucleotide, peptide nucleic acid (PNA) modified nucleotide, and 5′-(E)-vinylphosphonic acid (5′-VP) modified nucleotide.

[0046] In this invention, 2´-F, 2´-OME, and 2´-MOE modifications refer to the substitution of the 2′ hydroxyl group of the furanyl ring of the nucleotide by fluorine (F), methoxy (OMe), or methoxyethoxy (MOE), respectively. GNA modification refers to ethylene glycol nucleic acid nucleotides; UNA modification refers to open-ring ribonucleotides and / or ethylene glycol nucleic acid nucleotides. UNA and GNA are thermally unstable chemical modifications that can improve siRNA off-target effects (MK Schlegel et al., J. Am. Chem. Soc. 139, 8537-8546 (2017)).

[0047] PNA modification refers to peptide nucleic acids, where the glycosyl backbone of RNA is replaced with an amide-containing backbone. It is a type of DNA analog where the glycosyl backbone is replaced by a polypeptide backbone. The literature (M. Richter et al., ACS Chem. Biol. 18, 7-11(2022)) reported that replacing a single phosphodiester bond at positions 2-7 of the seed region of the antisense strand with an amide bond of PNA can effectively inhibit off-target side effects caused by microRNA.

[0048] 5´-VP modification refers to the modification of the 5´ end of dsRNA with vinyl-phosphate.

[0049] More preferably, the GNA modification is selected from adenosine-diol nucleic acid, cytidine-diol nucleic acid, thymidine-diol nucleic acid, and guanosine-diol nucleic acid.

[0050] In some embodiments, the oligonucleotide comprises at least one modified nucleotide and / or at least one interstrand modification.

[0051] In some embodiments, the interchain modifications include phosphothiophosphate (PS) modification, methylphosphonate (MP) modification, and methoxypropylphosphonate (MOP) modification.

[0052] In some embodiments, all nucleotides in the oligonucleotide are modified nucleotides.

[0053] In some embodiments, the oligonucleotide is siRNA. Preferably, the siRNA comprises a sense strand and an antisense strand.

[0054] In some embodiments, the nucleotide linkages in the siRNA are phosphodiester bonds and / or thiophosphate bonds.

[0055] In some embodiments, at least one strand of the siRNA comprises nucleotides selected from natural ribonucleotides and / or modified nucleotides.

[0056] Preferably, the modifying groups of the nucleotides are each independently selected from 2'-F, 2'-OME, 2'-MOE, GNA, UNA, PNA, or 5'-VP. More preferably, the modifying groups are each independently selected from 2'-F and 2'-OME.

[0057] In some embodiments, the lipophilic modifying group is covalently linked to the 3′ end of the oligonucleotide via the linking group.

[0058] Preferably, the lipophilic modification group is covalently linked to the 3′ end of the siRNA positive strand via the linker group.

[0059] In some embodiments, all nucleotides on the positive strand of the siRNA are modified nucleotides.

[0060] In some embodiments, all nucleotides on the antisense strand of the siRNA are modified nucleotides.

[0061] In some embodiments, the siRNA is a siRNA that targets tyrosinase (TYR).

[0062] Preferably, the siRNA targeting TYR comprises a sense strand and an antisense strand.

[0063] More preferably, the sense chain comprises the sequence shown in SEQ ID NO:1, and the antisense chain comprises the sequence shown in SEQ ID NO:2.

[0064] Specifically, the sequence shown in SEQ ID NO:1 is 5′ UGCCAAUUUCAGCUUUAGA 3′, the sequence shown in SEQ ID NO:2 is 5′ UCUAAAGCUGAAAUUGGCAdTdT 3′.

[0065] In some embodiments, the ceramide group is selected from any of the following structures: .

[0066] Secondly, the present invention provides a method for preparing the above-mentioned lipophilic modified small nucleic acid, comprising the following steps: S1. Couple the lipophilic modification group to the controllable porous glass (CPG) to obtain a lipophilic... CPG solid support; S2, with lipophilic properties CPG solid-phase support was used as a solid-phase carrier to synthesize the positive strand of siRNA using the phosphoramide method. After synthesis, the siRNA single-strand crude product was obtained by ammonolysis and deprotection. S3 and crude siRNA single strands were purified to obtain the sense and antisense strands; S4. Anneal equal molar amounts of the sense and antisense strands to form double-stranded siRNA.

[0067] In some embodiments, the synthesis method described in step S2 is as follows: using the phosphoramide method, the modified phosphoramide monomer is used sequentially to extend the chain according to the siRNA sequence.

[0068] Preferably, the synthesis method further includes selectively oxidizing or sulfiding to construct a phosphate backbone after each condensation step.

[0069] In some implementations, in step S4, the annealing temperature is 85-90°C, the annealing time is 3-5 minutes, and then the temperature is cooled to room temperature.

[0070] Thirdly, the present invention provides a composition comprising the above-described lipophilic modified small nucleic acid.

[0071] In some embodiments, the composition is a pharmaceutical composition or a cosmetic composition.

[0072] In some embodiments, the cosmetic composition is a cosmetic composition with whitening effects.

[0073] In some embodiments, the composition further comprises pharmaceutically and / or cosmetically acceptable excipients.

[0074] In some embodiments, the composition is a solid, liquid, or gel composition.

[0075] In some embodiments, the composition is a solution, lyophilized powder, gel, sponge, or cellulosic formulation.

[0076] In some embodiments, the pharmaceutical composition is an oral formulation, a topical formulation, an intravenous injection, a subcutaneous injection, or an intramuscular injection.

[0077] Any of the formulations can be used to deliver the lipophilic modified small nucleic acid for enhancements in skin permeability, cellular uptake, stability, or gene silencing. In some embodiments, the lipophilic modified small nucleic acid is formulated in a buffer solution, such as an aqueous phosphate-buffered saline solution, liposomes, micelle structures, and shells. The buffer solution may be selected from solutions of acetate, citrate, glutenin, carbonate, or phosphate, or any combination thereof. In some embodiments, the lipophilic modified small nucleic acid is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the lipophilic modified small nucleic acid is formulated in an alkaline buffer solution (e.g., PBS).

[0078] In some implementations, the formulation comprises lipid nanoparticles. Specifically, the excipient comprises liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need.

[0079] In some embodiments, the formulation comprises a gel, specifically a carbomer gel or a hyaluronic acid gel.

[0080] In some embodiments, the excipient imparts to the composition enhanced stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient.

[0081] In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or a medium (e.g., buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the lipophilic modified small nucleic acid is lyophilized to extend its shelf life and then prepared into a solution before use (e.g., administration to a subject). Therefore, the excipient in a composition comprising any of the lipophilic modified small nucleic acids described herein can be a lyophilization protectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0082] In some embodiments, the composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0083] Compositions suitable for injectable applications include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL.™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). These carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In many cases, isotonic agents, such as sugars, and polyols such as mannitol, sorbitol, and sodium chloride, are preferably included in the composition. Sterile injectable solutions can be prepared by incorporating the desired amount of lipophilic modified small nucleic acid with one or a combination of the above-listed ingredients into a selected solvent, followed by filtration sterilization.

[0084] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., a lipophilic modified small nucleic acid for reducing melanin), although the percentage of one or more active ingredients may be between about 1% and about 80% or more of the total composition by weight or volume. Those skilled in the art of preparing such formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore various dosages and treatment regimens may be desirable.

[0085] In some embodiments, the cosmetic composition is a topical product.

[0086] In some embodiments, the cosmetic composition is a beauty product or cosmetic product.

[0087] In some embodiments, the cosmetic composition is applied topically; preferably transdermal.

[0088] In some embodiments, the dosage form of the cosmetic composition is an emulsion, cream, mousse, gel, foam, lotion, mask, ointment, balm, solution, serum, spray, or face mask.

[0089] The actual dose level of the lipophilic modified small nucleic acid in the composition can vary, and the amount of the lipophilic modified small nucleic acid brings satisfactory results to a specific user without producing toxicity or side effects.

[0090] The composition may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses. A “unit dose” is a discrete amount of the composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is typically equal to the dose of the active ingredient administered to a subject and / or a convenient fraction of that dose (e.g., one-half or one-third of the dose).

[0091] As used herein, the term "treatment" means, unless otherwise stated, reversing or alleviating the condition or disease to which the term applies, or one or more symptoms of such condition or disease, inhibiting the progression of said condition or disease or one or more symptoms thereof, or preventing said condition or disease or one or more symptoms thereof. As used herein, the term "treatment" refers to a therapeutic act, as defined above.

[0092] The “effective amount” of the lipophilic modified small nucleic acid described in this invention refers to an amount sufficient to elicit the desired biological response. The effective amount of the lipophilic modified small nucleic acid described in this invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the administration pattern, and the age and health status of the subject. In some embodiments, the effective amount is a therapeutically effective amount. Alternatively, in a single method or use, this invention can be used for prophylactic treatment when indicated and effective. In some embodiments, the effective amount is the amount of the lipophilic modified small nucleic acid described herein in a single dose. In some embodiments, the effective amount is a combination of the lipophilic modified small nucleic acid described herein in multiple doses.

[0093] The term "therapeuticly effective amount" as used herein refers to an amount sufficient to provide therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with that condition. Therapeuticly effective amount of lipophilic modified small nucleic acids refers to an amount in which the therapeutic agent, alone or in combination with other therapies, provides therapeutic benefit in the treatment of a condition. The term "therapeuticly effective amount" may encompass amounts that improve overall therapy, reduce or avoid symptoms, signs, or causes of a condition, and / or enhance the therapeutic efficacy of another therapeutic agent. In some embodiments, the therapeutically effective amount is an amount sufficient to treat any of the diseases or conditions described.

[0094] It should also be noted that the specific dosage and method of administration of the lipophilic modified small nucleic acid for different patients depends on many factors, including the patient's age, weight, sex, natural health condition, nutritional status, drug activity intensity, administration time, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The effective dosage of the active ingredient depends at least on the nature of the condition to be treated, its toxicity, the method of delivery, and the pharmaceutical formulation, and will be determined through clinical studies using escalating doses based on standard practice.

[0095] Fourthly, the present invention provides the use of the above-mentioned lipophilic modified small nucleic acid or the above-mentioned composition in the preparation of pharmaceuticals, cosmetics or beauty products.

[0096] In some embodiments, the drug is a transdermal formulation.

[0097] In some embodiments, the cosmetic or beauty product is a cosmetic or beauty product with whitening effect.

[0098] The beneficial effects of this invention are as follows: Compared with existing technologies, this invention utilizes the natural and high affinity of ceramides to the stratum corneum lipid matrix to enable the modified small nucleic acid molecules to "disguise" themselves as the skin's own lipid components and actively "integrate" into the stratum corneum lipid bilayer structure. This solves the skin irritation and biosafety issues that may be caused by existing non-natural lipid modifiers. The lipophilic modified small nucleic acid of this invention has the advantages of higher biocompatibility, better safety, and significantly improved transdermal efficiency.

[0099] The effectiveness was clearly verified: all ceramide-modified siRNAs (compounds 1-2 to 1-5) showed significant inhibitory activity in both mouse melanocyte B16 and human melanocyte MNT1 cell models (inhibition rate in B16: 50.79-80.09%; inhibition rate in MNT1: 37.78-64.17%), which was far superior to the unmodified group, demonstrating the universality and high efficiency of this series of modification strategies.

[0100] Provides a basis for optimized design: different chain length modifications exhibit different activities (C 18 It performed best in B16 cells, with an inhibition rate of 80.09%, which provides clear design guidance and data support for flexibly selecting the optimal carbon chain length according to specific treatment needs (lesion depth, degree of skin barrier damage, etc.) to achieve the best transdermal efficiency and therapeutic effect. Attached Figure Description

[0101] Figure 1 A bar chart showing the results of quantitative real-time PCR detection of mouse melanoma B16 cells.

[0102] Figure 2 This is a bar chart showing the results of quantitative real-time PCR detection of human melanoma MNT1 cells. Detailed Implementation

[0103] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0104] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0105] Example 1: Preparation of delivery ligand Lp-01-CPG Synthetic compound 1

[0106] C8Ceramide (375 mg, 0.88 mmol) was dissolved in anhydrous pyridine (py, 2.5 mL). The reaction system was then purged under nitrogen atmosphere using a vacuum pump. 4,4'-bismethoxytriphenylmethylchloro(DMTrCl, 448 mg, 1.32 mmol, 1.5 equivalence) was added with stirring in an ice bath, and the mixture was heated to room temperature and stirred overnight. The pyridine was then removed by vacuum distillation, dissolved in dichloromethane, washed once with saturated sodium bicarbonate and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to obtain the crude product. Purification was achieved by rapid silica gel column chromatography (dichloromethane / methanol = 100:1–10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). The product was dried under vacuum to obtain compound 1, with a mass of 577 mg and a yield of 90%. The product was identified by LC-MS: MS m / z: C 47 H 69 NO5Na, [M+Na]+, theoretical value: 751.07, actual value: 751.11.

[0107] Synthetic compound 2

[0108] Compound 1 (577 mg, 0.79 mmol), succinic anhydride (158 mg, 1.58 mmol, 2.0 equivalence), and 4-dimethylaminopyridine (DMAP, 289 mg, 2.37 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (DCM, 5 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (DIPEA, 0.73 mL, 3.94 mmol, 5.0 equivalence) was added with stirring in an ice bath. The reaction was allowed to proceed overnight with stirring at room temperature. After complete reaction was confirmed by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once with saturated ammonium chloride solution and once with saturated brine solution. The mixture was dried over anhydrous sodium sulfate, filtered, and then distilled under reduced pressure and concentrated to obtain the crude product. Compound 2 was purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, and gradient elution). After vacuum pumping, the product was dried to obtain compound 2, with a mass of 478 mg and a yield of 73%. The product was identified by LC-MS: MS m / z: C 51 H 73 NO8Na, [M+Na]+, theoretical value: 851.14, actual value: 851.07.

[0109] Synthetic compound 3

[0110] Compound 2 (478 mg, 0.58 mmol) and HBTU (332 mg, 0.87 mmol, 1.5 equivalence) were dissolved in anhydrous N,N-dimethylformamide (10 mL), followed by the addition of triethylamine (132 μL, 1.16 mmol, 2.0 equivalence). After shaking for 30 min, aminolated CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 1000 Å, 80 μmol / g, 2.5 g) was added, and the reaction was shaken for another 24 h. After the reaction was completed, the mixture was filtered, and the CPG was washed three times each with N,N-dimethylformamide and dichloromethane, and then dried under vacuum. Then, Cap A (3 mL, manufacturer: Anage) and Cap B (3 mL, manufacturer: Anage) reagents were added to the CPG filter cake, and the mixture was shaken for 2 hours to cap the unlinked amino groups with acetyl groups. The mixture was then filtered again, and the CPG filter cake was washed three times each with N,N-dimethylformamide and dichloromethane, and then completely dried using a vacuum pump. Compound 3 was obtained, which is the CPG linked to ligand Lp-01, with a yield of 2.73 g. The loading was determined to be 41.19 μmol / g by detecting the absorbance of the 4,4'-dimethoxytriphenylmethyl group (DMTr).

[0111] Example 2: Preparation of delivery ligand Lp-02-CPG Synthetic compound 4

[0112] C 16 Ceramide (475 g, 0.88 mmol) was dissolved in anhydrous pyridine (Py, 2.5 mL), and the reaction system was then purged under nitrogen atmosphere using a vacuum pump. 4,4'-bismethoxytriphenylmethylchloro(DMTrCl, 448 mg, 1.32 mmol, 1.5 equivalence) was added under ice-bath stirring, and the mixture was heated to room temperature and stirred overnight. The pyridine was then removed by vacuum distillation, dissolved in dichloromethane, washed once with saturated sodium bicarbonate and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum distillation to obtain the crude product. Purification was achieved by rapid silica gel column chromatography (dichloromethane / methanol = 100:1–10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). The product was dried under vacuum to obtain compound 4, with a mass of 682 mg and a yield of 92%. The product was identified by LC-MS: MS m / z: C 55 H 88 NO5, [M+H]+, theoretical value: 843.30, actual value: 843.30.

[0113] Synthetic compound 5

[0114] Compound 4 (682 mg, 0.81 mmol), succinic anhydride (162 mg, 1.62 mmol, 2.0 equivalence), and 4-dimethylaminopyridine (DMAP, 297 mg, 2.43 mmol, 3.0 equivalence) were dissolved in anhydrous dichloromethane (DCM, 5 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (DIPEA, 0.75 mL, 4.05 mmol, 5.0 equivalence) was added with stirring in an ice bath. The reaction was allowed to proceed overnight with stirring at room temperature. After complete reaction was confirmed by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once with saturated ammonium chloride solution and once with saturated brine solution. The mixture was dried over anhydrous sodium sulfate, filtered, and then distilled under reduced pressure and concentrated to obtain the crude product. Purification was performed by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, using gradient elution). After vacuum pumping, compound 5 was obtained with a mass of 488 mg, yielding 64%. The product was identified by LC-MS: MS m / z: C 59 H 91 NO₈Na, [M+Na]⁺, theoretical value: 965.38, actual value: 965.35.

[0115] Synthetic compound 6

[0116] Compound 5 (488 mg, 0.52 mmol) and HBTU (297 mg, 0.78 mmol, 1.5 equivalence) were dissolved in anhydrous N,N-dimethylformamide (10 mL), followed by the addition of triethylamine (118 μL, 1.04 mmol, 2.0 equivalence). After shaking for 30 min, aminolated CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 1000 Å, 80 μmol / g, 2.5 g) was added, and the reaction was shaken for another 24 h. After the reaction was completed, the mixture was filtered, and the CPG was washed three times each with N,N-dimethylformamide and dichloromethane, and then dried under vacuum. Then, Cap A (3 mL, manufacturer: Anage) and Cap B (3 mL, manufacturer: Anage) reagents were added to the CPG filter cake, and the mixture was shaken for 2 hours to cap the unlinked amino groups with acetyl groups. The mixture was then filtered again, and the CPG filter cake was washed three times each with N,N-dimethylformamide and dichloromethane, and then completely dried using a vacuum pump. Compound 6 was obtained, which is the CPG linked to ligand Lp-02, with a yield of 2.79 g. The loading was determined to be 39.66 μmol / g by detecting the absorbance of the 4,4'-dimethoxytriphenylmethyl group (DMTr).

[0117] Example 3: Preparation of delivery ligand Lp-03-CPG Synthetic compound 7

[0118] C 18 Ceramide (498 mg, 0.88 mmol) was dissolved in anhydrous pyridine (Py, 2.5 mL), and the reaction system was purged under nitrogen atmosphere using a vacuum pump. Then, 4,4'-bismethoxytriphenylmethyl chloride (DMTrCl, 448 mg, 1.32 mmol, 1.5 equivalence) was added with stirring in an ice bath, and the mixture was heated to room temperature and stirred overnight. The pyridine was then removed by vacuum distillation, dissolved in dichloromethane, washed once with saturated sodium bicarbonate and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to obtain the crude product. Purification was achieved by rapid silica gel column chromatography (dichloromethane / methanol = 100:1–10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). The product was dried under vacuum to obtain compound 7, with a mass of 466 mg and a yield of 61%. The product was identified by LC-MS: MS m / z: C 57 H 89 NO5Na, [M+Na]+, theoretical value: 891.34, actual value: 891.30.

[0119] Synthetic compound 8

[0120] Compound 7 (466 mg, 0.54 mmol), succinic anhydride (108 mg, 1.08 mmol, 2.0 equivalences), and 4-dimethylaminopyridine (DMAP, 198 mg, 1.62 mmol, 3.0 equivalences) were dissolved in anhydrous dichloromethane (DCM, 5 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (DIPEA, 0.5 mL, 2.70 mmol, 5.0 equivalences) was added with stirring in an ice bath. The reaction was allowed to proceed overnight with stirring at room temperature. After complete reaction was confirmed by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once with saturated ammonium chloride solution and once with saturated brine solution. The mixture was dried over anhydrous sodium sulfate, filtered, and then distilled under reduced pressure and concentrated to obtain the crude product. Purification was performed by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, using gradient elution). After vacuum pumping, compound 8 was obtained with a mass of 214 mg, representing a yield of 41%. The product was identified by LC-MS: MS m / z: C 61 H 93 NO8Na, [M+Na]+, theoretical value: 991.41, actual value: 991.45.

[0121] Synthetic compound 9

[0122] Compound 8 (214 mg, 0.22 mmol) and HBTU (126 mg, 0.33 mmol, 1.5 equivalence) were dissolved in anhydrous N,N-dimethylformamide (10 mL), followed by the addition of triethylamine (50 μL, 0.44 mmol, 2.0 equivalence). After shaking for 30 min, aminolated CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 1000 Å, 80 μmol / g, 2.75 g) was added, and the reaction was shaken for another 24 h. After the reaction was completed, the mixture was filtered, and the CPG was washed three times each with N,N-dimethylformamide and dichloromethane, and then dried under vacuum. Then, Cap A (3 mL, manufacturer: Anage) and Cap B (3 mL, manufacturer: Anage) reagents were added to the CPG filter cake, and the mixture was shaken for 2 hours to cap the unlinked amino groups with acetyl groups. The mixture was then filtered again, and the CPG filter cake was washed three times each with N,N-dimethylformamide and dichloromethane, and then completely dried using a vacuum pump. Compound 9, ligand Lp-03-linked CPG, was obtained, with a yield of 2.67 g. The loading was determined to be 34.46 μmol / g by detecting the absorbance of the 4,4'-dimethoxytriphenylmethyl group (DMTr).

[0123] Example 4: Preparation of delivery ligand Lp-04-CPG Synthetic compound 10

[0124] C 24 Ceramide (574 mg, 0.88 mmol) was dissolved in anhydrous pyridine (Py, 2.5 mL), and the reaction system was purged under nitrogen atmosphere using a vacuum pump. Then, 4,4'-bismethoxytriphenylmethyl chloride (DMTrCl, 448 mg, 1.32 mmol, 1.5 equivalence) was added with stirring in an ice bath, and the mixture was heated to room temperature and stirred overnight. The pyridine was then removed by vacuum distillation, dissolved in dichloromethane, washed once with saturated sodium bicarbonate and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to obtain the crude product. Purification was achieved by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). The product was dried under vacuum to obtain compound 10, with a mass of 411 mg and a yield of 49%. The product was identified by LC-MS: MS m / z: C 63 H 103 NO5Na, [M+Na]+, theoretical value: 977.52, actual value: 977.50.

[0125] Synthetic compound 11

[0126] Compound 10 (411 mg, 0.43 mmol), succinic anhydride (86 mg, 0.86 mmol, 2.0 equivalences), and 4-dimethylaminopyridine (DMAP, 158 mg, 1.30 mmol, 3.0 equivalences) were dissolved in anhydrous dichloromethane (DCM, 5 mL). The reaction system was then purged under a nitrogen atmosphere using a vacuum pump. N,N-diisopropylethylamine (DIPEA, 0.4 mL, 2.16 mmol, 5.0 equivalences) was added with stirring in an ice bath. The reaction was allowed to proceed overnight with stirring at room temperature. After complete reaction was confirmed by LC-MS, the reaction was quenched with water. The reaction system was extracted three times with dichloromethane. The combined organic phases were washed once with saturated ammonium chloride solution and once with saturated brine solution. The mixture was dried over anhydrous sodium sulfate, filtered, and then distilled under reduced pressure and concentrated to obtain the crude product. Purification was performed by rapid silica gel column chromatography (dichloromethane / methanol = 100:1-10:1, with 0.5% triethylamine added to each of mobile phases A and B, gradient elution). After vacuum pumping, compound 11 was obtained with a mass of 381 mg, yielding 84%. The product was identified by LC-MS: MS m / z: C 67 H 108 NO8, [M+H]+, theoretical value: 1055.59, actual value: 1055.63.

[0127] Synthetic compound 12

[0128] Compound 11 (381 mg, 0.36 mmol) and HBTU (207 mg, 0.54 mmol, 1.5 equivalence) were dissolved in anhydrous N,N-dimethylformamide (10 mL), followed by the addition of triethylamine (82 μL, 0.72 mmol, 2.0 equivalence). After shaking for 30 min, aminolated CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 1000 Å, 80 μmol / g, 2.75 g) was added, and the mixture was shaken for another 24 h. After the reaction was completed, the mixture was filtered, and the CPG was washed three times each with N,N-dimethylformamide and dichloromethane, and then dried under vacuum. Then, Cap A (3 mL, manufacturer: Anage) and Cap B (3 mL, manufacturer: Anage) reagents were added to the CPG filter cake, and the mixture was shaken for 2 hours to cap the unlinked amino groups with acetyl groups. The mixture was then filtered again, and the CPG filter cake was washed three times each with N,N-dimethylformamide and dichloromethane, and then completely dried using a vacuum pump. Compound 12 was obtained, which is the CPG linked to ligand Lp-04, with a yield of 2.37 g. The loading was determined to be 37.01 μmol / g by detecting the absorbance of the 4,4'-dimethoxytriphenylmethyl group (DMTr).

[0129] Example 5: Synthesis of oligonucleotides and oligonucleotide conjugates The oligonucleotides and oligonucleotide conjugates of the present invention can be synthesized using phosphoramide solid-phase synthesis methods well known in the art. Nucleoside monomers are sequentially linked in a 3' to 5' direction according to the nucleotide type and sequence of the oligonucleotides. The linkage of each nucleoside monomer involves the same cycle, including four steps: deprotection, coupling, capping, oxidation, or sulfidation.

[0130] 1. Synthesis of the Justice Chain (SS Chain) and the Antisense Chain (AS Chain) The nucleoside monomers are sequentially linked in the 3' to 5' direction along the oligonucleotide sequence using a blank CPG solid-phase support or the aforementioned liposome ligands (Lp-01, Lp-02, Lp-03, Lp-04) linked to the present invention via a solid-phase phosphoramide synthesis method. Typically, the 3' end of the sense or antisense strand of the oligonucleotide is linked to the CNS-targeted delivery structure. In some specific embodiments, the nucleoside monomers are prepared as a 0.04 mol / L acetonitrile solution and dried through a 4 Å molecular sieve, with identical reaction conditions for each step. The deprotection agent is a 3% trichloroacetic acid solution in dichloromethane, deprotected three times for 30 s. The coupling agent is a 0.25 mol / L acetonitrile solution of 5-ethylthio-1H-tetrazole, coupled three times for 3 min. The coupling reaction is carried out in anhydrous acetonitrile. Capping is done using a 20% acetic anhydride-acetonitrile solution and an N-methylimidazolium / pyridine / acetonitrile solution (20 / 30 / 50). v / v / v ), capped twice; oxidation was performed using a pyridine / water solution of 0.05 mol / L iodine (90 / 10, v / v Thiolation was performed twice using a 0.20 mol / L solution of 5-imino-1,2,4-dithiazolidin-3-thione / pyridine.

[0131] 2. Purification and annealing of oligonucleotides 2.1 Ammonolysis The CPG carrier that has completed the synthesis step was added to a 5 mL centrifuge tube, along with 1 mL of 25-28% concentrated ammonia solution. The mixture was heated in a forced-air oven at 80°C for 2 hours or at 60°C for 6 hours. After filtration, the solid carrier was washed three times with water. The filtrate was concentrated using a concentrator to obtain the crude product for further purification.

[0132] 2.2 Deprotection When at least one 2'-TBDMS protection exists on the synthesized nucleotide sequence, the method further includes contacting the crude product after the ammonolysis step with triethylamine hydrofluoric acid to remove the 2'-TBDMS protection. The deprotection reagent uses an N-methylimidazole / triethylamine hydrofluoric acid / triethylamine solution (6 / 3 / 4). v / v / v The reaction was carried out at 65°C for 2.5 hours.

[0133] 2.3 Purification Purification and desalting methods are well known to those skilled in the art. For example, nucleic acids can be purified using a preparative ion chromatography column with gradient elution of NaBr or NaCl; after the products are collected and combined, desalting can be performed using a reversed-phase chromatography column. The fraction with a purity greater than 95% is collected, concentrated, dried, and prepared into a 100 μM PBS solution.

[0134] 2.4 Annealing In a centrifuge tube, the PBS solution containing the sense strand (SS strand) and the antisense strand (AS strand) was mixed at a molar ratio (SS strand / AS strand = 1.1 / 1), heated to 85°C, held for 3-5 minutes, and then allowed to cool naturally to room temperature. The system was then freeze-dried to obtain the product.

[0135] Oligonucleotide conjugates 1-2 to 1-5 were synthesized according to the above method. The sequence of TYR-siRNA is shown in Table 1. Information on the siRNA conjugates formed by the compounds of this invention and siRNA is shown in Table 2. In the table, 1-1 is a blank CPG solid-phase support, 1-2 is the delivery ligand Lp-01-CPG, 1-3 is the delivery ligand Lp-02-CPG, 1-4 is the delivery ligand Lp-03-CPG, and 1-5 is the delivery ligand Lp-04-CPG.

[0136] Table 1 Unmodified siRNA sequences

[0137] Table 2 Modified siRNA sequences

[0138] Note: Uppercase letters A, C, G, and U represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a nucleotide modified with 2'-methoxy; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a nucleotide modified with 2'-fluorine; s indicates that the connection between two adjacent nucleotides is a phosphate thioester group.

[0139] Example 6: Inhibitory effect of TYR siRNA on tyrosinase TYR expression in mouse melanocyte line B16 The mouse melanocyte cell line B16 (purchased from the Cell Bank of the Chinese Academy of Sciences) was selected for the experiment to detect the inhibitory effect of siRNA targeting TYR on tyrosinase (TYR) expression, as shown in Table 2. The experimental procedure was as follows: The B16 melanocyte cell line was digested with trypsin and seeded at an appropriate density into 24-well cell culture plates, with a density of 10 cells per well. 5Cells were cultured overnight at 37°C and 5% CO2 in DuPont modified Eagle (DMEM) medium containing 10% fetal bovine serum (FBS). The next day, the culture medium was replaced with serum-free medium, and siRNA targeting TYR (as shown in Table 2) was added to a final concentration of 10 µM, in triplicate. No siRNA was added to the negative control group. Cells were cultured for another 24 hours at 37°C and 5% CO2. After 24 hours, the serum-free medium was replaced with serum-containing medium, and the cells were cultured for another 24 hours at 37°C and 5% CO2. Cells were then harvested, washed with PBS, and RNA was extracted using Trizol (Invitrogen 15596018). The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara PrimeScript™ II 1st Strand cDNA Synthesis Kit: 6210A) according to the manufacturer's instructions. Next, the inhibition rate of TYR mRNA expression was detected using a quantitative real-time PCR kit (TB Green® Premix Ex Taq™ II, Takara, RR820A). GAPDH cDNA was used as an internal control in this quantitative real-time PCR method. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s.

[0140] The primer sequences are shown in Table 3.

[0141] Table 3 Sequences of mTYR detection primers

[0142] Data Analysis: Using comparative Ct ( △△ In the Ct method, siRNA inhibitory activity is measured by the remaining amount of TYR gene expression 2- △△ Ct indicates that △△ Ct = [(Target gene in Ct experimental group - Internal reference in Ct experimental group) – (Target gene in Ct negative control group - Internal reference in Ct negative control group)]. The mRNA inhibition rate is then calculated using the following formula: mRNA inhibition rate = (1 - Remaining mTYR gene expression level) × 100%.

[0143] Table 2 shows the inhibitory effects of siRNA conjugates on mTYR mRNA expression in mouse melanocyte line B16, such as... Figure 1 As shown in Table 4.

[0144] Table 4. Inhibition of mTYR mRNA expression in mouse melanocyte cell line B16 by siRNA conjugates

[0145] Example 7: Inhibitory effect of TYR siRNA on tyrosinase TYR expression in human melanocyte cell line MNT1 Human melanocyte cell line MNT1 (purchased from the Cell Bank of the Chinese Academy of Sciences) was selected for experiments to detect the inhibitory effect of TYR-targeting siRNA on tyrosinase (TYR) expression, as shown in Table 2. The experimental procedure was as follows: MNT1 melanocyte cells were digested with trypsin, adjusted to an appropriate density, and seeded into 24-well cell culture plates at a density of 10 cells per well. 5 Cells were cultured overnight at 37°C and 5% CO2 in DuPont modified Eagle (DMEM) medium containing 10% fetal bovine serum (FBS). The next day, the culture medium was replaced with serum-free medium, and siRNA targeting TYR (as shown in Table 2) was added to a final concentration of 10 µM, in triplicate. No siRNA was added to the negative control group. Cells were cultured for another 24 hours at 37°C and 5% CO2. After 24 hours, the serum-free medium was replaced with serum-containing medium, and the cells were cultured for another 24 hours at 37°C and 5% CO2. Cells were then harvested, washed with PBS, and RNA was extracted using Trizol (Invitrogen 15596018). The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara PrimeScript™ II 1st Strand cDNA Synthesis Kit: 6210A) according to the manufacturer's instructions. Next, the inhibition rate of TYR mRNA expression was detected using a quantitative real-time PCR kit (TB Green® Premix Ex Taq™ II, Takara, RR820A). GAPDH cDNA was used as an internal control in this quantitative real-time PCR method. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s.

[0146] The primer sequences are shown in Table 5 as hTYR.

[0147] Table 5 Sequences of hTYR detection primers

[0148] Data Analysis: Using comparative Ct ( △△ In the Ct method, siRNA inhibitory activity is measured by the remaining amount of TYR gene expression 2- △△ Ct indicates that △△Ct = [(Target gene in Ct experimental group - Internal reference in Ct experimental group) – (Target gene in Ct negative control group - Internal reference in Ct negative control group)]. The mRNA inhibition rate is then calculated using the following formula: mRNA inhibition rate = (1 - Remaining amount of hTYR gene expression) × 100%.

[0149] The siRNA conjugates shown in Table 2 inhibited hTYR mRNA expression in the human melanocyte cell line MNT1 as shown in Table 6. Figure 2 As shown.

[0150] Table 6. Inhibition of hTYR mRNA expression in human melanocyte cell line MNT1 by siRNA conjugates.

[0151] Example 8: In vitro cytotoxicity experiment of lipophilic modified siRNA This embodiment uses human immortalized keratinocytes (HaCaT cells, purchased from the Cell Bank of the Chinese Academy of Sciences) as an in vitro cell model. The CCK-8 assay was used to detect the changes in cell activity after treatment with different concentrations of lipophilic modified siRNA, to investigate the effect of lipophilic modified siRNA on the activity of normal skin cells, and to verify its biocompatibility and safety. At the same time, it provides a basis for subsequent siRNA transfection experiments and selection of transdermal drug delivery concentration.

[0152] The experimental procedure is as follows: HaCaT cells were cultured in DMEM complete medium containing 10% FBS and 1% P&S in a conventional incubator at 37℃, 5% CO2, and saturated humidity. Cells in the logarithmic growth phase were used for the experiment. The old medium in the culture dish was discarded, and the cells were washed with PBS. An appropriate amount of trypsin-EDTA digestion solution was added to digest the cells. When the cells became rounded and detached, complete medium was added to stop the digestion. The cells were gently pipetted to prepare a single-cell suspension, counted, and the cell density was adjusted. The cell density was adjusted to 5 × 10⁶ cells / year using complete medium. 4Cells were seeded at a rate of 100 μL per well in a 96-well cell culture plate (i.e., 5 × 10³ cells per well). To prevent edge effects, PBS buffer was added to the outermost ring of wells in the 96-well plate. The 96-well plate was incubated overnight (approximately 12-24 h) at 37°C with 5% CO2 to allow for full cell adhesion. After cell adhesion, the old culture medium was discarded, and the cells were treated according to the following groups, with 3 (or more) replicates per group: Blank control group (NC group): cells were seeded and an equal volume of complete culture medium without siRNA was added; siRNA treatment groups: lipophilic modified siRNA solutions were added to wells at final concentrations of 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 50 μM, respectively, with the same volume added per well as the blank control group; Blank wells: only complete culture medium was added, without seeding cells, used to subtract background. The final volume per well was kept consistent across all groups (100 μL). After adding the reagent, the 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours of siRNA treatment, cell morphology and adhesion were observed under an inverted microscope to confirm the absence of contamination. 10 μL of CCK-8 reagent was added to each well, and the plate was gently tapped to mix thoroughly with the culture medium. The 96-well plates were then incubated at 37°C in the dark for 1 hour. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader, and the OD450 was recorded.

[0153] Data Analysis: First, subtract the OD value of the blank well from the OD value of each experimental well to obtain the corrected OD value. Using the cell viability of the blank control group (NC group) as 100%, calculate the relative cell viability of each siRNA concentration treatment group: Relative cell viability (%) = (OD value of experimental group - OD value of blank well) / (OD value of NC control group - OD value of blank well) × 100% Where: The relative cell viability of HaCaT cells after treatment with different concentrations of lipophilic modified siRNA for 24 h is shown in Table 7.

[0154] Table 7. Effects of siRNA conjugates on HaCaT cell viability

[0155] Example 9: In vivo activity assay of lipophilic modified siRNA The inhibitory efficiency of the oligonucleotide conjugates (1-2, 1-3, 1-4 and 1-5), the control conjugate 1-1 (unmodified siRNA), and the solvent group 1×PBS on TYR mRNA expression in C57B / 6j mice was investigated.

[0156] All of the present invention, 1-1, 1-2, 1-3, 1-4 and 1-5, are formulated as gel preparations, with the siRNA concentration in the gel being 7 mg / mL; the gel matrix is ​​carbomer.

[0157] The C57B / 6j mice used in this example were purchased from Vital River, approximately 8-10 weeks old, and male.

[0158] First, C57B / 6j mice were divided into three groups: a negative control group, a positive control group, and an experimental group (4 groups), with three mice in each group. One day before drug administration, the hair on the backs of the mice was carefully removed using an electric shaver. If there were obvious stubble, a thin layer of depilatory cream could be applied, left to stand for 1-2 minutes, and then gently wiped clean with a cotton swab. The back skin was then washed with warm water and patted dry. Care should be taken to avoid damaging the stratum corneum. Drug administration was only administered after 24 hours of recovery following hair removal, provided that there was no redness, swelling, or breakage of the skin.

[0159] Each group weighed out the corresponding drug formulation and evenly applied it to the hairless area on the back of mice (approximately 2cm × 3cm). The dosage was 5mg / kg (based on siRNA content), with an application volume of 5mL / kg. The formulation was gently spread evenly using a sterile glass rod or fingertip, and massaged briefly to ensure full contact with the skin. Administered once daily. Mice were housed individually during the administration period to prevent licking and interference with drug retention in the treatment area. After 28 days of administration, the mice were euthanized, and the skin from the treatment area on the back was immediately harvested. An appropriate amount of skin tissue was homogenized using Trizol at 4°C, and total RNA was extracted from the skin tissue following standard procedures.

[0160] The expression level of TYR mRNA in mouse skin tissue was detected by real-time quantitative PCR. Total RNA was reverse transcribed into cDNA using a Takara PrimeScript™ II 1st Strand cDNA Synthesis Kit (6210A) according to the manufacturer's instructions. The inhibition rate of siRNA on TYR mRNA expression in skin tissue was then detected using a Thermo TaqMan™ FastAdvanced Master Mix (4444964). In this quantitative PCR method, the mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control gene. Primers targeting mouse TYR and mouse glyceraldehyde-3-phosphate dehydrogenase were used to detect mTTR and glyceraldehyde-3-phosphate dehydrogenase, respectively. The primer sequences are shown in Table 3.

[0161] In this quantitative real-time PCR method, siRNA inhibition activity is represented by the remaining amount of TYR gene expression 2 - ΔΔCt, where ΔΔCt = [(target gene in Ct experimental group - internal reference in Ct experimental group) – (target gene in Ct negative control group - internal reference in Ct negative control group)]. The mRNA inhibition rate is then calculated using the following formula: mRNA inhibition rate = (1 - remaining amount of mTYR gene expression) × 100%. The inhibition rate results for each experimental group are shown in Table 8.

[0162] Table 8. Inhibition of mTYR mRNA expression in mouse skin by siRNA conjugates

[0163] Verification of technical effectiveness and / or analysis of technical problem solving The results of Examples 6 and 7 show that all ceramide-modified siRNAs exhibited significant inhibitory activity in both mouse melanocyte B16 and human melanocyte MNT1 cell models (B16 inhibition rate: 50.79-80.09%; MNT1 inhibition rate: 37.78-64.17%), which was far superior to the unmodified group, demonstrating the universality and high efficiency of this series of modification strategies.

[0164] The results of Example 8 show that the lipophilic modified siRNA of the present invention exhibits good cytocompatibility with normal HaCaT keratinocytes in the concentration range of 50M, with no obvious toxic reactions and reliable safety. Therefore, lipophilic modification significantly enhances the transdermal delivery capability of siRNA without accompanying significant cytotoxic risks. The resulting siRNA is suitable for the development of transdermal formulations, consistent with the "high safety" technical effect of this invention.

[0165] As shown in the in vivo experimental data of Example 9, the oligonucleotide conjugates of the present invention can significantly inhibit the expression of TYR mRNA, and the inhibition rate is significantly higher than that of the control group. Among them, the effect of experimental groups 1-4 is particularly outstanding, with an inhibition efficiency as high as 67.01%.

[0166] In summary, the ceramide-modified siRNA groups showed significantly higher target gene knockdown efficiency than the unmodified siRNA control group, confirming that ceramide lipophilic modification effectively enhances the transdermal delivery efficiency and gene silencing activity of siRNA. Further analysis of the relationship between ceramide modification with different carbon chain lengths and gene knockdown efficiency revealed that both the transdermal delivery efficiency and target gene knockdown efficiency of siRNA increased with increasing ceramide carbon chain length. Specifically, the knockdown efficiency of the modified siRNA reached its highest level when the ceramide carbon chain length was 18. However, when the ceramide carbon chain length continued to increase beyond 18, both the transdermal delivery efficiency and target gene knockdown efficiency of siRNA decreased. These results indicate that the carbon chain length of ceramide has a significant impact on the transdermal delivery efficiency of modified siRNA, and there exists an optimal carbon chain length range (with a carbon chain length of 18 showing the best effect). This provides important experimental evidence for the design and optimization of the transdermal delivery system of this invention.

[0167] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A lipophilic modified small nucleic acid for transdermal formulations, characterized in that, The compound of general formula (I), its stereoisomers, its tautomers, its crystalline hydrates, its solvates, or its pharmaceutically acceptable salts are all of the same formula (I). Nuc–L–Cer(I); In the formula, Nuc is a small nucleic acid; L is a linker group; and Cer is a lipophilic modifying group.

2. The lipophilic modified small nucleic acid according to claim 1, characterized in that, The small nucleic acid is an oligonucleotide; preferably, the oligonucleotide is selected from small interfering RNA, microRNA, short hairpin RNA, antisense oligonucleotide, CpG oligodeoxynucleotide, aptamer, Dicer substrate interfering RNA or single-stranded siRNA. Preferably, the linking group is selected from amide bonds, ester bonds, disulfide bonds, carbonate bonds, hydrazone bonds, phosphate ester bonds, triazole groups, or carbamate bonds; Preferably, the lipophilic modifying group is selected from cholesterol, fatty acids, ceramides or choline, and more preferably a ceramide group.

3. The lipophilic modified small nucleic acid according to claim 2, characterized in that, The ceramide group has the structure shown in formula (II): (II); Where R1 is C7-C 25 Saturated or unsaturated alkyl chains; R2 is C 13 -C 17 Saturated or unsaturated alkyl chains; Preferably, R1 is C7-C 25 A saturated alkyl chain; more preferably, R1 is C7-C6. 15 saturated alkyl chain, C 15 -C 17 saturated alkyl chain or C 17 -C 23 saturated alkyl chains; Preferably, R2 is C 14 -C 16 Saturated or unsaturated alkyl chains.

4. The lipophilic modified small nucleic acid according to claim 2, characterized in that, The lipophilic modifying group is covalently linked to at least one site of the oligonucleotide via the linking group; preferably, the site is at least one of the 5' end, 3' end, ribose 2'-OH site, phosphate backbone site, pyrimidine base 5 position and purine base 8 position; more preferably, the lipophilic modifying group is covalently linked to the 3' end of the oligonucleotide via the linking group.

5. The lipophilic modified small nucleic acid according to claim 2, characterized in that, In the oligonucleotide, each nucleotide is independently either modified or unmodified. Preferably, the modified nucleotide is selected from at least one of the following: 2'-fluorine modified nucleotide, 2'-O-methyl modified nucleotide, glycerol nucleic acid modified nucleotide, open-ring nucleic acid modified nucleotide, peptide nucleic acid modified nucleotide, 5′-(E)-vinylphosphonic acid modified nucleotide, 2'-amino modified nucleotide, 2'-hydroxy modified nucleotide, 2'-O-allyl modified nucleotide, 2′-O-methoxyethyl modified nucleotide, and 5'-phosphate modified nucleotide; more preferably, it is 2′-fluorine modified nucleotide, 2′-O-methyl modified nucleotide, 2′-O-methoxyethyl modified nucleotide, glycerol nucleic acid modified nucleotide, open-ring nucleic acid modified nucleotide, peptide nucleic acid modified nucleotide, and 5′-(E)-vinylphosphonic acid modified nucleotide.

6. The lipophilic modified small nucleic acid according to claim 2, characterized in that, The oligonucleotide comprises at least one modified nucleotide and / or at least one interstrand modification; preferably, all nucleotides in the oligonucleotide are modified nucleotides. Preferably, the interchain modification includes thiophosphate modification, methylphosphonate modification, and methoxypropylphosphonate modification. Preferably, the oligonucleotide is a small interfering RNA; more preferably, the small interfering RNA comprises a sense strand and an antisense strand; More preferably, the nucleotide linkages in the small interfering RNA are phosphodiester bonds and / or thiophosphate bonds; More preferably, at least one strand of the small interfering RNA is composed of nucleotides selected from natural ribonucleotides and / or modified nucleotides; The lipophilic modification group is covalently linked to the 3′ end of the positive strand of the small interfering RNA via the linker group.

7. The lipophilic modified small nucleic acid according to claim 2, characterized in that, The small interfering RNA is a small interfering RNA that targets tyrosinase; preferably, the small interfering RNA that targets tyrosinase comprises a sense strand of the sequence shown in SEQ ID NO:1 and an antisense strand of the sequence shown in SEQ ID NO:

2. Preferably, the ceramide group is selected from any of the following structures: 。 8. The method for preparing lipophilic modified small nucleic acids according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Couple the lipophilic modification group to the controllable porous glass to obtain a lipophilic... Controllable-pore glass solid support; S2, with lipophilic properties A controllable porous glass solid-phase support was used as a solid-phase carrier to synthesize positive strands of small interfering RNA using the phosphoramide method. After synthesis, ammonolysis and deprotection were performed to obtain crude single-stranded small interfering RNA. S3. The crude single-stranded small interfering RNA was purified to obtain the sense and antisense strands. S4. Anneal equal molar amounts of the sense and antisense strands to form double-stranded small interfering RNA; Preferably, in step S4, the annealing temperature is 85-90°C, the annealing time is 3-5 min, and then the temperature is cooled to room temperature.

9. A composition, characterized in that, The composition comprises the lipophilic modified small nucleic acid according to any one of claims 1-7 or the lipophilic modified small nucleic acid obtained by the preparation method of claim 8; preferably, the composition is a pharmaceutical composition or a cosmetic composition; more preferably, the pharmaceutical composition is an oral preparation, a topical preparation, an intravenous injection, a subcutaneous injection or an intramuscular injection; the cosmetic composition is for topical application, preferably transdermal application.

10. The use of the lipophilic modified small nucleic acid according to any one of claims 1-7, the lipophilic modified small nucleic acid obtained by the preparation method of claim 8, or the composition according to claim 9 in the preparation of pharmaceuticals, cosmetics, or beauty products; preferably, the pharmaceutical is a transdermal formulation; the cosmetic or beauty product is a cosmetic or beauty product with whitening effect.

Images